( C ) Still images from a TIRF time - series of p135 ( green ) binding to either WT ( red ) , or CPA - treated porcine MTs ( blue ) . Right , standard deviation map of p135 binding shows no preference for either type of MT , and quantification of mean fluorescence intensity ( arbitrary units ) per μm MT for p135 bound to CPA - treated MTs relative to WT MTs in the same chamber ( n ≥ 10 MTs quantified for each condition from three independent experiments ) . Scale bars are 5 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Still images from a TIRF time-series of p135 (green) binding to either WT (red), or CPA-treated porcine MTs (blue). Right, standard deviation map of p135 binding shows no preference for either type of MT, and quantification of mean fluorescence intensity (arbitrary units) per μm MT for p135 bound to CPA-treated MTs relative to WT MTs in the same chamber (n ≥ 10 MTs quantified for each condition from three independent experiments). Scale bars are 5 μm.


( B ) Example of a chimeric MT generated from recombinant yeast tubulin containing tyrosinated ( red ) and detyrosinated ( blue ) sections . Most TMR - labeled DDB molecules ( green ) move processively through the annealed junction between the two types of MT lattice ( 65 % , n = 836 processive complexes , 65 MT - MT junctions , three independent experiments ) . Diffusive DDB molecules ( white arrow ) are primarily observed on the tyrosinated section of the MT and do not diffuse across the boundary . Right , video frames showing a processive DDB complex ( pink arrowhead ) moving across the junction ( yellow arrowhead ) of tyrosinated and detryosinated MT . The ( + ) and ( - ) signs denote MT polarity inferred from the directionality of DDB movement . Scale bars are 5 μm and 25 sec . See also Movie EV3 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Example of a chimeric MT generated from recombinant yeast tubulin containing tyrosinated (red) and detyrosinated (blue) sections. Most TMR-labeled DDB molecules (green) move processively through the annealed junction between the two types of MT lattice (65%, n=836 processive complexes, 65 MT-MT junctions, three independent experiments). Diffusive DDB molecules (white arrow) are primarily observed on the tyrosinated section of the MT and do not diffuse across the boundary. Right, video frames showing a processive DDB complex (pink arrowhead) moving across the junction (yellow arrowhead) of tyrosinated and detryosinated MT. The (+) and (-) signs denote MT polarity inferred from the directionality of DDB movement. Scale bars are 5 μm and 25 sec. See also Movie EV3.


( C ) TIRF images of low ( 0 . 3 nM ) and high ( 10 nM ) concentrations of TMR - labeled p150 ( green ) bound to chimeric yeast MTs containing tyrosinated ( red ) and detyrosinated ( blue ) sections of MT . Representative kymographs below show diffusive behavior of p150 molecules on the MT . Note that p150 rarely diffuses into the detyrosinated section of MT , even at high concentrations . Scale bars are 5 μm and 10 sec .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) TIRF images of low (0.3 nM) and high (10 nM) concentrations of TMR-labeled p150 (green) bound to chimeric yeast MTs containing tyrosinated (red) and detyrosinated (blue) sections of MT. Representative kymographs below show diffusive behavior of p150 molecules on the MT. Note that p150 rarely diffuses into the detyrosinated section of MT, even at high concentrations. Scale bars are 5 μm and 10 sec.


( D - E ) TIRFimages and kymographs of annealed WT and CPA - treated porcineMTs ( D ) , or annealed WT MTs ( E ) . The majority of DDB complexes ( 78 % , n = 960 processive complexes , 45 MT - MT junctions , three independent experiments ) move processively across the boundary between WT and CPA - MTs ( D ) . Note TMR - labeled DDB complexes ( green ) in ( E ) traverse the annealed boundary without pausing ( 93 % , n = 677 processive complexes , 45 MT - MT junctions , three independent experiments ) , suggesting that the annealed junction does not affect DDB motility .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D-E) TIRFimages and kymographs of annealed WT and CPA-treated porcineMTs(D), or annealed WT MTs(E). The majority of DDB complexes (78%, n=960 processive complexes, 45 MT-MT junctions, three independent experiments) move processively across the boundary between WT and CPA-MTs(D). Note TMR-labeled DDB complexes (green) in (E) traverse the annealed boundary without pausing (93%, n=677 processive complexes, 45 MT-MT junctions, three independent experiments), suggesting that the annealed junction does not affect DDB motility.


( F ) TIRF image and kymograph from a chimeric MT composed of WT ( red ) and subtilisin - treated porcine MTs ( blue ) . Note some DDB complexes ( pink arrows ) traverse the boundary and continue moving along the subtilisin - treated section of the MT ( 41 % , n = 709 processive complexes , 56 MT - MT junctions , 3 independent experiments ) . All scale bars in D - F are 5 μm and 25 sec .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) TIRF image and kymograph from a chimeric MT composed of WT (red) and subtilisin-treated porcine MTs (blue). Note some DDB complexes (pink arrows) traverse the boundary and continue moving along the subtilisin-treated section of the MT (41%, n=709 processive complexes, 56 MT-MT junctions, 3 independent experiments). All scale bars in D-F are 5 μm and 25 sec.


A . Heatmap of IC50 values for cell viability . Human dermal fibroblasts ( HDFB ) controls and 4 MCC cell lines ( PeTa , MKL - 1 , WaGa , MS - 1 ) were treated with the 43 indicated small molecules targeting epigenetic modifiers . IC50 values are depicted as log10 ( IC50 ) in [ mM ] . n / a , IC50 values could not be calculated . n = 4 technical replicates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. Heatmap of IC50 values for cell viability. Human dermal fibroblasts (HDFB) controls and 4 MCC cell lines (PeTa, MKL-1, WaGa, MS-1) were treated with the 43 indicated small molecules targeting epigenetic modifiers. IC50 values are depicted as log10(IC50) in [mM]. n/a, IC50 values could not be calculated. n = 4 technical replicates.


B . Dose - response curves of PeTa cells and control HDFB cells after 6 days of treatment with GSK - LSD1 or ORY - 1001 . Dose - response curves of 3 other MCC cell lines are displayed in Fig EV1A . n = 4 technical replicates . Data are represented as means ± SD .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B. Dose-response curves of PeTa cells and control HDFB cells after 6 days of treatment with GSK-LSD1 or ORY-1001. Dose-response curves of 3 other MCC cell lines are displayed in Fig EV1A. n = 4 technical replicates. Data are represented as means ± SD.


D . In vitro competition assay of the 3 MCC cell lines MKL - 2 , PeTa , and WaGa transduced with either shLSD1 . 1 , shLSD1 . 2 , shRenilla ( negative control ) , or shRPS15 ( positive control ) . Individual graphs are displayed in Fig EV1E .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D. In vitro competition assay of the 3 MCC cell lines MKL-2, PeTa, and WaGa transduced with either shLSD1.1, shLSD1.2, shRenilla (negative control), or shRPS15 (positive control). Individual graphs are displayed in Fig EV1E.


E . Dependency plot depicting the mean dependency of the 3 MCC cell lines PeTa , MKL - 1 , MKL - 2 of the genes targeted by the compound library in Fig 1A . A score of 0 indicates that a gene is not essential ; correspondingly - 1 is comparable to the median of all pan - essential genes . Data obtained from DepMap ; dependencies for the individual cell lines are displayed in Fig EV1F .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E. Dependency plot depicting the mean dependency of the 3 MCC cell lines PeTa, MKL-1, MKL-2 of the genes targeted by the compound library in Fig 1A. A score of 0 indicates that a gene is not essential; correspondingly -1 is comparable to the median of all pan-essential genes. Data obtained from DepMap; dependencies for the individual cell lines are displayed in Fig EV1F.


F . Violin plot depicting the LSD1 dependency score in MCC compared to cancer types from 23 tissues , ordered according to mean dependency score . Red horizontal line depicts the median . Data obtained from DepMap RNAi screen . Blood , hematopoietic and lymphoid tissue ; U . aerodigestive , upper aerodigestive tract ; A . ganglia , autonomic ganglia ; CNS , central nervous system .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F. Violin plot depicting the LSD1 dependency score in MCC compared to cancer types from 23 tissues, ordered according to mean dependency score. Red horizontal line depicts the median. Data obtained from DepMap RNAi screen. Blood, hematopoietic and lymphoid tissue; U. aerodigestive, upper aerodigestive tract; A. ganglia, autonomic ganglia; CNS, central nervous system.


B . Individual tumor growth in GSK - LSD1 ( n = 9 ) or vehicle - treated ( n = 8 ) mice . Red arrow : start of therapy , day 22

O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O

B. Individual tumor growth in GSK-LSD1 (n = 9) or vehicle-treated (n = 8) mice. Red arrow: start of therapy, day 22


C . Representative picture of mice treated with vehicle or GSK - LSD1 at day 41 after PeTa cell injection . Tumor location is indicated with a circle

O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O

C. Representative picture of mice treated with vehicle or GSK-LSD1 at day 41 after PeTa cell injection. Tumor location is indicated with a circle


D . Kaplan - Meier curve of GSK - LSD1 ( n = 9 ) or vehicle - treated ( n = 8 ) mice . Mice were sacrificed when tumors reached a volume ≥ 1 . 5cm3 or the greatest dimension was ≥ 1 . 5cm . Red arrow : start of therapy , day 22 . * * * * p < 0 . 0001 ( log - rank Mantel - Cox test )

O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D. Kaplan-Meier curve of GSK-LSD1 (n = 9) or vehicle-treated (n = 8) mice. Mice were sacrificed when tumors reached a volume ≥ 1.5cm3 or the greatest dimension was ≥ 1.5cm. Red arrow: start of therapy, day 22. ****p < 0.0001 (log-rank Mantel-Cox test)


F . Individual tumor growth in GSK - LSD1 ( n = 8 ) or vehicle - treated ( n = 8 ) mice . Red arrow : start of therapy , day 1

F. Individual tumor growth in GSK-LSD1 (n = 8) or vehicle-treated (n = 8) mice. Red arrow: start of therapy, day 1


G . Relative mouse weight ( % ) during treatment of GSK - LSD1 ( n = 8 ) or vehicle - treated ( n = 8 ) mice

O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O

G. Relative mouse weight (%) during treatment of GSK-LSD1 (n = 8) or vehicle-treated (n = 8) mice


H . Kaplan - Meier curve of GSK - LSD1 ( n = 8 ) or vehicle - treated ( n = 8 ) mice . Mice were sacrificed when tumors reached a volume ≥ 1 . 5cm3 or greatest dimension ≥ 1 . 5cm . Red arrow : start of therapy , day 1 . * * * * p < 0 . 0001 ( log - rank Mantel - Cox test )

O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

H. Kaplan-Meier curve of GSK-LSD1 (n = 8) or vehicle-treated (n = 8) mice. Mice were sacrificed when tumors reached a volume ≥ 1.5cm3 or greatest dimension ≥ 1.5cm. Red arrow: start of therapy, day 1. ****p < 0.0001 (log-rank Mantel-Cox test)


A . Ki - 67 staining of PeTa cells treated for 3 days with GSK - LSD1 ( 1 μM ) or ORY - 1001 ( 1 μM ) . n = 3 biological replicates . Data are represented as means ± SD . * * * p < 0 . 001 ( DMSO vs GSK - LSD1 p = 0 . 0002 ; DMSO vs ORY - 1001 p = 0 . 0004 ; unpaired Student ' s t - test ) .

O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O

A. Ki-67 staining of PeTa cells treated for 3 days with GSK-LSD1 (1 μM) or ORY-1001 (1 μM). n = 3 biological replicates. Data are represented as means ±SD. ***p < 0.001 (DMSO vs GSK-LSD1 p = 0.0002; DMSO vs ORY-1001 p = 0.0004; unpaired Student's t-test).


B . Representative FACS readout of EdU / Propidium iodide cell cycle staining of PeTa cells after 6 days of 100 nM GSK - LSD1 or DMSO treatment . C . Quantification of EdU / Propidium iodide staining depicted in Fig 3B . n = 3 biological replicates . Data are represented as means ± SD . * * p < 0 . 01 ; * * * * p < 0 . 0001 ; ns , non - significant ( G1 phase : DMSO vs GSK - LSD1 p = 0 . 0059 ; S phase : DMSO vs GSL - LSD1 p < 0 . 0001 ; G2 / M phase : DMSO vs GSK - LSD1 p = 0 . 2338 ; unpaired Student ' s t - test ) .

O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O

B. Representative FACS readout of EdU/Propidium iodide cell cycle staining of PeTa cells after 6 days of 100 nM GSK-LSD1 or DMSO treatment. C. Quantification of EdU/Propidium iodide staining depicted in Fig 3B. n = 3 biological replicates. Data are represented as means ±SD. **p<0.01; ****p<0.0001; ns, non-significant (G1 phase: DMSO vs GSK-LSD1 p = 0.0059; S phase: DMSO vs GSL-LSD1 p<0.0001; G2/M phase: DMSO vs GSK-LSD1 p = 0.2338; unpaired Student's t-test).


D . Immunoblot of PARP1 cleavage of PeTa cells after 6 days of indicated treatment . Etoposide serves as positive control for apoptosis , H3 serves as loading control . c . PARP1 , cleaved PARP1 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D. Immunoblot of PARP1 cleavage of PeTa cells after 6 days of indicated treatment. Etoposide serves as positive control for apoptosis, H3 serves as loading control. c.PARP1, cleaved PARP1.


E . Caspase - 3 / 7 cleavage activity for PeTa cell line after 6 days of indicated treatment . Etoposide serves as a positive control for apoptosis . n = 3 technical replicates . Data are represented as means ± SD . RLU , relative luminescence units .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E. Caspase-3/7 cleavage activity for PeTa cell line after 6 days of indicated treatment. Etoposide serves as a positive control for apoptosis. n = 3 technical replicates. Data are represented as means ±SD. RLU, relative luminescence units.


F . Representative FACS readout of tetramethylrhodamine ethyl ester ( TMRE ) stained cells upon 100 nM GSK - LSD1 or DMSO treatment after 6 days . G . Quantification of tetramethylrhodamine ethyl ester ( TMRE ) stained cells upon 100 nM GSK - LSD1 or DMSO treatment at indicated time points .

O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O

F. Representative FACS readout of tetramethylrhodamine ethyl ester (TMRE) stained cells upon 100 nM GSK-LSD1 or DMSO treatment after 6 days. G. Quantification of tetramethylrhodamine ethyl ester (TMRE) stained cells upon 100 nM GSK-LSD1 or DMSO treatment at indicated time points.


H . Representative FACS readout of Annexin V / DAPI staining of PeTa cells after 6 days of 100 nM GSK - LSD1 DMSO treatment . I . Quantification of Annexin V / DAPI staining depicted in Fig 3H . n = 3 biological replicates . Data are represented as means ± SD . * * * * p < 0 . 0001 ( Early , Late , Total ; unpaired Student ' s t - test ) .

O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

H. Representative FACS readout of Annexin V/DAPI staining of PeTa cells after 6 days of 100 nM GSK-LSD1 DMSO treatment. I. Quantification of Annexin V/DAPI staining depicted in Fig 3H. n = 3 biological replicates. Data are represented as means ±SD. ****p<0.0001 (Early, Late, Total; unpaired Student's t-test).


J . Quantification of in vitro TUNEL signal of PeTa cells after 6 days of 100 nM GSK - LSD1 or vehicle treatment . Representative images in Appendix Fig S1B . n = 8 , * * * p = 0 . 001 ( Vehicle vs GSK - LSD1 ; unpaired Student ' s t - test ) . ROI , region of interest .

O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O

J. Quantification of in vitro TUNEL signal of PeTa cells after 6 days of 100 nM GSK-LSD1 or vehicle treatment. Representative images in Appendix Fig S1B. n = 8, ***p = 0.001 (Vehicle vs GSK-LSD1; unpaired Student's t-test). ROI, region of interest.


K . Top . Representative images of immunofluorescent Ki - 67 staining of tumor slides after 10 days of in vivo GSK - LSD1 or vehicle treatment . Upper right scale bar represents 100 μm , insert scale bar represents 20 μm . Bottom . Quantification of Ki - 67 signal of tumor slides from mice treated with GSK - LSD1 or DMSO for 1 day , 10 days or until experiment endpoint . n = 15 , * * p < 0 . 01 , * * * * p < 0 . 0001 ( D1 vehicle vs D1 GSK - LSD1 p = 0 . 0015 , unpaired Student ' s t - test ; D10 vehicle vs D10 GSK - LSD1 p < 0 . 0001 , unpaired Student ' s t - test ; Endpoint vehicle vs Endpoint GSK - LSD1 p = 0 . 0012 , Mann - Whitney test ) . ROI , region of interest .

O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O

K. Top. Representative images of immunofluorescent Ki-67 staining of tumor slides after 10 days of in vivo GSK-LSD1 or vehicle treatment. Upper right scale bar represents 100 μm, insert scale bar represents 20 μm. Bottom. Quantification of Ki-67 signal of tumor slides from mice treated with GSK-LSD1 or DMSO for 1 day, 10 days or until experiment endpoint. n = 15, **p<0.01, ****p<0.0001 (D1 vehicle vs D1 GSK-LSD1 p = 0.0015, unpaired Student's t-test; D10 vehicle vs D10 GSK-LSD1 p<0.0001, unpaired Student's t-test; Endpoint vehicle vs Endpoint GSK-LSD1 p = 0.0012, Mann-Whitney test). ROI, region of interest.


L . Top . Representative images of immunofluorescent TUNEL staining of tumor slides after 10 days of in vivo GSK - LSD1 or vehicle treatment . Upper right scale bar represents 100 μm , insert scale bar represents 20 μm . Bottom . Quantification of TUNEL signal of tumor slides from mice treated with GSK - LSD1 or DMSO for 1 day , 10 days or until experiment endpoint . n = 15 , * * * p < 0 . 001 ( D1 vehicle vs D1 GSK - LSD1 p = 0 . 0009 ; D10 vehicle vs D10 GSK - LSD1 p = 0 . 0001 ; Endpoint vehicle vs Endpoint GSK - LSD1 p = 0 . 001 ; unpaired Student ' s t - test with Welch ' s correction ) . ROI , region of interest .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O

L. Top. Representative images of immunofluorescent TUNEL staining of tumor slides after 10 days of in vivo GSK-LSD1 or vehicle treatment. Upper right scale bar represents 100 μm, insert scale bar represents 20 μm. Bottom. Quantification of TUNEL signal of tumor slides from mice treated with GSK-LSD1 or DMSO for 1 day, 10 days or until experiment endpoint. n = 15, ***p<0.001 (D1 vehicle vs D1 GSK-LSD1 p = 0.0009; D10 vehicle vs D10 GSK-LSD1 p = 0.0001; Endpoint vehicle vs Endpoint GSK-LSD1 p = 0.001; unpaired Student's t-test with Welch's correction). ROI, region of interest.


M . Representative images of hematoxylin and eosin ( H & E ) staining of tumor slides from mice treated with GSK - LSD1 or DMSO for 1 day ( D1 , top ) , 10 days ( D10 , middle ) or until experiment endpoint ( bottom ) . Upper right scale bar represents 50 μm , insert scale bar represents 10 μm .

O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

M. Representative images of hematoxylin and eosin (H&E) staining of tumor slides from mice treated with GSK-LSD1 or DMSO for 1 day (D1, top), 10 days (D10, middle) or until experiment endpoint (bottom). Upper right scale bar represents 50 μm, insert scale bar represents 10 μm.


A . Photomicrographs of the MCC cell line PeTa after 6 days of 100 nM GSK - LSD1 or DMSO treatment . Scale bar , 100 μm .

O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O

A. Photomicrographs of the MCC cell line PeTa after 6 days of 100 nM GSK-LSD1 or DMSO treatment. Scale bar, 100 μm.


B . Metagene plots showing the average CUT & RUN signal for H3K4me1 , H3K27ac , and H3K27me3 for differentially expressed genes after 6 days of LSD1i or DMSO treatment . kb , kilobase ; TSS , transcription start site ; TES transcription end site .

O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B. Metagene plots showing the average CUT&RUN signal for H3K4me1, H3K27ac, and H3K27me3 for differentially expressed genes after 6 days of LSD1i or DMSO treatment. kb, kilobase; TSS, transcription start site; TES transcription end site.


C . ATAC - seq peak profile around TSS of differentially expressed genes after 6 days of LSD1i or DMSO treatment . kb , kilobase ; TSS , transcription start

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C. ATAC-seq peak profile around TSS of differentially expressed genes after 6 days of LSD1i or DMSO treatment. kb, kilobase; TSS, transcription start site


D . Volcano plot showing the - log10 ( adjusted p - value ) and log2 fold - change ( log2FC ) for transcripts detected by RNAseq analysis of PeTa cells treated with 100 nM GSK - LSD1 or DMSO for 6 days . Significantly down - regulated genes ( FDR ≤ 0 . 05 ; log2FC ≤ - 1 ) involved in mitotic cell cycle ( GO : 0000278 ) are highlighted in blue . E . Upstream promoter motif enrichment of the downregulated genes from the dataset in Fig 4D . Top - 5 enriched motifs are depicted . TF , transcription factor .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D. Volcano plot showing the -log10 (adjusted p-value) and log2 fold-change (log2FC) for transcripts detected by RNAseq analysis of PeTa cells treated with 100 nM GSK-LSD1 or DMSO for 6 days. Significantly down-regulated genes (FDR≤0.05; log2FC≤-1) involved in mitotic cell cycle (GO:0000278) are highlighted in blue. E. Upstream promoter motif enrichment of the downregulated genes from the dataset in Fig 4D. Top-5 enriched motifs are depicted. TF, transcription factor.


O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O


B . MA - plot of the mRNA changes detected by SLAMseq after 1h and 6h of 4sU labelling . Significantly up - and downregulated genes ( FDR ≤ 0 . 05 ; abs ( log2FC ) ≥ 0 . 5 ) are marked in red and blue , respectively . Genes belonging to neuronal differentiation ( GO : 0048666 ) are labelled . FC , fold - change ; CMP , counts per million .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B. MA-plot of the mRNA changes detected by SLAMseq after 1h and 6h of 4sU labelling. Significantly up- and downregulated genes (FDR≤0.05; abs(log2FC)≥0.5) are marked in red and blue, respectively. Genes belonging to neuronal differentiation (GO:0048666) are labelled. FC, fold-change; CMP, counts per million.


H . Gene set enrichment analysis ( GSEA ) of the generated Merkel cell signature on the data in Fig 4B . NES , normalized enrichment score ; FDR , false discovery rate .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

H. Gene set enrichment analysis (GSEA) of the generated Merkel cell signature on the data in Fig 4B. NES, normalized enrichment score; FDR, false discovery rate.


A . Schematics and immunoblot of LSD1 co - immunoprecipitation ( coIP ) from PeTa cells . IB , Immunoblot ; MS , mass spectrometry .

O O O O O O O O O O O O O O O O O O O O O O O O O O

A. Schematics and immunoblot of LSD1 co-immunoprecipitation (coIP) from PeTa cells. IB, Immunoblot; MS, mass spectrometry.


B . Volcano plot displaying the protein - protein binding partners of LSD1 . Proteins belonging to the LSD1 complex are marked in red , protein localization in blue and other protein interaction partners in green . FC , fold - change . C . Protein - protein interaction ( PPI ) mapping of the identified LSD1 binding partners . Individual complexes and p - values are displayed in Fig EV 6A .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B. Volcano plot displaying the protein-protein binding partners of LSD1. Proteins belonging to the LSD1 complex are marked in red, protein localization in blue and other protein interaction partners in green. FC, fold-change. C. Protein-protein interaction (PPI) mapping of the identified LSD1 binding partners. Individual complexes and p-values are displayed in Fig EV 6A.


D . Volcano plot depicting the protein - protein binding partners of LSD1 depleting in PeTa cells treated with ORY - 1001 versus DMSO . Proteins belonging to the LSD1 complex are marked in red , protein localization in blue and other protein interaction partners in green . FC , fold - change .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D. Volcano plot depicting the protein-protein binding partners of LSD1 depleting in PeTa cells treated with ORY-1001 versus DMSO. Proteins belonging to the LSD1 complex are marked in red, protein localization in blue and other protein interaction partners in green. FC, fold-change.


E . Violin plot depicting the HMG20B dependency in MCC compared to cancer types from 23 tissues , ordered according to mean . Red horizontal line depicts the median . Data obtained from DepMap RNAi screen . Blood , hematopoietic and lymphoid tissue ; U . aerodigestive , upper aerodigestive tract ; A . ganglia , autonomic ganglia ; CNS , central nervous system .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E. Violin plot depicting the HMG20B dependency in MCC compared to cancer types from 23 tissues, ordered according to mean. Red horizontal line depicts the median. Data obtained from DepMap RNAi screen. Blood, hematopoietic and lymphoid tissue; U. aerodigestive, upper aerodigestive tract; A. ganglia, autonomic ganglia; CNS, central nervous system.


G . Volcano plot depicting the differentially regulated proteins obtained from Fig 6F . Proteins members of the LSD1 - CoREST complex are labeled and highlighted in red . Proteins significantly upregulated ( - log10 ( p - adjusted ) ≥ 1 ; log2FC ≥ 0 . 75 ) and involved in nervous system development ( GO : 0007399 ) are labelled . FC , fold - change .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G. Volcano plot depicting the differentially regulated proteins obtained from Fig 6F. Proteins members of the LSD1-CoREST complex are labeled and highlighted in red. Proteins significantly upregulated (-log10(p-adjusted)≥1; log2FC≥0.75) and involved in nervous system development (GO:0007399) are labelled. FC, fold-change.


H . Left . Immunoblot confirmation of HMG20B downregulation . H3 serves as nuclear fraction control and α - Tubulin as loading control . Right . Immunoblot quantification . HMG20B protein levels are normalized to H3 and relative to DMSO control .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

H. Left. Immunoblot confirmation of HMG20B downregulation. H3 serves as nuclear fraction control and α-Tubulin as loading control. Right. Immunoblot quantification. HMG20B protein levels are normalized to H3 and relative to DMSO control.


K . Bar graph of HMG20B rescue experiment in PeTa cells transduced with either a shRNA targeting HMG20B ( shHMG20B ) or negative control ( shRenilla ) and with an overexpression construct expressing either of the HMG20B mutant that cannot be targeted by the shRNA . Cell survival is depicted at day 8 after transduction and normalized to shRenilla . EV , empty vector control , WT , wild - type ; ΔALPHA , alpha - helices deletion ; ΔCC , coiled - coil domain deletion ; ΔHMG , HMG box deletion .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

K. Bar graph of HMG20B rescue experiment in PeTa cells transduced with either a shRNA targeting HMG20B (shHMG20B) or negative control (shRenilla) and with an overexpression construct expressing either of the HMG20B mutant that cannot be targeted by the shRNA. Cell survival is depicted at day 8 after transduction and normalized to shRenilla. EV, empty vector control, WT, wild-type; ΔALPHA, alpha-helices deletion; ΔCC, coiled-coil domain deletion; ΔHMG, HMG box deletion.


B . RT - qPCR quantification of neuronal genes at the different time points and conditions depicted in Fig 7A . Data are relative to the housekeeping gene HPRT1 and normalized to the respective DMSO control . n = 4 technical replicates . Bar graphs represent mean ± SD .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B. RT-qPCR quantification of neuronal genes at the different time points and conditions depicted in Fig 7A. Data are relative to the housekeeping gene HPRT1 and normalized to the respective DMSO control. n = 4 technical replicates. Bar graphs represent mean ± SD.


C . Immunoblot probing for sustainability of LSD1 activity inhibition at the different time points and conditions depicted in Fig 7A . α - Tubulin serves as loading control . pSMAD1 / 5 / 9 , phospho - SMAD1 / 5 / 9 . D . Immunoblot probing for LSD1 - CoREST complex members at the different time points and conditions depicted in Fig 7A . α - Tubulin serves as loading control .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C. Immunoblot probing for sustainability of LSD1 activity inhibition at the different time points and conditions depicted in Fig 7A. α-Tubulin serves as loading control. pSMAD1/5/9, phospho-SMAD1/5/9. D. Immunoblot probing for LSD1-CoREST complex members at the different time points and conditions depicted in Fig 7A. α-Tubulin serves as loading control.


E . Representative FACS readout of EdU / Propidium iodide cell cycle staining of PeTa cells 8 days after drug wash - out . F . Quantification of EdU / Propidium iodide staining depicted in Fig 7E . n = 3 biological replicates . Data are represented as means ± SD . * p < 0 . 05 , * * p < 0 . 01 ; * * * p < 0 . 001 ( G1 phase : DMSO vs GSK - LSD1 p = 0 . 0104 ; S phase : DMSO vs GSK - LSD1 p = 0 . 0001 ; G2 / M phase : DMSO vs GSK - LSD1 p = 0 . 0062 ; unpaired Student ' s t - test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O

E. Representative FACS readout of EdU/Propidium iodide cell cycle staining of PeTa cells 8 days after drug wash-out. F. Quantification of EdU/Propidium iodide staining depicted in Fig 7E. n = 3 biological replicates. Data are represented as means ±SD. *p<0.05, **p<0.01; ***p<0.001 (G1 phase: DMSO vs GSK-LSD1 p = 0.0104; S phase: DMSO vs GSK-LSD1 p = 0.0001; G2/M phase: DMSO vs GSK-LSD1 p = 0.0062; unpaired Student's t-test).


G . Quantification of FACS Annexin V / DAPI staining of PeTa cells 8 days after drug wash - out . n = 3 biological replicates . Data are represented as means ± SD . * * * * p < 0 . 0001 ( unpaired Student ' s t - test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G. Quantification of FACS Annexin V/DAPI staining of PeTa cells 8 days after drug wash-out. n = 3 biological replicates. Data are represented as means ±SD. ****p<0.0001 (unpaired Student's t-test).


I . Subcutaneous tumor growth of pretreated cells ( untreated , n = 8 ; D1 , n = 9 ; D3 , n = 9 ; D6 , n = 4 ) Data are represented as means ± SEM . * p < 0 . 05 , * * * p < 0 . 001 ( untreated vs D1 p = 0 . 023 ; untreated vs D3 p = 0 . 023 ; untreated vs D6 p = 0 . 0007 ; Kruskal - Wallis test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

I. Subcutaneous tumor growth of pretreated cells (untreated, n = 8; D1, n = 9; D3, n = 9; D6, n = 4) Data are represented as means ±SEM. *p<0.05, ***p<0.001 (untreated vs D1 p = 0.023; untreated vs D3 p = 0.023; untreated vs D6 p = 0.0007; Kruskal-Wallis test).


C - F . Polysome profiling of yeast cells with ( C - E ) or without ( F ) cycloheximide ( CHX ) treatment , immunoblots for Stm1 and Rpl3 in polysome fractions , and quantification of Stm1 distribution across the polysome profiles of cells grown under nutrient - rich condition ( C , F ) , rapamycin treatment for 1 hour ( D ) , or nitrogen starvation for 1 hour ( E ) . Individual polysome profiles were fractionated on a 5 - 50 % sucrose density gradient containing 150 mM KCl into 9 fractions , precipitated and immunoblotted for Stm1 and Rpl3 . Rpl3 is used as a loading control . Each experiment was repeated at least three times .

O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C-F. Polysome profiling of yeast cells with (C-E) or without (F) cycloheximide (CHX) treatment, immunoblots for Stm1 and Rpl3 in polysome fractions, and quantification of Stm1 distribution across the polysome profiles of cells grown under nutrient-rich condition (C, F), rapamycin treatment for 1 hour (D), or nitrogen starvation for 1 hour (E). Individual polysome profiles were fractionated on a 5-50% sucrose density gradient containing 150 mM KCl into 9 fractions, precipitated and immunoblotted for Stm1 and Rpl3. Rpl3 is used as a loading control. Each experiment was repeated at least three times.


G . Polysome profiles of stm1 ∆ cells containing either full - length or N - terminally deleted or C - terminally deleted Stm1 mutants upon nitrogen starvation for 1 hour .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G. Polysome profiles of stm1∆ cells containing either full-length or N-terminally deleted or C-terminally deleted Stm1 mutants upon nitrogen starvation for 1 hour.


H . Binding of full - length or deletion mutants of Stm1 across the polysome profile fractions shown in Figure 2E . 9 equal volume fractions of each polysome profile were precipitated and analyzed by immunoblot for Stm1 and Rpl3 . At least two biological replicates are used for each mutant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

H. Binding of full-length or deletion mutants of Stm1 across the polysome profile fractions shown in Figure 2E. 9 equal volume fractions of each polysome profile were precipitated and analyzed by immunoblot for Stm1 and Rpl3. At least two biological replicates are used for each mutant.


A - B . In vitro kinase assay ( A ) and its quantitation ( B ) using recombinant mTORC1 and recombinant Stm1 or Stm1AAA as a substrate in the presence of 32P - γ - ATP . Immunoblots for Stm1 and Raptor are shown . Asterisk indicates the auto - phosphorylation of a mTORC1 component . INK128 is used as an inhibitor of mTOR . Minimum of three replicates are used for statistical analysis by unpaired t - test . Data are presented as the mean ± SD .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A-B. In vitro kinase assay (A) and its quantitation (B) using recombinant mTORC1 and recombinant Stm1 or Stm1AAA as a substrate in the presence of 32P-γ-ATP. Immunoblots for Stm1 and Raptor are shown. Asterisk indicates the auto-phosphorylation of a mTORC1 component. INK128 is used as an inhibitor of mTOR. Minimum of three replicates are used for statistical analysis by unpaired t-test. Data are presented as the mean ± SD.


C . Polysome profiles of wild - type and stm1 ∆ cells under nitrogen starvation for 1 hour . D . Polysome profiles of wild - type and STM1AAA cells under nitrogen starvation for 1 hour . E . Polysome profiles of wild - type and STM1EEE cells under nitrogen starvation for 1 hour . F -

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C. Polysome profiles of wild-type and stm1∆ cells under nitrogen starvation for 1 hour. D. Polysome profiles of wild-type and STM1AAA cells under nitrogen starvation for 1 hour. E. Polysome profiles of wild-type and STM1EEE cells under nitrogen starvation for 1 hour. F-


A - B . Ribosomal subunit profiles ( A ) and quantitation of ribosome content ( B ) of stm1 ∆ , wild - type , STM1AAA , and STM1EEE cells treated with rapamycin for 24 hours . For the ribosomal subunit profile analyses , the cell extracts were treated with 50 mM EDTA and separated on 5 - 25 % sucrose density gradients . At least five biological replicates are used for calculation of p - values from multiple unpaired t - tests are indicated in the graphs .

A-B. Ribosomal subunit profiles (A) and quantitation of ribosome content (B) of stm1∆, wild-type, STM1AAA, and STM1EEE cells treated with rapamycin for 24 hours. For the ribosomal subunit profile analyses, the cell extracts were treated with 50 mM EDTA and separated on 5-25% sucrose density gradients. At least five biological replicates are used for calculation of p-values from multiple unpaired t-tests are indicated in the graphs.


E - G . Immunoblot of Rpl24 - GFP and free GFP ( E ) , quantification of total GFP relative to Pgk1 ( F ) , and free GFP relative to Rpl24 - GFP ( G ) in stm1 ∆ , wild - type , STM1AAA , and STM1EEE cells treated with rapamycin for 24 hours . At least six biological replicates are subjected to statistical analysis using multiple unpaired t - tests . ( s . e . for short exposure ; l . e . for long exposure ) .

E-G. Immunoblot of Rpl24-GFP and free GFP (E), quantification of total GFP relative to Pgk1 (F), and free GFP relative to Rpl24-GFP (G) in stm1∆, wild-type, STM1AAA, and STM1EEE cells treated with rapamycin for 24 hours. At least six biological replicates are subjected to statistical analysis using multiple unpaired t-tests. (s.e. for short exposure; l.e. for long exposure).


J - K . Immunoblot ( J ) and the quantification ( K ) of Rpl3 in wild - type and stm1 ∆ cells without rapamycin , or with rapamycin for 24 hours along with or without bortezomib treatment for 8 hours . At least six biological replicates are subjected to statistical analysis using multiple unpaired t - tests .

O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O

J-K. Immunoblot (J) and the quantification (K) of Rpl3 in wild-type and stm1∆ cells without rapamycin, or with rapamycin for 24 hours along with or without bortezomib treatment for 8 hours. At least six biological replicates are subjected to statistical analysis using multiple unpaired t-tests.


A - B . Puromycin incorporation ( A ) and its quantification ( B ) in stm1 ∆ , wild - type , STM1AAA , and STM1EEE cells starved of nitrogen for 24 hours and restimulated with amino acids ( + N ) for 10 , 20 , or 30 minutes . Immunoblots for puromycin , Stm1 , Rpl3 , and Pgk1 are shown . Relative puromycin incorporation for at least five biological replicates is subjected to multiple unpaired t - tests .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A-B. Puromycin incorporation (A) and its quantification (B) in stm1∆, wild-type, STM1AAA, and STM1EEE cells starved of nitrogen for 24 hours and restimulated with amino acids (+ N) for 10, 20, or 30 minutes. Immunoblots for puromycin, Stm1, Rpl3, and Pgk1 are shown. Relative puromycin incorporation for at least five biological replicates is subjected to multiple unpaired t-tests.


D - E . Representative fluorescent microscopic images for expression of Rpl24 - GFP ( D ) and its intensity quantification ( E ) in tetrads of control ( RPL24GFP ) and stm1 ∆ RPL24GFP cells ( n = 100 , each ) . Experiments are performed at least three times . Statistical analysis is done by unpaired t - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D-E. Representative fluorescent microscopic images for expression of Rpl24-GFP (D) and its intensity quantification (E) in tetrads of control (RPL24GFP) and stm1∆ RPL24GFP cells (n=100, each). Experiments are performed at least three times. Statistical analysis is done by unpaired t-test.


A - C . Polysome profile and immunoblotting of SERBP1 , RPS6 , and RPL7a across the polysome fractions of HEK293T cells treated with DMSO ( A ) , INK128 ( B ) , or harringtonine ( C ) for 1 hour . Polysome profiles were performed using 5 - 50 % sucrose density gradients containing 150 mM KCl .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O

A-C. Polysome profile and immunoblotting of SERBP1, RPS6, and RPL7a across the polysome fractions of HEK293T cells treated with DMSO (A), INK128 (B), or harringtonine (C) for 1 hour. Polysome profiles were performed using 5-50% sucrose density gradients containing 150 mM KCl.


I - J . Immunoblot for ubiquitin ( I ) and its quantification ( J ) in the supernatant and ribosomal pellet obtained from HEK293T cells treated with control sgRNA ( sgCtrl ) or SERBP1 sgRNA ( sgSERBP1 ) along with INK128 treatment for 48 hours . At least four biological replicates are analyzed by unpaired t - test . Data are presented as the mean ± SD .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

I-J. Immunoblot for ubiquitin (I) and its quantification (J) in the supernatant and ribosomal pellet obtained from HEK293T cells treated with control sgRNA (sgCtrl) or SERBP1 sgRNA (sgSERBP1) along with INK128 treatment for 48 hours. At least four biological replicates are analyzed by unpaired t-test. Data are presented as the mean ± SD.


A Control animals ( + / w ; UAS - ralIR / + ) , n = 20 , and those with salivary gland - specific knockdown of ral ( fkh - GAL4 / w ; UAS - ralIR / + ) , n = 20 , were analyzed by histology for the presence of salivary gland material ( red dotted circle ) 24 hours after puparium formation . B Quantification of data from ( A ) . Data are represented as means . Statistical significance was determined using a Chi - square test . C Control animals ( + / w ; UAS - ralS25N / + ) , n = 20 , and those with salivary gland - specific expression of dominant - negative Ral ( fkh - GAL4 / w ; UAS - ralS25N / + ) , n = 20 , were analyzed by histology for the presence of salivary gland material ( red dotted circle ) 24 hours after puparium formation . D Quantification of data from ( C ) . Data are represented as means . Statistical significance was determined using a Chi - square test . E Control animals ( ral35d / + ) , n = 25 , and ral hypomorph mutants ( ral35d / Y ) , n = 23 , were analyzed by histology for the presence of salivary gland material ( red dotted circle ) 24 hours after puparium formation . F Quantification of data from ( E ) . Data are represented as means . Statistical significance was determined using a Chi - square test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A Control animals (+/w; UAS-ralIR/+), n= 20, and those with salivary gland-specific knockdown of ral (fkh-GAL4/w; UAS-ralIR/+), n=20, were analyzed by histology for the presence of salivary gland material (red dotted circle) 24 hours after puparium formation.B Quantification of data from (A). Data are represented as means. Statistical significance was determined using a Chi-square test.C Control animals (+/w; UAS-ralS25N/+), n= 20, and those with salivary gland-specific expression of dominant-negative Ral (fkh-GAL4/w; UAS-ralS25N/+), n= 20, were analyzed by histology for the presence of salivary gland material (red dotted circle) 24 hours after puparium formation.D Quantification of data from (C). Data are represented as means. Statistical significance was determined using a Chi-square test.E Control animals (ral35d/+), n= 25, and ral hypomorph mutants (ral35d/Y), n= 23, were analyzed by histology for the presence of salivary gland material (red dotted circle) 24 hours after puparium formation.F Quantification of data from (E). Data are represented as means. Statistical significance was determined using a Chi-square test.


G Control animals ( + / w ; UAS - rglIR / + ) , n = 19 , and those with salivary gland - specific knockdown of rgl ( fkh - GAL4 / w ; UAS - rglIR / + ) , n = 20 , were analyzed by histology for the presence of salivary gland material ( red dotted circle ) 24 hours after puparium formation . H Quantification of data from ( G ) . Data are represented as means . Statistical significance was determined using a Chi - square test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G Control animals (+/w; UAS-rglIR/+), n= 19, and those with salivary gland-specific knockdown of rgl (fkh-GAL4/w; UAS-rglIR/+), n=20, were analyzed by histology for the presence of salivary gland material (red dotted circle) 24 hours after puparium formation.H Quantification of data from (G). Data are represented as means. Statistical significance was determined using a Chi-square test.


A ral hypomorph mutants ( ral35d / Y ; ; UAS - p35 / + ) , n = 20 , animals with salivary gland - specific expression of p35 ( ral35d / + ; ; UAS - p35 / fkh - GAL4 ) , n = 21 , and ral hypomorph mutants with salivary gland - specific expression of p35 ( ral35d / Y ; ; UAS - p35 / fkh - GAL4 ) , n = 20 , were analyzed by histology for the presence of salivary gland material ( red dotted circles ) 24 hours after puparium formation . B Quantification of data from ( A ) . Data are represented as means . Statistical significance was determined using a Chi - square test comparing the percentages of gland fragments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A ral hypomorph mutants (ral35d/Y;; UAS-p35/+), n= 20, animals with salivary gland-specific expression of p35 (ral35d/+;; UAS-p35/fkh-GAL4), n= 21, and ral hypomorph mutants with salivary gland-specific expression of p35 (ral35d/Y;; UAS-p35/fkh-GAL4), n= 20, were analyzed by histology for the presence of salivary gland material (red dotted circles) 24 hours after puparium formation.B Quantification of data from (A). Data are represented as means. Statistical significance was determined using a Chi-square test comparing the percentages of gland fragments.


C Salivary glands dissected from 0 hour after puparium formation control animals ( + / w ; UAS - ralIR / + ) and those with salivary gland - specific knockdown of ral ( fkh - GAL4 / w ; UAS - ralIR / + ) and stained with cleaved Caspase - 3 antibody ( green ) and DAPI ( blue ) . Scale bars represent 20µm . D Salivary glands dissected from 13 hour after puparium formation control animals ( + / w ; UAS - ralIR / + ) and those with salivary gland - specific knockdown of ral ( fkh - GAL4 / w ; UAS - ralIR / + ) and stained with cleaved Caspase - 3 antibody ( green ) and DAPI ( blue ) . Scale bars represent 20µm . E Quantification of data from ( C and D ) . Data are represented as means ± SEM ; n ≥ 10 . Statistical significance was determined using a Student ' s t test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C Salivary glands dissected from 0 hour after puparium formation control animals (+/w; UAS-ralIR/+) and those with salivary gland-specific knockdown of ral (fkh-GAL4/w; UAS-ralIR/+) and stained with cleaved Caspase-3 antibody (green) and DAPI (blue). Scale bars represent 20µm.D Salivary glands dissected from 13 hour after puparium formation control animals (+/w; UAS-ralIR/+) and those with salivary gland-specific knockdown of ral (fkh-GAL4/w; UAS-ralIR/+) and stained with cleaved Caspase-3 antibody (green) and DAPI (blue). Scale bars represent 20µm.E Quantification of data from (C and D). Data are represented as means ± SEM; n≥ 10. Statistical significance was determined using a Student's t test.


A ral hypomorph mutants ( ral35d / Y ; ; UAS - Atg6IR / + ) , n = 27 , animals with salivary gland - specific expression of Atg6IR ( ral35d / + ; ; UAS - Atg6IR / fkh - GAL4 ) , n = 20 , and ral hypomorph mutants with salivary gland - specific expression of Atg6IR ( ral35d / Y ; ; UAS - Atg6IR / fkh - GAL4 ) , n = 24 , were analyzed by histology for the presence of salivary gland material ( red dotted circles ) 24 hours after puparium formation . B Quantification of data from ( A ) . Data are represented as means . Statistical significance was determined using a Chi - square test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A ral hypomorph mutants (ral35d/Y;; UAS-Atg6IR/+), n= 27, animals with salivary gland-specific expression of Atg6IR (ral35d/+;; UAS-Atg6IR/fkh-GAL4), n= 20, and ral hypomorph mutants with salivary gland-specific expression of Atg6IR (ral35d/Y;; UAS-Atg6IR/fkh-GAL4), n= 24, were analyzed by histology for the presence of salivary gland material (red dotted circles) 24 hours after puparium formation.B Quantification of data from (A). Data are represented as means. Statistical significance was determined using a Chi-square test.


C Salivary glands dissected from 14 hours after puparium formation animals expressing mCherry - Atg8a in all cells , and ralIR specifically in GFP - marked clone cells ( yw , hsflp / w ; pmCherry - Atg8a / UAS - ralIR ; act < FRT , cd2 , FRT > GAL4 , UAS - GFP / + ) analyzed for mCherry - Atg8a puncta . GFP - marked clone ralIR - expressing cells contain a yellow asterisk . Scale bars represent 20µm . D Quantification of data from ( C ) . Data are represented as means ± SEM ; n ≥ 10 . Statistical significance was determined using a Student ' s t test . E Salivary glands dissected from 14 hours after puparium formation animals that have ral loss of function ( ralPG89 ) specifically in non - RFP cells , and that express GFP - Atg8a in all cells ( yw , hs - Flp , FRT19A , Ubi - RFP / ralPG89 , FRT19A ; pGFP - Atg8a ) analyzed for GFP - Atg8a puncta . ralPG89 mutant cells are marked with a yellow asterisk . Scale bars represent 20µm . F Quantification of data from ( E ) . Data are represented as means ± SEM ; n ≥ 10 . Statistical significance was determined using a Student ' s t test . G Salivary glands dissected from 14 hours after puparium formation animals that have ral loss of function ( ralPG89 ) specifically in non - RFP cells ( yw , hs - Flp , FRT19A , Ubi - RFP / ralPG89 , FRT19A ) and are stained for Ref ( 2 ) p ( green ) and DAPI ( blue ) . ralPG89 mutant cells are marked with a yellow asterisk . Scale bars represent 20µm . H Quantification of data from ( G ) . Data are represented as means ± SEM ; n ≥ 10 . Statistical significance was determined using a Student ' s t test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C Salivary glands dissected from 14 hours after puparium formation animals expressing mCherry-Atg8a in all cells, and ralIR specifically in GFP-marked clone cells (yw, hsflp/w; pmCherry-Atg8a/UAS-ralIR; act< FRT, cd2, FRT> GAL4, UAS-GFP/+) analyzed for mCherry-Atg8a puncta. GFP-marked clone ralIR-expressing cells contain a yellow asterisk. Scale bars represent 20µm.D Quantification of data from (C). Data are represented as means ± SEM; n≥ 10. Statistical significance was determined using a Student's t test.E Salivary glands dissected from 14 hours after puparium formation animals that have ral loss of function (ralPG89) specifically in non-RFP cells, and that express GFP-Atg8a in all cells (yw, hs-Flp, FRT19A, Ubi-RFP/ ralPG89,FRT19A; pGFP-Atg8a) analyzed for GFP-Atg8a puncta. ralPG89 mutant cells are marked with a yellow asterisk. Scale bars represent 20µm.F Quantification of data from (E). Data are represented as means ± SEM; n≥ 10. Statistical significance was determined using a Student's t test.G Salivary glands dissected from 14 hours after puparium formation animals that have ral loss of function (ralPG89) specifically in non-RFP cells (yw, hs-Flp, FRT19A, Ubi-RFP/ ralPG89,FRT19A) and are stained for Ref(2)p (green) and DAPI (blue). ralPG89 mutant cells are marked with a yellow asterisk. Scale bars represent 20µm.H Quantification of data from (G). Data are represented as means ± SEM; n≥ 10. Statistical significance was determined using a Student's t test.


I Fat bodies dissected from 4 hour starved animals that have ral loss of function ( ralPG89 ) specifically in non - RFP cells , and that express GFP - Atg8a in all cells ( yw , hs - Flp , FRT19A , Ubi - RFP / ralPG89 , FRT19A ; pGFP - Atg8a ) analyzed for GFP - Atg8a puncta . ralPG89 mutant cells are marked with a yellow asterisk . Scale bars represent 20µm . J Quantification of data from ( I ) . Data are represented as means ± SEM ; n ≥ 10 . Statistical significance was determined using a Student ' s t test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

I Fat bodies dissected from 4 hour starved animals that have ral loss of function (ralPG89) specifically in non-RFP cells, and that express GFP-Atg8a in all cells (yw, hs-Flp, FRT19A, Ubi-RFP/ ralPG89,FRT19A; pGFP-Atg8a) analyzed for GFP-Atg8a puncta. ralPG89 mutant cells are marked with a yellow asterisk. Scale bars represent 20µm.J Quantification of data from (I). Data are represented as means ± SEM; n≥ 10. Statistical significance was determined using a Student's t test.


A , C , E , G , I Fat bodies dissected from 4 hour starved animals expressing mCherry - Atg8a in all cells , and ( A ) sec5IR , ( C ) sec15IR , ( E ) sec3IR , ( G ) sec8IR , ( I ) exo84IR specifically in GFP - marked clone cells ( yw , hsflp / w ; pmCherry - Atg8a / + ; act < FRT , cd2 , FRT > GAL4 , UAS - GFP / + ) analyzed for mCherry - Atg8a puncta . Scale bars represent 20µm . B , D , F , H , J Quantification of data from ( A , C , E , G , I ) . Data are represented as means ± SEM ; n ≥ 10 . Statistical significance was determined using a Student ' s t test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A, C, E, G, I Fat bodies dissected from 4 hour starved animals expressing mCherry-Atg8a in all cells, and (A) sec5IR, (C) sec15IR, (E) sec3IR, (G) sec8IR, (I) exo84IR specifically in GFP-marked clone cells (yw, hsflp/w; pmCherry-Atg8a/+; act< FRT, cd2, FRT> GAL4, UAS-GFP/+) analyzed for mCherry-Atg8a puncta. Scale bars represent 20µm.B, D, F, H, J Quantification of data from (A, C, E, G, I). Data are represented as means ± SEM; n≥ 10. Statistical significance was determined using a Student's t test.


A Salivary glands dissected from 14 hours after puparium formation ( top left ) control ( fkh - GAL4 / + ; pmCherry - Atg8a / + ) , and those with salivary gland - specific knockdown of ( middle left ) sec5 ( fkh - GAL4 / w ; pmCherry - Atg8a / + ; UAS - sec5IR / + ) , ( bottom left ) sec15 ( fkh - GAL4 / w ; pmCherry - Atg8a / + ; UAS - sec15IR / + ) , ( top right ) sec3 ( fkh - GAL4 / w ; pmCherry - Atg8a / UAS - sec3IR ) , ( middle right ) sec8 ( fkh - GAL4 / w ; pmCherry - Atg8a / UAS - sec8IR ) , ( bottom right ) exo84 ( fkh - GAL4 / w ; pmCherry - Atg8a / UAS - exo84IR ) analyzed for mCherry - Atg8a puncta . Scale bars represent 20µm . B Quantification of data from ( A ) . Data are represented as means ± SEM ; n ≥ 10 . Statistical significance was determined using a Student ' s t test ( * * p < 0 . 001 , * * * p < 0 . 0001 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A Salivary glands dissected from 14 hours after puparium formation (top left) control (fkh-GAL4/+; pmCherry-Atg8a/+), and those with salivary gland-specific knockdown of (middle left) sec5 (fkh-GAL4/w; pmCherry-Atg8a/+; UAS-sec5IR/+), (bottom left) sec15 (fkh-GAL4/w; pmCherry-Atg8a/+; UAS-sec15IR/+), (top right) sec3 (fkh-GAL4/w; pmCherry-Atg8a/UAS-sec3IR), (middle right) sec8 (fkh-GAL4/w; pmCherry-Atg8a/UAS-sec8IR), (bottom right) exo84 (fkh-GAL4/w; pmCherry-Atg8a/UAS-exo84IR) analyzed for mCherry-Atg8a puncta. Scale bars represent 20µm.B Quantification of data from (A). Data are represented as means ± SEM; n≥ 10. Statistical significance was determined using a Student's t test (** p< 0.001, *** p< 0.0001).


A Control animals ( + / w ; UAS - sec5IR / + ) , n = 19 , and those with salivary gland - specific knockdown of sec5 ( fkh - GAL4 / w ; UAS - sec5IR / + ) , n = 20 , were analyzed by histology for the presence of salivary gland material ( red dotted circle ) 24 hours after puparium formation . B Quantification of data from ( A ) . Data are represented as means . Statistical significance was determined using a Chi - square test . C Control animals ( + / w ; UAS - sec15IR / + ) , n = 20 , and those with salivary gland - specific expression sec15 ( fkh - GAL4 / w ; UAS - sec15IR / + ) , n = 20 , were analyzed by histology for the presence of salivary gland material ( red dotted circle ) 24 hours after puparium formation . D Quantification of data from ( C ) . Data are represented as means . Statistical significance was determined using a Chi - square test . E Control animals ( + / w ; UAS - sec3IR / + ) , n = 20 , and those with salivary gland - specific expression sec3 ( fkh - GAL4 / w ; UAS - sec3IR / + ) , n = 20 , were analyzed by histology for the presence of salivary gland material ( red dotted circle ) 24 hours after puparium formation . F Quantification of data from ( E ) . Data are represented as means . Statistical significance was determined using a Chi - square test . G Control animals ( + / w ; UAS - sec8IR / + ) , n = 20 , and those with salivary gland - specific knockdown of sec8 ( fkh - GAL4 / w ; UAS - sec8IR / + ) , n = 19 , were analyzed by histology for the presence of salivary gland material ( red dotted circle ) 24 hours after puparium formation . H Quantification of data from ( G ) . Data are represented as means . Statistical significance was determined using a Chi - square test . I Control animals ( + / w ; UAS - exo84IR / + ) , n = 19 , and those with salivary gland - specific knockdown of exo84 ( fkh - GAL4 / w ; UAS - exo84IR / + ) , n = 20 , were analyzed by histology for the presence of salivary gland material ( red dotted circle ) 24 hours after puparium formation . J Quantification of data from ( I ) . Data are represented as means . Statistical significance was determined using a Chi - square test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A Control animals (+/w; UAS-sec5IR/+), n= 19, and those with salivary gland-specific knockdown of sec5 (fkh-GAL4/w; UAS-sec5IR/+), n=20, were analyzed by histology for the presence of salivary gland material (red dotted circle) 24 hours after puparium formation.B Quantification of data from (A). Data are represented as means. Statistical significance was determined using a Chi-square test.C Control animals (+/w; UAS-sec15IR/+), n= 20, and those with salivary gland-specific expression sec15 (fkh-GAL4/w; UAS-sec15IR/+), n= 20, were analyzed by histology for the presence of salivary gland material (red dotted circle) 24 hours after puparium formation.D Quantification of data from (C). Data are represented as means. Statistical significance was determined using a Chi-square test.E Control animals (+/w; UAS-sec3IR/+), n= 20, and those with salivary gland-specific expression sec3 (fkh-GAL4/w; UAS-sec3IR/+), n= 20, were analyzed by histology for the presence of salivary gland material (red dotted circle) 24 hours after puparium formation.F Quantification of data from (E). Data are represented as means. Statistical significance was determined using a Chi-square test.G Control animals (+/w; UAS-sec8IR/+), n= 20, and those with salivary gland-specific knockdown of sec8 (fkh-GAL4/w; UAS-sec8IR/+), n=19, were analyzed by histology for the presence of salivary gland material (red dotted circle) 24 hours after puparium formation.H Quantification of data from (G). Data are represented as means. Statistical significance was determined using a Chi-square test.I Control animals (+/w; UAS-exo84IR/+), n= 19, and those with salivary gland-specific knockdown of exo84 (fkh-GAL4/w; UAS-exo84IR/+), n=20, were analyzed by histology for the presence of salivary gland material (red dotted circle) 24 hours after puparium formation.J Quantification of data from (I). Data are represented as means. Statistical significance was determined using a Chi-square test.


A Salivary glands dissected from late 3rd instar larvae ( Gbe Su ( H ) - lac - Z / w - ; + ; Fkh - GAL4 / + ) show less notch activity ( left ) compared to glands from 14 hours after puparium formation ( APF ) animals ( middle ) . Down - regulation of ral in salivary glands from 14 hours APF animals ( Gbe Su ( H ) - lac - Z / w - ; UAS - ralIR / + ; Fkh - GAL4 / + ) reduces the levels of notch activity ( right ) . Scale bar represents 20µm . B Quantification of data from ( A ) . Data are represented as means ± SEM ; n ≥ 4 . Statistical significance was determined using a Student ' s t test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A Salivary glands dissected from late 3rd instar larvae (Gbe Su(H)-lac-Z/w-;+;Fkh-GAL4/+) show less notch activity (left) compared to glands from 14 hours after puparium formation (APF) animals (middle). Down-regulation of ral in salivary glands from 14 hours APF animals (Gbe Su(H)-lac-Z/w-;UAS-ralIR/+;Fkh-GAL4/+) reduces the levels of notch activity (right). Scale bar represents 20µm.B Quantification of data from (A). Data are represented as means ± SEM; n≥ 4. Statistical significance was determined using a Student's t test.


C Salivary glands dissected from 14 hours after puparium formation animals expressing mCherry - Atg8a in all cells , and notchIR specifically in GFP - marked clone cells ( yw , hsflp / w , UAS - notchIR ; pmCherry - Atg8a / + ; act < FRT , cd2 , FRT > GAL4 , UAS - GFP / + ) analyzed for mCherry - Atg8a puncta . Scale bar represents 20µm . D Quantification of data from ( C ) . Data are represented as means ± SEM ; n > 10 . Statistical significance was determined using a Student ' s t test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C Salivary glands dissected from 14 hours after puparium formation animals expressing mCherry-Atg8a in all cells, and notchIR specifically in GFP-marked clone cells (yw, hsflp/w,UAS-notchIR; pmCherry-Atg8a/+; act< FRT, cd2, FRT> GAL4, UAS-GFP/+) analyzed for mCherry-Atg8a puncta. Scale bar represents 20µm.D Quantification of data from (C). Data are represented as means ± SEM; n> 10. Statistical significance was determined using a Student's t test.


D Zygotic grk2i283 mutant at 4dpf compared to wild type ( N = 80 ) . Arrows indicate the position of swim bladder . Scale bar ,

O O O O O O O O O O O O O O O O O O O O O O O O O O O

D Zygotic grk2i283 mutant at 4dpf compared to wild type (N=80). Arrows indicate the position of swim bladder. Scale bar, 1mm


A Western blot analysis of the different forms of Gli2a in MZgrk2 embryos compared to wild type , shh mRNA and dnPKA mRNA injected wild type and cyclopamine ( Cyc ( A ) ) treated wild type 20hpf embryos . Gli2a - FL levels are low relative to Gli2aR levels in wild type embryos , but are elevated in response to pathway activation ( shh and dnPKA mRNA injected ) . Gli2aR levels are increased while Gli2a - FL is undetectable in CycA treated and MZgrk2 mutant embryos . Probing the same blot with rabbit anti - GRK3 ( which recognizes the zebrafish Grk2 protein ) reveals a complete loss of full length Grk2 in MZgrk2 embryos . Probing with anti - γ - tubulin was performed as a loading control . Three biological replicates of this analysis were performed .

O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A Western blot analysis of the different forms of Gli2a in MZgrk2 embryos compared to wild type, shh mRNA and dnPKA mRNA injected wild type and cyclopamine (Cyc(A)) treated wild type 20hpf embryos. Gli2a-FL levels are low relative to Gli2aR levels in wild type embryos, but are elevated in response to pathway activation (shh and dnPKA mRNA injected). Gli2aR levels are increased while Gli2a-FL is undetectable in CycA treated and MZgrk2 mutant embryos. Probing the same blot with rabbit anti-GRK3 (which recognizes the zebrafish Grk2 protein) reveals a complete loss of full length Grk2 in MZgrk2 embryos. Probing with anti-γ-tubulin was performed as a loading control. Three biological replicates of this analysis were performed.


B Phenotype of wild type , MZgrk2 , and grk2 - GFP mRNA injected MZgrk2 embryos at 24hpf ( n = 20 for each sample ) . The white lines indicate the shape of the somites ( middle panels ) and the separation of the eyes ( right hand panels ) . Scale bar ,

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B Phenotype of wild type, MZgrk2, and grk2-GFP mRNA injected MZgrk2 embryos at 24hpf (n=20 for each sample). The white lines indicate the shape of the somites (middle panels) and the separation of the eyes (right hand panels). Scale bar, 200μm


C In situ hybridisation of ptch2 , nkx2 . 2a and olig2 transcripts in wild type , MZgrk2 and grk2 - GFP injected MZgrk2 24hpf embryos . Each panel shows a full view of the embryo on the left and a cross sectional view of a somite on the right . ( n = 30 for each sample ) . Scale bars , 200μm ( whole mounts ) , 50μm ( sections ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C In situ hybridisation of ptch2, nkx2.2a and olig2 transcripts in wild type, MZgrk2 and grk2-GFP injected MZgrk2 24hpf embryos. Each panel shows a full view of the embryo on the left and a cross sectional view of a somite on the right. (n=30 for each sample). Scale bars, 200μm (whole mounts), 50μm (sections).


D Expression of Prox1a and Engrailed proteins ( Eng ) in somites of wild - type , MZgrk2 and grk2 - GFP mRNA injected MZgrk2 embryos at 30hpf . Each panel shows Prox1a in green , Eng in red and the merge images with DAPI staining in blue . ( n = 10 for each sample ) . Scale bar , 50μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D Expression of Prox1a and Engrailed proteins (Eng) in somites of wild-type, MZgrk2 and grk2-GFP mRNA injected MZgrk2 embryos at 30hpf. Each panel shows Prox1a in green, Eng in red and the merge images with DAPI staining in blue. (n=10 for each sample). Scale bar, 50μm.


A Prox1a and Eng expression in the myotome of 30hpf wild type and MZgrk2 embryos injected with mRNAs encoding Shh , dnPKA or mSmoA1 - GFP . Each panel shows Prox1a in green , Eng in red , and the merged images with DAPI staining in blue . The co - labelling for Prox1a and Eng ( orange ) is indicative of MP differentiation ( n = 30 for each sample ) Scale bar , 50μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A Prox1a and Eng expression in the myotome of 30hpf wild type and MZgrk2 embryos injected with mRNAs encoding Shh, dnPKA or mSmoA1-GFP. Each panel shows Prox1a in green, Eng in red, and the merged images with DAPI staining in blue. The co-labelling for Prox1a and Eng (orange) is indicative of MP differentiation (n=30 for each sample) Scale bar, 50μm.


B Western blot showing levels of Gli2a - FL and Gli2a - R forms in 20hpf wild type and MZgrk2 embryos injected with mRNA encoding Shh , dnPKA or mSmoA1 - GFP , respectively . γ - tubulin was used as loading control . Three biological replicates of this analysis were performed .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B Western blot showing levels of Gli2a-FL and Gli2a-R forms in 20hpf wild type and MZgrk2 embryos injected with mRNA encoding Shh, dnPKA or mSmoA1-GFP, respectively. γ-tubulin was used as loading control. Three biological replicates of this analysis were performed.


A Western blot analysis of GRK2 , GLI1 , PTCH1 , and GLI3 protein levels in Flp - In - 3T3 cells with lentiCRISPR of 4 different guide RNAs in presence or absence of SAG . α - tubulin was used as loading control . Guide RNA 2 was found to remove Grk2 efficiently and used for subsequent experiments . GLI3 panel shows both full - length ( GLI3FL ) and repressor form of GLI3 ( GLI3R ) . ( n = 3 )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A Western blot analysis of GRK2, GLI1, PTCH1, and GLI3 protein levels in Flp-In-3T3 cells with lentiCRISPR of 4 different guide RNAs in presence or absence of SAG. α-tubulin was used as loading control. Guide RNA 2 was found to remove Grk2 efficiently and used for subsequent experiments. GLI3 panel shows both full-length (GLI3FL) and repressor form of GLI3 (GLI3R). (n=3)


B , C Hh reporter activity assay for wild type and Grk2 - / - cells . Cells were treated with PKA peptide inhibitor ( PKI ) and mutant form of PKA peptide inhibitor ( PKI - M ) respectively , in presence or absence of SHH ( B ) Cells were transfected with mSmoA1 - GFP expressing constructs in the presence of SHH or SAG ( C ) . Data represent the mean and ± SD ( n = 3 ) . Unpaired Students t - test was used for analysis . * * * P < 0 . 001 ; * * P < 0 . 01 ; * P < 0 . 05 and n . s . ( not significant ) .

O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B, C Hh reporter activity assay for wild type and Grk2-/- cells. Cells were treated with PKA peptide inhibitor (PKI) and mutant form of PKA peptide inhibitor (PKI-M) respectively, in presence or absence of SHH (B) Cells were transfected with mSmoA1-GFP expressing constructs in the presence of SHH or SAG (C). Data represent the mean and ± SD (n=3). Unpaired Students t-test was used for analysis. ***P<0.001; **P<0.01; *P<0.05 and n.s. (not significant).


A Expression of Prox1a ( green ) and the eng2a : GFP reporter ( red ) in otherwise wild - type 30hpf embryos injected with mRNA encoding wild type and mutant forms of mouse Smo . Ectopic MPs are indicated by fibres co - labelled with Prox1a and GFP ; ectopic MFFs are labeled only with GFP . Images are representative of embryos in the following proportions of each sample : 28 / 28 ( mSmo ; mSmoA1 ; mSmoSD ; mSmoKRA ) ; 9 / 28 ( mSmoA1SA ) ; 12 / 28 ( mSmoA1SD ) . Scale bar , 50μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A Expression of Prox1a (green) and the eng2a:GFP reporter (red) in otherwise wild-type 30hpf embryos injected with mRNA encoding wild type and mutant forms of mouse Smo. Ectopic MPs are indicated by fibres co-labelled with Prox1a and GFP; ectopic MFFs are labeled only with GFP. Images are representative of embryos in the following proportions of each sample: 28/28 (mSmo; mSmoA1; mSmoSD; mSmoKRA); 9/28 (mSmoA1SA); 12/28 (mSmoA1SD). Scale bar, 50μm.


B Average number of Prox1a + ve slow fibres in wild type embryos injected with mRNA encoding different forms of mSmo . Prox1 positive cells were quantified in 4 somites in each of 4 embryos for each sample . The error bars indicate SD . Unpaired Student ' s t - test was used to determine the statistical significance between uninjected embryos and the various Smo mutants ( black asterisk ) ; and between mSmoA1 and mSmoA1SA or mSmoA1SD ( red asterisk ) . * * * P < 0 . 001 ; * * P < 0 . 01 ; * P < 0 . 05 and n . s . ( not significant ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B Average number of Prox1a+ve slow fibres in wild type embryos injected with mRNA encoding different forms of mSmo. Prox1 positive cells were quantified in 4 somites in each of 4 embryos for each sample. The error bars indicate SD. Unpaired Student's t-test was used to determine the statistical significance between uninjected embryos and the various Smo mutants (black asterisk); and between mSmoA1 and mSmoA1SA or mSmoA1SD (red asterisk). ***P<0.001; **P<0.01; *P<0.05 and n.s. (not significant).


C Prox1a and Eng expression in smohi1640 mutant embryos injected with mRNA encoding mSmo ( n = 10 ) , mSmoSA ( n = 9 ) and mSmo14SA ( n = 6 ) . Note the full recovery of SSFs and MPs compared to the un - injected controls . Scale bar , 50μm .

C Prox1a and Eng expression in smohi1640 mutant embryos injected with mRNA encoding mSmo (n=10), mSmoSA (n=9) and mSmo14SA (n=6). Note the full recovery of SSFs and MPs compared to the un-injected controls. Scale bar, 50μm.


D In situ hybridisation for transcripts of ptch2 , olig2 and nkx2 . 2 in 24hpf smohi1640 mutant embryos injected with mRNA encoding mSmo and mSmoSA ( n = 6 for each sample ) . Scale bars , are 100μm ( lateral view ) , 50μm ( sections ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D In situ hybridisation for transcripts of ptch2, olig2 and nkx2.2 in 24hpf smohi1640 mutant embryos injected with mRNA encoding mSmo and mSmoSA (n=6 for each sample). Scale bars, are 100μm (lateral view), 50μm (sections).


E Hh reporter assay of the activity of wild type and mutant forms of mSmo in Smo - / - MEFs in response to Shh or SAG stimulation . Note that mSmoA1 shows constitutive activity in the absence of either Shh or SAG whereas mSmoKRA dose not ; all mutants affecting phosphorylation failed to restore the response to Shh or SAG . Data represent the mean and ± SD ( n = 3 ) . Unpaired Students t - test was used for analysis . * * * * P < 0 . 0001 ; * * P < 0 . 01 ; and n . s . ( not significant ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E Hh reporter assay of the activity of wild type and mutant forms of mSmo in Smo-/- MEFs in response to Shh or SAG stimulation. Note that mSmoA1 shows constitutive activity in the absence of either Shh or SAG whereas mSmoKRA dose not; all mutants affecting phosphorylation failed to restore the response to Shh or SAG. Data represent the mean and ± SD (n=3). Unpaired Students t-test was used for analysis. ****P<0.0001; **P<0.01; and n.s. (not significant).


A Wild type 18hpf embryos injected with mRNA encoding GFP - tagged mSmo ( green ) showing localisation to the PC of myotomal cells labeled with anti - Acetylated tubulin ( AcTub ; red ) , stimulated in response to Shh injection ( n = 4 ) . Differences in PC distribution are due to morphological changes in the myotome induced by ectopic expression of Shh as revealed by the distribution of nuclei ( DAPI stained ; blue ) . Scale bar , 10μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A Wild type 18hpf embryos injected with mRNA encoding GFP-tagged mSmo (green) showing localisation to the PC of myotomal cells labeled with anti-Acetylated tubulin (AcTub; red), stimulated in response to Shh injection (n = 4). Differences in PC distribution are due to morphological changes in the myotome induced by ectopic expression of Shh as revealed by the distribution of nuclei (DAPI stained; blue). Scale bar, 10μm.


B Notochord cells of wild type embryos expressing GFP tagged wild - type and mutant forms of mSmo . Note the localisation to the PC ( labeled with anti - AcTub ; red ) in each case ( n = 4 for each sample ) . Scale bar , 10μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B Notochord cells of wild type embryos expressing GFP tagged wild-type and mutant forms of mSmo. Note the localisation to the PC (labeled with anti-AcTub; red) in each case (n=4 for each sample). Scale bar, 10μm.


C MZgrk2 18hpf embryos injected with mRNA encoding GFP - tagged wild type mSmo or mSmoSA ( green ) showing localisation to the PC ( labeled with anti - AcTub ; red ) in myotomal cells . More Smo is localised to the PC in MZgrk2 mutants compared to wild type ( panel A ) . Scale bar , 10μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C MZgrk2 18hpf embryos injected with mRNA encoding GFP-tagged wild type mSmo or mSmoSA (green) showing localisation to the PC (labeled with anti- AcTub; red) in myotomal cells. More Smo is localised to the PC in MZgrk2 mutants compared to wild type (panel A). Scale bar, 10μm.


B Prox1a ( green ) and Eng ( red ) expression in the myotome of 30hpf MZgrk2 embryos that were injected with mRNA encoding : Grk2K220R - GFP ; Grk2K220M - GFP ; Grk2D335N - GFP ; Grk2ΔN7aa - GFP and mGrk2L4AV7AL8A - GFP . Merged panels show nuclear staining with DAPI in blue . ( n = 20 for each sample ) . Scale bar , 50μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B Prox1a (green) and Eng (red) expression in the myotome of 30hpf MZgrk2 embryos that were injected with mRNA encoding: Grk2K220R-GFP; Grk2K220M-GFP; Grk2D335N-GFP; Grk2ΔN7aa-GFP and mGrk2L4AV7AL8A-GFP. Merged panels show nuclear staining with DAPI in blue. (n=20 for each sample). Scale bar, 50μm.


C Hh reporter activity assay in wild type and Grk2 - / - 3T3 cells transfected with the equivalent wild type and mutant kinase domain forms of mouse Grk2 , Grk2K220R , Grk2K220M and Grk2D335N after 24h Shh - N and SAG treatment . Data represent the mean and ± SD ( n = 3 ) . Unpaired Students t - test was used for analysis . * * * * P < 0 . 0001 ; * * P < 0 . 01 ; * P < 0 . 05 ; and n . s . ( not significant ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C Hh reporter activity assay in wild type and Grk2-/- 3T3 cells transfected with the equivalent wild type and mutant kinase domain forms of mouse Grk2, Grk2K220R, Grk2K220M and Grk2D335N after 24h Shh-N and SAG treatment. Data represent the mean and ± SD (n=3). Unpaired Students t-test was used for analysis. ****P<0.0001; **P<0.01; *P<0.05; and n.s. (not significant).


Immunoblot analysis of protein lysates from ARPE - 19 cells expressing TFEB - WT - FLAG or TFEB - S401A - FLAG treated with 200 μM NaAsO2 or 250 nM Torin - 1 for 1 h . Two different rabbit polyclonal antibodies raised against a TFEB - S401 phospho - specific peptide were tested .

O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O

Immunoblot analysis of protein lysates from ARPE-19 cells expressing TFEB-WT-FLAG or TFEB-S401A-FLAG treated with 200 μM NaAsO2 or 250 nM Torin-1 for 1 h. Two different rabbit polyclonal antibodies raised against a TFEB-S401 phospho-specific peptide were tested.


Immunoblot analysis of protein lysates from ARPE - 19 cells expressing TFEB - WT - FLAG , TFEB - S3A / R4A - FLAG or TFEB - R245 - 247A - FLAG treated with 200 μM NaAsO2 for 1 h . Immunoblots are representative of at least three independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O

Immunoblot analysis of protein lysates from ARPE-19 cells expressing TFEB-WT-FLAG, TFEB-S3A/R4A-FLAG or TFEB-R245-247A-FLAG treated with 200 μM NaAsO2 for 1 h. Immunoblots are representative of at least three independent experiments.


Immunoblot analysis of protein lysates from HeLa cells stably expressing TFEB - WT - FLAG incubated with the indicated kinase inhibitors for 1 h prior to the addition of 200 μM NaAsO2 for 1 h . Quantification of immunoblot data shown in ( A ) . Data are presented as mean ± SD using one - way ANOVA ( unpaired ) followed by Tukey ' s multiple comparisons test , ( ns ) not significant , and ( * * * * ) p < 0 . 0001 from three independent experiments . Data information : n = 3 biological replicates ( each dot represents a biological replicate ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Immunoblot analysis of protein lysates from HeLa cells stably expressing TFEB-WT-FLAG incubated with the indicated kinase inhibitors for 1 h prior to the addition of 200 μM NaAsO2 for 1 h. Quantification of immunoblot data shown in (A). Data are presented as mean ± SD using one-way ANOVA (unpaired) followed by Tukey's multiple comparisons test, (ns) not significant, and (****)p<0.0001 from three independent experiments. Data information: n = 3 biological replicates (each dot represents a biological replicate).


Immunoblot analysis of protein lysates from HeLa cells stably expressing TFEB - WT - FLAG incubated with either 200 μM NaAsO2 for 1 h , EBSS for 4 h , 100 ng / ml EGF for the indicated times or 37 μM Anisomycin for 1 h . Before the addition of EGF or Anisomycin , cells were serum starved for 8 h .

O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O

Immunoblot analysis of protein lysates from HeLa cells stably expressing TFEB-WT-FLAG incubated with either 200 μM NaAsO2 for 1 h, EBSS for 4 h, 100 ng/ml EGF for the indicated times or 37 μM Anisomycin for 1 h. Before the addition of EGF or Anisomycin, cells were serum starved for 8 h.


Immunoblot analysis of protein lysates from HeLa cells stably expressing TFEB - WT - FLAG exposed to 30J / m2 of UV - C and allowed to recover in complete medium for the indicated times . Cells were incubated with p38 MAPK inhibitor ( 20 μM , SB203580 ) for 1 h before UV - C irradiation and allowed to recover for 30 min in the presence of the inhibitor .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O

Immunoblot analysis of protein lysates from HeLa cells stably expressing TFEB-WT-FLAG exposed to 30J/m2 of UV-C and allowed to recover in complete medium for the indicated times. Cells were incubated with p38 MAPK inhibitor (20 μM, SB203580) for 1 h before UV-C irradiation and allowed to recover for 30 min in the presence of the inhibitor.


Immunoblot analysis of protein lysates from HeLa cells stably expressing TFEB - WT - FLAG depleted of either p38 MAPK ( α + β ) or JNK1 or JNK2 or JNK ( 1 + 2 ) and incubated with 200 μM NaAsO2 for 1 h . Immunoblots are representative of at least three independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O

Immunoblot analysis of protein lysates from HeLa cells stably expressing TFEB-WT-FLAG depleted of either p38 MAPK (α+β) or JNK1 or JNK2 or JNK(1+2) and incubated with 200 μM NaAsO2 for 1 h. Immunoblots are representative of at least three independent experiments.


Immunoblot analysis of in vitro p38 MAPK kinase assay using GST - TFEB - PRD as substrate in the presence or absence of either recombinant human active p38α MAPK or ATP . Quantification of immunoblot data shown in ( F ) . Data are presented as mean ± SD using one - way ANOVA ( unpaired ) followed by Tukey ' s multiple comparisons test , ( * ) p < 0 . 05 from three independent experiments . Data information : n = 3 biological replicates ( each dot represents a biological replicate ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Immunoblot analysis of in vitro p38 MAPK kinase assay using GST-TFEB-PRD as substrate in the presence or absence of either recombinant human active p38α MAPK or ATP. Quantification of immunoblot data shown in (F). Data are presented as mean ± SD using one-way ANOVA (unpaired) followed by Tukey's multiple comparisons test, (*)p<0.05 from three independent experiments. Data information: n = 3 biological replicates (each dot represents a biological replicate).


Immunoblot analysis of protein lysates from Raw 264 . 7 cells stably expressing TFEB - WT - FLAG incubated with 1μg / ml LPS for the indicated times .

O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O

Immunoblot analysis of protein lysates from Raw 264.7 cells stably expressing TFEB-WT-FLAG incubated with 1μg/ml LPS for the indicated times.


Immunoblot analysis of protein lysates from Raw 264 . 7 cells stably expressing TFEB - WT - FLAG incubated with either 20 μM SB203580 or 250 nM Torin - 1 for 1 h prior to the addition of 1 μg / ml LPS for 30 min .

O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O

Immunoblot analysis of protein lysates from Raw 264.7 cells stably expressing TFEB-WT-FLAG incubated with either 20 μM SB203580 or 250 nM Torin-1 for 1 h prior to the addition of 1 μg/ml LPS for 30 min.


Quantification of immunoblot data shown in ( B ) . Data are presented as mean ± SD using one - way ANOVA ( unpaired ) followed by Tukey ' s multiple comparisons test , ( ns ) not significant , ( * ) p < 0 . 05 and ( * * ) p < 0 . 01 from three independent experiments . Data information : n = 3 biological replicates ( each dot represents a biological replicate ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Quantification of immunoblot data shown in (B). Data are presented as mean ± SD using one-way ANOVA (unpaired) followed by Tukey's multiple comparisons test, (ns) not significant, (*)p<0.05 and (**)p<0.01 from three independent experiments. Data information: n = 3 biological replicates (each dot represents a biological replicate).


Immunoblot analysis of protein lysates from THP1 cells incubated with 1 μg / ml LPS for the indicated times .

O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O

Immunoblot analysis of protein lysates from THP1 cells incubated with 1 μg/ml LPS for the indicated times.


Immunoblot analysis of protein lysates from THP1 cells incubated with either vehicle ( DMSO ) or 20 μM SB203580 for 1 h prior to the addition of 1μg / ml LPS for the indicated times .

O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O

Immunoblot analysis of protein lysates from THP1 cells incubated with either vehicle (DMSO) or 20 μM SB203580 for 1 h prior to the addition of 1μg/ml LPS for the indicated times.


Immunoblot analysis of protein lysates from THP1 cells depleted of p38 MAPK ( α ) , p38 MAPK ( β ) or p38 MAPK ( α + β ) and incubated with 1 μg / ml LPS for 1 h . Immunoblots are representative of at least three independent experiments . Quantification of immunoblot data shown in ( F ) . Data are presented as mean ± SD using one - way ANOVA ( unpaired ) followed by Tukey ' s multiple comparisons test , ( * ) p < 0 . 05 from three independent experiments . Data information : n = 3 biological replicates ( each dot represents a biological replicate ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Immunoblot analysis of protein lysates from THP1 cells depleted of p38 MAPK (α), p38 MAPK (β) or p38 MAPK (α+β) and incubated with 1 μg/ml LPS for 1 h. Immunoblots are representative of at least three independent experiments. Quantification of immunoblot data shown in (F). Data are presented as mean ± SD using one-way ANOVA (unpaired) followed by Tukey's multiple comparisons test, (*)p<0.05 from three independent experiments. Data information: n = 3 biological replicates (each dot represents a biological replicate).


Immunoblot analysis of protein lysates from naïve THP1 - WT or TFEB - S401A knock - in ( clone M17 ) cells treated with 50 ng / ml PMA for the indicated times , PMA - differentiated THP1 ( Rested ) cells treated without or with 250 nM Torin - 1 for 1 h .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O

Immunoblot analysis of protein lysates from naïve THP1-WT or TFEB-S401A knock-in (clone M17) cells treated with 50 ng/ml PMA for the indicated times, PMA-differentiated THP1 (Rested) cells treated without or with 250 nM Torin-1 for 1 h.


Immunoblot analysis of proteins from nuclear and cytosolic fractions from naïve THP1 - WT or TFEB - S401A knock - ins ( clones I11 and M17 ) cells treated with 50 ng / ml PMA for the indicated times and PMA - differentiated THP1 ( Rested ) cells .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O

Immunoblot analysis of proteins from nuclear and cytosolic fractions from naïve THP1-WT or TFEB-S401A knock-ins (clones I11 and M17) cells treated with 50 ng/ml PMA for the indicated times and PMA-differentiated THP1 (Rested) cells.


Immunoblot analysis of proteins from nuclear and cytosolic fractions from naïve THP1 - WT or TFEB - S401A knock - in ( clone I11 ) cells treated with 50 ng / ml PMA for the indicated times .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O

Immunoblot analysis of proteins from nuclear and cytosolic fractions from naïve THP1-WT or TFEB-S401A knock-in (clone I11) cells treated with 50 ng/ml PMA for the indicated times.