( D ) Negative stain electron micrograph of GraFixed sc - mApe1 showing dodecameric particles ( circles ) . ( E ) Class averages of sc - mApe1 dodecamers including views of two - fold and three - fold symmetry . ( F ) Fourier shell correlation of the EM reconstruction of the sc - mApe1 dodecamer indicates a resolution of 24 Å .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Negative stain electron micrograph of GraFixed sc-mApe1 showing dodecameric particles (circles). (E) Class averages of sc-mApe1 dodecamers including views of two-fold and three-fold symmetry. (F) Fourier shell correlation of the EM reconstruction of the sc-mApe1 dodecamer indicates a resolution of 24 Å.


( G ) C . thermophilum mApe1 crystal structure fitted into electron microscopy ( EM ) map of sc - mApe1 dodecamer . Each of the 12 chains is depicted by a different color . A black sphere represents the N - terminus where the propeptide would emanate from the dodecamer . From left to right : two - fold axis view from the edge , three - fold axis view from the facet and three - fold axis view from the vertex .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) C. thermophilum mApe1 crystal structure fitted into electron microscopy (EM) map of sc-mApe1 dodecamer. Each of the 12 chains is depicted by a different color. A black sphere represents the N-terminus where the propeptide would emanate from the dodecamer. From left to right: two-fold axis view from the edge, three-fold axis view from the facet and three-fold axis view from the vertex.


( A ) Electron micrograph of negatively stained Ams1 with particles and higher - order assemblies ( arrows ) . Class averages exhibit two distinct two - fold views consistent with D2 symmetry . Scale bar 10 nm . ( B ) Electron cryomicrograph of Ams1 with particles highlighted ( circles ) . ( C ) Selected particles ( top row ) are shown with corresponding class averages ( bottom row ) . Scale bar 10 nm . ( D ) Fourier shell correlation indicates a resolution of 6 . 3 Å according to the 0 . 143 criterion .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Electron micrograph of negatively stained Ams1 with particles and higher-order assemblies (arrows). Class averages exhibit two distinct two-fold views consistent with D2 symmetry. Scale bar 10 nm. (B) Electron cryomicrograph of Ams1 with particles highlighted (circles). (C) Selected particles (top row) are shown with corresponding class averages (bottom row). Scale bar 10 nm. (D) Fourier shell correlation indicates a resolution of 6.3 Å according to the 0.143 criterion.


( E ) Top view of the cryo - EM structure , four Ams1 molecules were segmented and colored ( dark blue , beige , red , light blue ) .

O O O O O O O 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) Top view of the cryo-EM structure, four Ams1 molecules were segmented and colored (dark blue, beige, red, light blue).


( F ) Molecular architecture of Ams1 monomer . Primary structure , two map segments with fitted pseudo - atomic models are shown . Left . The N - terminal portion was built from distant homology models : a four - helix bundle ( 17 - 27 , 209 - 271 ) in yellow and a jelly - roll fold ( 45 - 203 ) in red . Right . The α - mannosidase core ( 287 - 1083 ) with an α / β barrel ( 287 - 573 ) in blue , a three - helix bundle ( 574 - 671 ) in green , and the β - sandwich domain ( 672 - 1083 ) in purple .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Molecular architecture of Ams1 monomer. Primary structure, two map segments with fitted pseudo-atomic models are shown. Left. The N-terminal portion was built from distant homology models: a four-helix bundle (17 - 27, 209 - 271) in yellow and a jelly-roll fold (45 - 203) in red. Right. The α-mannosidase core (287 - 1083) with an α/β barrel (287 - 573) in blue, a three-helix bundle (574 - 671) in green, and the β-sandwich domain (672 - 1083) in purple.


( G ) Density section through the Ams1 monomer with three - helix bundle in the center .

(G) Density section through the Ams1 monomer with three-helix bundle in the center.


( H ) Complete atomic model of Ams1 with active site indicated as yellow sphere fitted into the cryo - EM map . Corresponding 3D structure in Movie EV1 .

O O O O O O 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) Complete atomic model of Ams1 with active site indicated as yellow sphere fitted into the cryo-EM map. Corresponding 3D structure in Movie EV1.


( J ) The β - sandwich domain ( purple ) makes up the transverse interface between chain A and chain B .

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

(J) The β-sandwich domain (purple) makes up the transverse interface between chain A and chain B.


( K ) The N - terminal four - helix bundle ( orange ) mediates the longitudinal interface . Asterisk denotes position of W234E mutation between chain A and C / D .

O O O O O O O O O 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) The N-terminal four-helix bundle (orange) mediates the longitudinal interface. Asterisk denotes position of W234E mutation between chain A and C/D.


( L ) Negatively stained W234E Ams1 mutant reveals predominantly smaller particles when compared with wild - type tetramers . Representative class averages show a two - lobed structure consistent with reprojections of chain A and B ( dark blue and beige ) dimers depicted in ribbon and surface representation below . Scale bar 10 nm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Negatively stained W234E Ams1 mutant reveals predominantly smaller particles when compared with wild-type tetramers. Representative class averages show a two-lobed structure consistent with reprojections of chain A and B (dark blue and beige) dimers depicted in ribbon and surface representation below. Scale bar 10 nm.


( A ) SEC - MALS profile of Atg19 yields a molecular weight estimate of 142 kDa ( measured in triplicate ) .

(A) SEC-MALS profile of Atg19 yields a molecular weight estimate of 142 kDa (measured in triplicate).


( B ) Right . In vitro pull - down binding assay and subsequent SDS - PAGE . Amylose beads are used as a bait to demonstrate interaction of purified prApe1 dodecamers with MBP - Atg19 ( lane 1 ) whereas purified mApe1 dodecamers are not capable of interacting with MBP - Atg19 ( lane 3 ) . As controls , MBP does not bind prApe1 and mApe1 ( lanes 2 and 4 ) . Left . SDS - PAGE showing input of the corresponding pull - down assay .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Right. In vitro pull-down binding assay and subsequent SDS-PAGE. Amylose beads are used as a bait to demonstrate interaction of purified prApe1 dodecamers with MBP-Atg19 (lane 1) whereas purified mApe1 dodecamers are not capable of interacting with MBP-Atg19 (lane 3). As controls, MBP does not bind prApe1 and mApe1 (lanes 2 and 4). Left. SDS-PAGE showing input of the corresponding pull-down assay.


( C ) Co - expression of MBP - Atg19 and prApe1 in Sf21 cells ; SDS - PAGE of eluate from amylose beads .

O 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) Co-expression of MBP-Atg19 and prApe1 in Sf21 cells; SDS-PAGE of eluate from amylose beads.


( D ) Size - exclusion chromatography profile ( Superose 6 column ) of the prApe1 / Atg19 complex after anti MBP - Atg19 purification and cleavage of the MBP tag ( top ) . SDS - PAGE of peak fractions of the gel filtration profile ( bottom ) . Samples were run on two separate gels .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Size-exclusion chromatography profile (Superose 6 column) of the prApe1/Atg19 complex after anti MBP-Atg19 purification and cleavage of the MBP tag (top). SDS-PAGE of peak fractions of the gel filtration profile (bottom). Samples were run on two separate gels.


( E ) Negative stain electron micrograph corresponding to the size - exclusion chromatography peak at 10 ml retention volume of ( D ) . Several prApe1 dodecamers possess extra density corresponding to Atg19 and are highlighted in the insets .

O O O O O O O O O O O O O O O O O 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) Negative stain electron micrograph corresponding to the size-exclusion chromatography peak at 10 ml retention volume of (D). Several prApe1 dodecamers possess extra density corresponding to Atg19 and are highlighted in the insets.


( F ) Comparison between class averages of purified prApe1 dodecamers with class averages of prApe1 dodecamers that were co - purified with Atg19 .

(F) Comparison between class averages of purified prApe1 dodecamers with class averages of prApe1 dodecamers that were co-purified with Atg19.


( G ) Bar plot of occurrence of single , double , triple and multiple dodecamers from the prApe1 and the prApe1 / Atg19 sample . Data information : n = 3 , 186 particles were counted in the case of prApe1 and n = 3 , 340 particles were counted in the case of prApe1 / Atg19 . Particles were picked from 20 different micrographs . * * * P < 2 . 2x10 - 16 ( Pearson ' s Chi - squared 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

(G) Bar plot of occurrence of single, double, triple and multiple dodecamers from the prApe1 and the prApe1/Atg19 sample. Data information: n=3,186 particles were counted in the case of prApe1 and n=3,340 particles were counted in the case of prApe1/Atg19. Particles were picked from 20 different micrographs. ***P < 2.2x10-16 (Pearson's Chi-squared test).


H ) Epifluorescence microscopy images of the prApe1 - GFP / Atg19 - mCherry / ypt7Δ , prApe1 - GFP / Atg19Δ / ypt7Δ S . cerevisiae cells that were used to analyze the fluorescence intensity of all prApe1 - GFP spots . BF represents bright - field images .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Epifluorescence microscopy images of the prApe1-GFP/Atg19-mCherry/ypt7Δ, prApe1-GFP/Atg19Δ/ypt7Δ S. cerevisiae cells that were used to analyze the fluorescence intensity of all prApe1-GFP spots. BF represents bright-field images.


I ) Bar plot showing the ratio between the GFP intensity of prApe1 spots in prApe1 - GFP / Atg19 - mCherry / ypt7Δ versus prApe1 - GFP / Atg19Δ / ypt7Δ S . cerevisiae cells . The intensity of Ape1 - GFP was normalized over the average fluorescence intensity of Nuf2 - GFP measured in kinetochores as performed in [ 36 ] . Data information : a total of 54 prApe1 - GFP spots and 80 Nuf2 - GFP spots were analyzed in prApe1 - GFP / Atg19Δ / ypt7Δ cells , whereas 52 prApe1 - GFP spots and 98 Nuf2 - GFP spots were analyzed in prApe1 - GFP / Atg19 - mCherry / ypt7Δ control cells . Error bars indicate standard error of the mean ( SEM ) . * P = 0 . 03 ( Z 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

I) Bar plot showing the ratio between the GFP intensity of prApe1 spots in prApe1-GFP/Atg19-mCherry/ypt7Δ versus prApe1-GFP/Atg19Δ/ypt7Δ S. cerevisiae cells. The intensity of Ape1-GFP was normalized over the average fluorescence intensity of Nuf2-GFP measured in kinetochores as performed in [36]. Data information: a total of 54 prApe1-GFP spots and 80 Nuf2-GFP spots were analyzed in prApe1-GFP/Atg19Δ/ypt7Δ cells, whereas 52 prApe1-GFP spots and 98 Nuf2-GFP spots were analyzed in prApe1-GFP/Atg19-mCherry/ypt7Δ control cells. Error bars indicate standard error of the mean (SEM). *P = 0.03 (Z test).


Figure 4 : Quantification of the Ape1 , Ams1 , Atg19 protein abundances in living cells . ( A ) Epifluorescence microscopy images of the prApe1 - GFP / Atg19 - mCherry / ypt7Δ , Ams1 - GFP / Atg19 - mCherry / ypt7Δ , Atg19 - GFP / Ape1 - mCherry / ypt7Δ S . cerevisiae cells that were used to quantify the fluorescence intensity of GFP spots . Only co - localizing GFP and RFP spots were considered . BF represents bright - field images . ( B ) Bar plot showing the number of molecules of prApe1 , Ams1 and Atg19 found in Cvt vesicles in living cells . Data information : data are representative of two independent experiments in which a total of 217 prApe1 - GFP , 181 Ams1 - GFP and 233 Atg19 - GFP spots were analyzed . Error bars indicate standard deviation ( SD ) . * * * P < 0 . 001 ; * P = 0 . 0176 ( Z 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

Figure 4: Quantification of the Ape1, Ams1, Atg19 protein abundances in living cells. (A) Epifluorescence microscopy images of the prApe1-GFP/Atg19-mCherry/ypt7Δ, Ams1-GFP/Atg19-mCherry/ypt7Δ, Atg19-GFP/Ape1-mCherry/ypt7Δ S. cerevisiae cells that were used to quantify the fluorescence intensity of GFP spots. Only co-localizing GFP and RFP spots were considered. BF represents bright-field images. (B) Bar plot showing the number of molecules of prApe1, Ams1 and Atg19 found in Cvt vesicles in living cells. Data information: data are representative of two independent experiments in which a total of 217 prApe1-GFP, 181 Ams1-GFP and 233 Atg19-GFP spots were analyzed. Error bars indicate standard deviation (SD). ***P < 0.001; *P = 0.0176 (Z test).


( A ) Fluorescence microscopy image of a resin section of prApe1 - GFP / Atg19 - mCherry / ypt7Δ cells , merge of green ( prApe1 - GFP ) and red ( Atg19 - mcherry ) channels . Tetraspeck beads are detected in the blue channel . ( B ) Electron tomography slice of the boxed area in ( A ) . The outer and inner dashed green circles of 160 and 60 nm radius represent estimated 50 % and 90 % localization accuracy , respectively . Insets show close - up views of a double ( top ) and single ( bottom ) membrane . Corresponding tomogram Movie EV2 . ( C ) Plot of the x and y dimensions of the Cvt vesicles obtained from localization correlation . The average dimensions give rise to spheroids with an average ellipticity of 11 % ( shown in dark grey ) . Both prApe1 - GFP and prApe1 - GFP / Atg19 - mCherry datasets were included . The cases representing autophagosomes were excluded .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Fluorescence microscopy image of a resin section of prApe1-GFP/Atg19-mCherry/ypt7Δ cells, merge of green (prApe1-GFP) and red (Atg19-mcherry) channels. Tetraspeck beads are detected in the blue channel. (B) Electron tomography slice of the boxed area in (A). The outer and inner dashed green circles of 160 and 60 nm radius represent estimated 50% and 90% localization accuracy, respectively. Insets show close-up views of a double (top) and single (bottom) membrane. Corresponding tomogram Movie EV2. (C) Plot of the x and y dimensions of the Cvt vesicles obtained from localization correlation. The average dimensions give rise to spheroids with an average ellipticity of 11 % (shown in dark grey). Both prApe1-GFP and prApe1-GFP/Atg19-mCherry datasets were included. The cases representing autophagosomes were excluded.


( D ) Dense Cvt aggregate that is not surrounded by membrane . Corresponding tomogram Movie EV3 . ( E ) Cvt aggregate that is partially enwrapped by double membrane . Corresponding tomogram Movie EV4 . ( F ) Cvt vesicle fully enclosed by double membrane . Bottom : schematic representation . White arrowheads indicate the double membrane . Corresponding tomogram Movie EV5 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Dense Cvt aggregate that is not surrounded by membrane. Corresponding tomogram Movie EV3. (E) Cvt aggregate that is partially enwrapped by double membrane. Corresponding tomogram Movie EV4. (F) Cvt vesicle fully enclosed by double membrane. Bottom: schematic representation. White arrowheads indicate the double membrane. Corresponding tomogram Movie EV5.


Figure 6 : Model of the Cvt pathway . ( A ) prApe1 the major Cvt cargo forms homo - oligomers as dodecamers that ( B ) assemble into higher - order assemblies mediated by the N - terminal propeptide . ( C ) These assemblies are capable of binding Atg19 that forms a trimer in solution . ( D ) Ams1 , which forms a homo - oligomer as a tetramer , joins the Cvt assembly by binding to Atg19 via a site that is distinct from the Ape1 binding site . ( E ) prApe1 dodecamers , secondary cargo enzymes such as Ams1 tetramers and Atg19 trimers are the main constituents of the Cvt aggregate in the cytosol . ( F ) The Cvt aggregate becomes enwrapped by a double membrane via Atg8 and Atg19 interaction to form ( G ) the Cvt vesicle . Cvt assembly stages in ( E ) , ( F ) and ( G ) are drawn respecting the determined average size of the vesicles , the dimensions and stoichiometry of the major cargo prApe1 and secondary cargo Ams1 found in this article . ( H ) After fusion with the vacuole , prApe1 is subjected to maturation while maintaining the dodecamer organization . ( I ) Ams1 does not undergo a maturation process in the vacuole .

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

Figure 6: Model of the Cvt pathway. (A) prApe1 the major Cvt cargo forms homo-oligomers as dodecamers that (B) assemble into higher-order assemblies mediated by the N-terminal propeptide. (C) These assemblies are capable of binding Atg19 that forms a trimer in solution. (D) Ams1, which forms a homo-oligomer as a tetramer, joins the Cvt assembly by binding to Atg19 via a site that is distinct from the Ape1 binding site. (E) prApe1 dodecamers, secondary cargo enzymes such as Ams1 tetramers and Atg19 trimers are the main constituents of the Cvt aggregate in the cytosol. (F) The Cvt aggregate becomes enwrapped by a double membrane via Atg8 and Atg19 interaction to form (G) the Cvt vesicle. Cvt assembly stages in (E), (F) and (G) are drawn respecting the determined average size of the vesicles, the dimensions and stoichiometry of the major cargo prApe1 and secondary cargo Ams1 found in this article. (H) After fusion with the vacuole, prApe1 is subjected to maturation while maintaining the dodecamer organization. (I) Ams1 does not undergo a maturation process in the vacuole.


Detection of native and GFP - fused TgAtg8 . Protein extracts corresponding to 107 tachyzoites from parental RHΔHX and transgenic GFP - TgAtg8 cell lines were separated by SDS - PAGE and analysed by Western blot using anti - TgAtg8 or anti - GFP antibodies . Overexpressed GFP - TgAtg8 and native TgAtg8 are indicated by arrows .

Detection of native and GFP-fused TgAtg8.Protein extracts corresponding to 107 tachyzoites from parental RHΔHX and transgenic GFP-TgAtg8 cell lines were separated by SDS-PAGE and analysed by Western blot using anti-TgAtg8 or anti-GFP antibodies. Overexpressed GFP-TgAtg8 and native TgAtg8 are indicated by arrows.


A . Autophagic vesicles marker GFP - TgAtg8 was found in the cytosol of tachyzoites and occasionally in punctate vesicles in recently egressed parasites .

A. Autophagic vesicles marker GFP-TgAtg8 was found in the cytosol of tachyzoites and occasionally in punctate vesicles in recently egressed parasites.


B . Extracellular tachyzoites were put to starve in HBSS medium for increased time periods and the proportions of cells displaying punctate or cytosolic GFP - TgAtg8 signals were assessed . Data are mean from n = 4 independent experiments ± SEM .

O O O O O O O O O O O O O O O O O O O O O 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. Extracellular tachyzoites were put to starve in HBSS medium for increased time periods and the proportions of cells displaying punctate or cytosolic GFP-TgAtg8 signals were assessed. Data are mean from n = 4 independent experiments ±SEM.


A . Extracellular tachyzoites were starved for 8 hours in HBSS medium and observed ( 2 , 3 ) . Unstarved control is shown for comparison ( 1 ) . Several vacuoles ( V ) can be seen in starved parasites , with also cytosol - and organelle - sequestrating membranous structures resembling autophagosomes ( Ap ) , as well as vacuoles containing partially digested material corresponding to autophagic degradative vacuoles ( Av ) . 2 ′ , 3 ′ and 3 " pictures are a magnification of squared regions . Scale bar = 1 µm in initial micrographs ( 1 , 2 , 3 ) and 0 . 2 µm in magnifications .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Extracellular tachyzoites were starved for 8 hours in HBSS medium and observed (2, 3). Unstarved control is shown for comparison (1). Several vacuoles (V) can be seen in starved parasites, with also cytosol- and organelle-sequestrating membranous structures resembling autophagosomes (Ap), as well as vacuoles containing partially digested material corresponding to autophagic degradative vacuoles (Av). 2′, 3′ and 3" pictures are a magnification of squared regions. Scale bar = 1 µm in initial micrographs (1, 2, 3) and 0.2 µm in magnifications.


B . Partial co - localisation of GFP - TgAtg8 - labeled autophagosomes and Toxoplasma mitochondrion . Extracellular tachyzoites expressing GFP - TgAtg8 were incubated in complete medium with Mitotracker Red to label the mitochondrion ; partial co - localisation could be observed in some parasites displaying autophagic vesicles .

B. Partial co-localisation of GFP-TgAtg8-labeled autophagosomes and Toxoplasma mitochondrion. Extracellular tachyzoites expressing GFP-TgAtg8 were incubated in complete medium with Mitotracker Red to label the mitochondrion; partial co-localisation could be observed in some parasites displaying autophagic vesicles.


C . Identification of autophagic vesicles in extracellular tachyzoites by immuno - electron microscopy . Extracellular tachyzoites expressing GFP - TgAtg8 were starved for 8 hours in HBSS medium , fixed , cryosectioned and stained with an anti - GFP antibody , then with gold - conjugated secondary antibody and observed . Gold particles can be seen around cytosol - sequestrating membranous structures resembling autophagosomes ( Ap ) , as well as larger vacuoles containing partially digested material corresponding to autophagic degradative vacuoles ( Av ) . Scale bar = 200 nm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Identification of autophagic vesicles in extracellular tachyzoites by immuno-electron microscopy. Extracellular tachyzoites expressing GFP-TgAtg8 were starved for 8 hours in HBSS medium, fixed, cryosectioned and stained with an anti-GFP antibody, then with gold-conjugated secondary antibody and observed. Gold particles can be seen around cytosol-sequestrating membranous structures resembling autophagosomes (Ap), as well as larger vacuoles containing partially digested material corresponding to autophagic degradative vacuoles (Av). Scale bar = 200 nm.


A . Protein extracts corresponding to tachyzoites incubated in the same starvation conditions as in Figure 2B were separated by urea SDS - PAGE and analysed with an anti - GFP antibody to detect TgAtg8 and the lipidated TgAtg8 - PE form . GFP - TgAtg8 parasites extracts were analysed with anti GFP antibody ( left ) , while parental cell line was analysed with anti - TgAtg8 antibody ( right ) . Anti - ROP5 was used as a 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 O O O O O O O O O

A. Protein extracts corresponding to tachyzoites incubated in the same starvation conditions as in Figure 2B were separated by urea SDS-PAGE and analysed with an anti-GFP antibody to detect TgAtg8 and the lipidated TgAtg8-PE form. GFP-TgAtg8 parasites extracts were analysed with anti GFP antibody (left), while parental cell line was analysed with anti-TgAtg8 antibody (right). Anti-ROP5 was used as a loading control.


B . Cell lysates from GFP - TgAtg8 parasites were subjected to a centrifugation at 100 , 000 g to separate the soluble fraction ( high speed supernatant , HSS ) from the membrane fraction ( high speed pellet , HSP ) . The faster migrating form of GFP - TgAtg8 is exclusively present in the membrane fraction as revealed by Western blot analysis after urea SDS - PAGE using anti -

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Cell lysates from GFP-TgAtg8 parasites were subjected to a centrifugation at 100,000 g to separate the soluble fraction (high speed supernatant, HSS) from the membrane fraction (high speed pellet, HSP). The faster migrating form of GFP-TgAtg8 is exclusively present in the membrane fraction as revealed by Western blot analysis after urea SDS-PAGE using anti-GFP


C . The HSP fraction described in B . was extracted with various agents and new HSS and HSP fractions were separated and analysed by Western blot after urea SDS - PAGE . Only a treatment with a detergent ( DOC ) resulted in GFP - TgAtg8 solubilisation from the HSP fraction .

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

C. The HSP fraction described in B. was extracted with various agents and new HSS and HSP fractions were separated and analysed by Western blot after urea SDS-PAGE. Only a treatment with a detergent (DOC) resulted in GFP-TgAtg8 solubilisation from the HSP fraction.


D . GFP - TgAtg8 - expressing parasites were grown in host cells in the presence of 3H - ethanolamine and then starved in HBSS for 8 hours , still in the presence of the radioactive PE precursor . Tachyzoites were then lysed and GFP - TgAtg8 was immunoprecipitated using anti - TgAtg8 antibody and analysed by urea SDS - PAGE . The immunoprecipitated forms of GFP - TgAtg8 were detected by Western blot using anti - GFP antibody and radioactive ethanolamine was found to be incorporated into immunoprecipitated GFP - TgAtg8 , as detected by fluorography ( 3H - etn ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. GFP-TgAtg8-expressing parasites were grown in host cells in the presence of 3H-ethanolamine and then starved in HBSS for 8 hours, still in the presence of the radioactive PE precursor. Tachyzoites were then lysed and GFP-TgAtg8 was immunoprecipitated using anti-TgAtg8 antibody and analysed by urea SDS-PAGE. The immunoprecipitated forms of GFP-TgAtg8 were detected by Western blot using anti-GFP antibody and radioactive ethanolamine was found to be incorporated into immunoprecipitated GFP-TgAtg8, as detected by fluorography (3H-etn).


A . Fluorescence microscopy analysis of extracellular tachyzoites expressing GFP - TgAtg8 or its glycine mutant version , before or after induction of autophagy for 8 hours in HBSS . Autophagosomes are labelled by GFP - TgAtg8 in control parasites ( arrowheads ) , but not by the glycine mutant version of the protein . B . Extracellular tachyzoites expressing GFP - TgAtg8 or its glycine mutant version were put to starve in HBSS medium for increased lengths of time and the proportions of cells displaying punctate or cytosolic GFP signals were assessed . Data are mean from n = 4 independent experiments ± SEM .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Fluorescence microscopy analysis of extracellular tachyzoites expressing GFP-TgAtg8 or its glycine mutant version, before or after induction of autophagy for 8 hours in HBSS. Autophagosomes are labelled by GFP-TgAtg8 in control parasites (arrowheads), but not by the glycine mutant version of the protein. B. Extracellular tachyzoites expressing GFP-TgAtg8 or its glycine mutant version were put to starve in HBSS medium for increased lengths of time and the proportions of cells displaying punctate or cytosolic GFP signals were assessed. Data are mean from n = 4 independent experiments ±SEM.


C . Protein extracts corresponding to tachyzoites expressing GFP - TgAtg8 and glycine mutant version incubated in HBSS for up to 8 hours , were separated by urea SDS - PAGE and analysed with an anti - GFP antibody to detect GFP - TgAtg8 and lipidated GFP - TgAtg8 - PE forms . Anti - ROP5 was used as a 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

C. Protein extracts corresponding to tachyzoites expressing GFP-TgAtg8 and glycine mutant version incubated in HBSS for up to 8 hours, were separated by urea SDS-PAGE and analysed with an anti-GFP antibody to detect GFP-TgAtg8 and lipidated GFP-TgAtg8-PE forms. Anti-ROP5 was used as a loading control.


A . Intracellular tachyzoites expressing GFP - TgAtg8 were used to invade host HFF and the proportions of cells displaying punctate or cytosolic GFP signals were assessed along time during development . Data are mean from n = 5 independent experiments ± SEM .

O O O O O O O O O O O O O O O O O O O O O O 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. Intracellular tachyzoites expressing GFP-TgAtg8 were used to invade host HFF and the proportions of cells displaying punctate or cytosolic GFP signals were assessed along time during development. Data are mean from n = 5 independent experiments ±SEM.


B . Fluorescence microscopy pictures of GFP - TgAtg8 - expressing tachyzoites during their intracellular development and division . The arrowhead indicates the residual body made of maternal material left after endodyogeny .

O O O O O O O O O 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. Fluorescence microscopy pictures of GFP-TgAtg8-expressing tachyzoites during their intracellular development and division. The arrowhead indicates the residual body made of maternal material left after endodyogeny.


C . Detection of GFP - TgAtg8 - labelled autophagosomes in dividing parasites . Progression of daughter cells division was followed by expression of an IMC1 - RFP construct .

O O O O O O 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. Detection of GFP-TgAtg8-labelled autophagosomes in dividing parasites. Progression of daughter cells division was followed by expression of an IMC1-RFP construct.


A . Schematic representation of the strategy . Recombineered cosmid bearing chloramphenicol acetyl transferase ( CAT ) gene for selection of resistant parasites , flanked by homologous regions from TgAtg3 , was used to replace endogenous genomic locus in the cell line expressing a regulatable extra copy of TgAtg3 ( imyc - sTgAtg3 ) . Mutants obtained after double homologous recombination events were selected with chloramphenicol . PCR amplifications and Southern blot strategy used for subsequent characterisation of the mutant are indicated in red and green , respectively . B . PCR verification of the removal of TgAtg3 in the mutant ( imyc - sTgAtg3 - ΔAtg3 , A ) compared to the parental cell line ( imyc - sTgAtg3 , B )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Schematic representation of the strategy. Recombineered cosmid bearing chloramphenicol acetyl transferase (CAT) gene for selection of resistant parasites, flanked by homologous regions from TgAtg3, was used to replace endogenous genomic locus in the cell line expressing a regulatable extra copy of TgAtg3 (imyc-sTgAtg3). Mutants obtained after double homologous recombination events were selected with chloramphenicol. PCR amplifications and Southern blot strategy used for subsequent characterisation of the mutant are indicated in red and green, respectively. B. PCR verification of the removal of TgAtg3 in the mutant (imyc-sTgAtg3-ΔAtg3, A) compared to the parental cell line (imyc-sTgAtg3, B)


C . Southern blot verification of the TgAtg3 gene replacement . ∼ 4 µg of genomic DNA from mutant ( A ) and parental ( B ) tachyzoites were digested by HindIII / AvrII , transferred on a nylon membrane and probed with 32P - labeled 5 ′ and 3 ′ DNA probes located as schematised in A .

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

C. Southern blot verification of the TgAtg3 gene replacement. ∼4 µg of genomic DNA from mutant (A) and parental (B) tachyzoites were digested by HindIII/AvrII, transferred on a nylon membrane and probed with 32P-labeled 5′ and 3′ DNA probes located as schematised in A.


A . The vector for the expression of the ATc - regulatable sTgAtg3 extra copy allows myc - epitope tagging of this protein . Anti - myc detection of myc - tagged TgAtg3 protein in intracellular parasites either without treatment with ATc , or with a continuous 2 days treatment with 1 . 5 µg / ml ATc , or after reinvasion following a continuous 4 days treatment . DIC : differential interference contrast .

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 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

A. The vector for the expression of the ATc-regulatable sTgAtg3 extra copy allows myc-epitope tagging of this protein. Anti-myc detection of myc-tagged TgAtg3 protein in intracellular parasites either without treatment with ATc, or with a continuous 2 days treatment with 1.5 µg/ml ATc, or after reinvasion following a continuous 4 days treatment. DIC: differential interference contrast.


B . Western blot detection of the myc - tagged sTgAtg3 protein ( arrowed ) in parasite lysates corresponding to the induction conditions described in A . Anti - SAG1 antibody was used as a 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

B. Western blot detection of the myc-tagged sTgAtg3 protein (arrowed) in parasite lysates corresponding to the induction conditions described in A. Anti-SAG1 antibody was used as a loading control.


C . Western blot analysis following urea SDS - PAGE of TgAtg3 - depleted parasites extracts , showing an absence of upregulation of the autophasome - bound TgAtg8 - PE form . Anti - ROP5 was used as a 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

C. Western blot analysis following urea SDS-PAGE of TgAtg3-depleted parasites extracts, showing an absence of upregulation of the autophasome-bound TgAtg8-PE form. Anti-ROP5 was used as a loading control.


A . Confluent monolayers of fibroblasts were infected either with extra copy - expressing imyc - sTgAtg3 parental cell line in the presence of ATc ( a ) , imyc - sTgAtg3 - ΔAtg3 mutant cell line without ATc ( b ) , mutant cell line in the presence of ATc ( c ) , or mutant cell line pre - incubated with ATc before start of the experiment and maintained in the presence of ATc . Plaques ( arrowheads ) resulting from the lysis of host cells due to the multiplication of the parasites are only visible when TgAtg3 is still expressed . B . Mean plaque area comparisons between fibroblast layers infected with controls and TgAtg3 - depleted parasites ( a , b , c : legend as in A ) . Plaques observed with the mutant in the presence of ATc were significantly smaller than with control cell lines ( * p < 0 . 005 , Student ' s T test ) . Data are mean from n = 3 independent experiments ± SEM .

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 B-CONTROLLED_VAR 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 B-CONTROLLED_VAR 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 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

A. Confluent monolayers of fibroblasts were infected either with extra copy-expressing imyc-sTgAtg3 parental cell line in the presence of ATc (a), imyc-sTgAtg3-ΔAtg3 mutant cell line without ATc (b), mutant cell line in the presence of ATc (c), or mutant cell line pre-incubated with ATc before start of the experiment and maintained in the presence of ATc. Plaques (arrowheads) resulting from the lysis of host cells due to the multiplication of the parasites are only visible when TgAtg3 is still expressed. B. Mean plaque area comparisons between fibroblast layers infected with controls and TgAtg3-depleted parasites (a, b, c: legend as in A). Plaques observed with the mutant in the presence of ATc were significantly smaller than with control cell lines (* p<0.005, Student's T test). Data are mean from n = 3 independent experiments ±SEM.


C . Numbers of parasites per vacuole are significantly lower in TgAtg3 - depleted cell line ( pre - incubated for 4 days with ATc ) compared with controls 24 or 48 hours post invasion . Data are mean from n = 3 independent experiments ± SEM .

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

C. Numbers of parasites per vacuole are significantly lower in TgAtg3-depleted cell line (pre-incubated for 4 days with ATc) compared with controls 24 or 48 hours post invasion. Data are mean from n = 3 independent experiments ±SEM.


TgAtg3 - depleted parasites show a defect in mitochondrion morphology . Mitochondrion labelled with specific antibodies ( see results section ) was found as a reduced structure , or even absent , in TgAtg3 - depleted cell lines ( middle series of micrographs , kept for 2 days in the presence of ATc or pre - incubated with ATc for 4 days before invasion , respectively ) . Parasites left to develop for two days in the presence of ATc to progressively extinguish the expression of TgAtg3 showed an accumulation of mitochondrial marker at the residual body . TgAtg3 depletion was verified by detecting myc - tagged regulatable extra - copy with specific antibody . TgAtg3 - expressing cell line imyc - sTgAtg3cultivated in the presence of ATc for 4 days was used as a control for mitochondrial morphology ( bottom ) . DNA was labelled with DAPI .

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

TgAtg3-depleted parasites show a defect in mitochondrion morphology.Mitochondrion labelled with specific antibodies (see results section) was found as a reduced structure, or even absent, in TgAtg3-depleted cell lines (middle series of micrographs, kept for 2 days in the presence of ATc or pre-incubated with ATc for 4 days before invasion, respectively). Parasites left to develop for two days in the presence of ATc to progressively extinguish the expression of TgAtg3 showed an accumulation of mitochondrial marker at the residual body. TgAtg3 depletion was verified by detecting myc-tagged regulatable extra-copy with specific antibody. TgAtg3-expressing cell line imyc-sTgAtg3cultivated in the presence of ATc for 4 days was used as a control for mitochondrial morphology (bottom). DNA was labelled with DAPI.


Altered mitochondrial ultrastructure in TgAtg3 - depleted tachyzoites observed by electron microscopy . Ultrathin section of a TgAtg3 - depleted ( 3 days of ATc treatment ) tachyzoite showing the dramatic alteration of the mitochondrion , where cristae remnants are still visible ( arrows ) , but which is filled up with membranous profiles ( arrowheads ) , whereas Golgi ( G ) , apicoplast ( A ) , rhoptries ( R ) and dense granule ( D ) look normal .

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

Altered mitochondrial ultrastructure in TgAtg3-depleted tachyzoites observed by electron microscopy.Ultrathin section of a TgAtg3-depleted (3 days of ATc treatment) tachyzoite showing the dramatic alteration of the mitochondrion, where cristae remnants are still visible (arrows), but which is filled up with membranous profiles (arrowheads), whereas Golgi (G), apicoplast (A), rhoptries (R) and dense granule (D) look normal.


About 25 μg of membrane ‐ enriched lysate from the mass spectrometry experiments was immunoblotted with anti ‐ PINK1 antibody . TOMM40 and GAPDH serve as markers for mitochondria and cytoplasm , respectively .

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

About 25 μg of membrane‐enriched lysate from the mass spectrometry experiments was immunoblotted with anti‐PINK1 antibody. TOMM40 and GAPDH serve as markers for mitochondria and cytoplasm, respectively.


A - CConfirmation by mass spectrometry that Rab8A ( A ) , Rab8B ( B ) and Rab13 ( C ) Ser111 is phosphorylated upon PINK1 activation after CCCP treatment . Flp ‐ In T ‐ Rex HEK293 cells expressing empty ‐ FLAG , WT PINK1 ‐ FLAG and KI ( D384A ) PINK1 ‐ FLAG were transfected either with HA ‐ Rab8A ( A ) , HA ‐ Rab8B ( B ) or HA ‐ Rab13 ( C ) induced with doxycycline and stimulated with 10 μM of CCCP for 3 h . Whole ‐ cell lysates ( 10 mg ) were immunoprecipitated with anti ‐ HA agarose , resolved by SDS - PAGE and stained with colloidal Coomassie blue ( second panel ) . Coomassie ‐ stained bands migrating with expected molecular mass of HA ‐ Rabs were excised , in ‐ gel digested with trypsin and subjected to high ‐ performance liquid chromatography with LC ‐ MS / MS on an LTQ ‐ Orbitrap mass spectrometer . Upper panel shows the extracted ion chromatogram ( XIC ) analysis of Ser111 ‐ containing phosphopeptides ( 8A , NIEEHApSADVEK ; 8B , NIEEHApSSDVER ; 13 , SIKENApSAGVER ) with the combined signal intensity of the 2 + and 3 + forms of the peptide indicated on the y ‐ axis . Note that the Ser111 phosphopeptide was only detected in samples from WT PINK1 ‐ FLAG ‐ expressing cells following CCCP stimulation .

O O O O 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-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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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

A-CConfirmation by mass spectrometry that Rab8A (A), Rab8B (B) and Rab13 (C) Ser111 is phosphorylated upon PINK1 activation after CCCP treatment. Flp‐In T‐Rex HEK293 cells expressing empty‐FLAG, WT PINK1‐FLAG and KI (D384A) PINK1‐FLAG were transfected either with HA‐Rab8A (A), HA‐Rab8B (B) or HA‐Rab13 (C) induced with doxycycline and stimulated with 10 μM of CCCP for 3 h. Whole‐cell lysates (10 mg) were immunoprecipitated with anti‐HA agarose, resolved by SDS-PAGE and stained with colloidal Coomassie blue (second panel). Coomassie‐stained bands migrating with expected molecular mass of HA‐Rabs were excised, in‐gel digested with trypsin and subjected to high‐performance liquid chromatography with LC‐MS/MS on an LTQ‐Orbitrap mass spectrometer. Upper panel shows the extracted ion chromatogram (XIC) analysis of Ser111‐containing phosphopeptides (8A, NIEEHApSADVEK; 8B, NIEEHApSSDVER; 13, SIKENApSAGVER) with the combined signal intensity of the 2+ and 3+ forms of the peptide indicated on the y‐axis. Note that the Ser111 phosphopeptide was only detected in samples from WT PINK1‐FLAG‐expressing cells following CCCP stimulation.


DCharacterisation of Rab8A , Rab8B and Rab13 phospho ‐ Ser111 antibodies . Flp ‐ In T ‐ Rex HEK293 cells expressing empty ‐ FLAG , WT PINK1 ‐ FLAG and KI ( D384A ) PINK1 ‐ FLAG were transfected with either WT or Ser111Ala ‐ mutant ( S111A ) HA ‐ Rab8A , HA ‐ Rab8B or HA ‐ Rab13 , induced with doxycycline and stimulated with 10 μM of CCCP for 3 h . Whole ‐ cell lysates ( 0 . 25 mg ) were immunoprecipitated with anti ‐ HA agarose and immunoblotted with Rab8A , Rab8B or Rab13 phospho ‐ Ser111 antibodies . Part of the immunoprecipitates was used to immunoblot for HA antibody as loading controls .

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

DCharacterisation of Rab8A, Rab8B and Rab13 phospho‐Ser111 antibodies. Flp‐In T‐Rex HEK293 cells expressing empty‐FLAG, WT PINK1‐FLAG and KI (D384A) PINK1‐FLAG were transfected with either WT or Ser111Ala‐mutant (S111A) HA‐Rab8A, HA‐Rab8B or HA‐Rab13, induced with doxycycline and stimulated with 10 μM of CCCP for 3 h. Whole‐cell lysates (0.25 mg) were immunoprecipitated with anti‐HA agarose and immunoblotted with Rab8A, Rab8B or Rab13 phospho‐Ser111 antibodies. Part of the immunoprecipitates was used to immunoblot for HA antibody as loading controls.


PINK1 is essential for CCCP ‐ mediated Rab8A Ser111 phosphorylation . WT and PINK1 KO HeLa cells were transfected with either WT or Ser111Ala ( S111A ) mutant constructs of HA ‐ Rab8A , HA ‐ Rab8B or HA ‐ Rab13 . Some PINK1 KO HeLa cells were reintroduced with PINK1 by transfection of WT PINK1 ‐ 3xFLAG or KI ( D384A ) PINK1 ‐ 3xFLAG as indicated . After transfection for at least 24 h , cells were treated with DMSO as a vehicle control or CCCP for 20 h . Whole ‐ cell lysates ( 1 mg ) were immunoprecipitated with anti ‐ HA agarose and immunoblotted with Rab8A , Rab8B or Rab13 phospho ‐ Ser111 antibody . Part of the immunoprecipitates was used to immunoblot for HA antibody as loading controls . For the lower panel , whole ‐ cell lysates ( 30 μg ) were immunoblotted with total PINK1 antibody to confirm PINK1 expression and with GAPDH as loading controls .

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 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

PINK1 is essential for CCCP‐mediated Rab8A Ser111 phosphorylation. WT and PINK1 KO HeLa cells were transfected with either WT or Ser111Ala (S111A) mutant constructs of HA‐Rab8A, HA‐Rab8B or HA‐Rab13. Some PINK1 KO HeLa cells were reintroduced with PINK1 by transfection of WT PINK1‐3xFLAG or KI (D384A) PINK1‐3xFLAG as indicated. After transfection for at least 24 h, cells were treated with DMSO as a vehicle control or CCCP for 20 h. Whole‐cell lysates (1 mg) were immunoprecipitated with anti‐HA agarose and immunoblotted with Rab8A, Rab8B or Rab13 phospho‐Ser111 antibody. Part of the immunoprecipitates was used to immunoblot for HA antibody as loading controls. For the lower panel, whole‐cell lysates (30 μg) were immunoblotted with total PINK1 antibody to confirm PINK1 expression and with GAPDH as loading controls.


Endogenous Rab8A Ser111 phosphorylation is PINK1 dependent . WT and PINK1 KO HeLa cells were treated with DMSO as a vehicle control or CCCP for 20 h . Some PINK1 KO HeLa cells were reintroduced with PINK1 by transfection of WT PINK1 ‐ 3xFLAG or KI ( D384A ) PINK1 ‐ 3xFLAG as indicated for at least 24 h before CCCP treatment . Whole ‐ cell lysates ( 1 mg ) were immunoprecipitated with anti ‐ Rab8A pre ‐ bound with protein A agarose followed by immunoblot with Rab8A phospho ‐ Ser111 antibody . Part of the immunoprecipitates was used to immunoblot with anti ‐ total Rab8A antibody as loading controls .

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 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

Endogenous Rab8A Ser111 phosphorylation is PINK1 dependent. WT and PINK1 KO HeLa cells were treated with DMSO as a vehicle control or CCCP for 20 h. Some PINK1 KO HeLa cells were reintroduced with PINK1 by transfection of WT PINK1‐3xFLAG or KI (D384A) PINK1‐3xFLAG as indicated for at least 24 h before CCCP treatment. Whole‐cell lysates (1 mg) were immunoprecipitated with anti‐Rab8A pre‐bound with protein A agarose followed by immunoblot with Rab8A phospho‐Ser111 antibody. Part of the immunoprecipitates was used to immunoblot with anti‐total Rab8A antibody as loading controls.


Absence of Rab8A Ser111 phosphorylation in human mutant PINK1 patient fibroblasts . Primary skinfibroblasts were derived from a patient with homozygous PINK1 Q456X mutation or unaffected control . Cells were incubated with DMSO or CCCP for 20 h , and whole ‐ cell lysates ( 1 mg ) were immunoprecipitated with anti ‐ Rab8A antibody conjugated to protein A agarose and immunoblotted with total or Rab8A phospho ‐ Ser111 antibody . Lysates ( 1 mg ) were also subjected to immunoprecipitation with polyclonal anti ‐ PINK1 antibody and immunoblotted with monoclonal PINK1 antibody . Equal loading of protein extracts was confirmed by GAPDH .

O O O O O O O O O O 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

Absence of Rab8A Ser111 phosphorylation in human mutant PINK1 patient fibroblasts. Primary skinfibroblasts were derived from a patient with homozygous PINK1 Q456X mutation or unaffected control. Cells were incubated with DMSO or CCCP for 20 h, and whole‐cell lysates (1 mg) were immunoprecipitated with anti‐Rab8A antibody conjugated to protein A agarose and immunoblotted with total or Rab8A phospho‐Ser111 antibody. Lysates (1 mg) were also subjected to immunoprecipitation with polyclonal anti‐PINK1 antibody and immunoblotted with monoclonal PINK1 antibody. Equal loading of protein extracts was confirmed by GAPDH.


Absence of Rab8A Ser111 phosphorylation in PINK1 knockout MEFs . MEFs were derived from PINK1 knockout embryos or wild ‐ type controls ( see Materials and Methods ) . Cells were incubated with DMSO or CCCP for 20 h , and whole ‐ cell lysates ( 1 mg ) were immunoprecipitated with anti ‐ Rab8A antibody conjugated to protein A agarose and immunoblotted with total or Rab8A phospho ‐ Ser111 antibody . Lysates ( 1 mg ) were also subjected to immunoprecipitation with a polyclonal anti ‐ mouse ‐ specific PINK1 antibody and immunoblotted with a different anti ‐ mouse ‐ specific PINK1 antibody . Equal loading of protein extracts was confirmed by GAPDH .

O O O O O O O O O O O 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

Absence of Rab8A Ser111 phosphorylation in PINK1 knockout MEFs. MEFs were derived from PINK1 knockout embryos or wild‐type controls (see Materials and Methods). Cells were incubated with DMSO or CCCP for 20 h, and whole‐cell lysates (1 mg) were immunoprecipitated with anti‐Rab8A antibody conjugated to protein A agarose and immunoblotted with total or Rab8A phospho‐Ser111 antibody. Lysates (1 mg) were also subjected to immunoprecipitation with a polyclonal anti‐mouse‐specific PINK1 antibody and immunoblotted with a different anti‐mouse‐specific PINK1 antibody. Equal loading of protein extracts was confirmed by GAPDH.


Rab8A is not required for the activation of Parkin E3 ligase activity at mitochondria in response to PINK1 activation by CCCPWild ‐ type ( WT ) or Rab8A knockout ( KO ) HeLa cells were transfected with WT or Cys431Phe ( C341F ) ‐ mutant Parkin . After transfection for 24 h , cells were treated with DMSO as a vehicle control or 10 μM CCCP for 6 h . Mitochondrial enriched extracts ( mitochondrial lysate ) were incubated with ubiquitin ‐ binding resins derived from his ‐ halo ‐ ubiquilin1 UBA domain tetramer ( UBAUBQLN1 ) . Captured ubiquitylated proteins were subject to immunoblotting with anti ‐ CISD1 and anti ‐ mitofusin 2 antibodies . Mitochondrial lysate and total lysate were also subjected to immunoblotting with indicated antibodies for loading and protein expression controls . Phospho ‐ Ser111 Rab8A was detected after Rab8A immunoprecipitation from 200 μg of mitochondrial lysate with anti ‐ Rab8A antibody .

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

Rab8A is not required for the activation of Parkin E3 ligase activity at mitochondria in response to PINK1 activation by CCCPWild‐type (WT) or Rab8A knockout (KO) HeLa cells were transfected with WT or Cys431Phe (C341F)‐mutant Parkin. After transfection for 24 h, cells were treated with DMSO as a vehicle control or 10 μM CCCP for 6 h. Mitochondrial enriched extracts (mitochondrial lysate) were incubated with ubiquitin‐binding resins derived from his‐halo‐ubiquilin1 UBA domain tetramer (UBAUBQLN1). Captured ubiquitylated proteins were subject to immunoblotting with anti‐CISD1 and anti‐mitofusin 2 antibodies. Mitochondrial lysate and total lysate were also subjected to immunoblotting with indicated antibodies for loading and protein expression controls. Phospho‐Ser111 Rab8A was detected after Rab8A immunoprecipitation from 200 μg of mitochondrial lysate with anti‐Rab8A antibody.


In vitro phosphorylation analysis of Rab8A by PINK1 . WT or S111A ‐ mutant Rab8A ( 1 . 2 μg ) was incubated in the presence of MBP ‐ fused WT or KI ( D359A ) TcPINK1 ( 1 . 1 μg ) and Mg2 + ‐ [ γ ‐ 32P ] ATP for the indicated time . Samples were subjected to SDS - PAGE , and proteins were detected by colloidal Coomassie blue staining ( lower panel ) . The [ γ ‐ 32P ] incorporation to substrate was detected by autoradiography ( upper panel ) . Cerenkov counting was used to calculate the stoichiometry of substrate phosphorylation as mol of [ γ ‐ 32P ] incorporation / mol of substrate . Ubiquitin was used as a positive control of the TcPINK1 substrate .

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

In vitro phosphorylation analysis of Rab8A by PINK1. WT or S111A‐mutant Rab8A (1.2 μg) was incubated in the presence of MBP‐fused WT or KI (D359A) TcPINK1 (1.1 μg) and Mg2+‐[γ‐32P] ATP for the indicated time. Samples were subjected to SDS-PAGE, and proteins were detected by colloidal Coomassie blue staining (lower panel). The [γ‐32P] incorporation to substrate was detected by autoradiography (upper panel). Cerenkov counting was used to calculate the stoichiometry of substrate phosphorylation as mol of [γ‐32P] incorporation/mol of substrate. Ubiquitin was used as a positive control of the TcPINK1 substrate.


Time ‐ course comparison of PINK1 ‐ mediated Rab8A , Rab8B and Rab13 Ser111 phosphorylation vs . Parkin Ser65 phosphorylation . Flp ‐ In T ‐ Rex HEK293 cells expressing WT PINK1 ‐ FLAG were transfected with either WT or Ser111Ala ‐ ( S111A ) mutant HA ‐ Rab8A , HA ‐ Rab8B or HA ‐ Rab13 , induced with doxycycline and stimulated with CCCP for the indicated time . In parallel , Flp ‐ In T ‐ Rex HEK293 cells expressing WT PINK1 ‐ FLAG were transfected with either WT or Ser65 Ala ( S65A ) ‐ mutant Parkin . Whole ‐ cell lysates ( 0 . 25 mg ) were immunoprecipitated with anti ‐ HA agarose and immunoblotted with indicated phospho ‐ Ser111 antibodies . Part of the immunoprecipitates was used to immunoblot for HA antibody as loading controls . For the lower panel , whole ‐ cell lysates ( 30 μg ) were immunoblotted with indicated antibodies .

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

Time‐course comparison of PINK1‐mediated Rab8A, Rab8B and Rab13 Ser111 phosphorylation vs. Parkin Ser65 phosphorylation. Flp‐In T‐Rex HEK293 cells expressing WT PINK1‐FLAG were transfected with either WT or Ser111Ala‐(S111A) mutant HA‐Rab8A, HA‐Rab8B or HA‐Rab13, induced with doxycycline and stimulated with CCCP for the indicated time. In parallel, Flp‐In T‐Rex HEK293 cells expressing WT PINK1‐FLAG were transfected with either WT or Ser65 Ala (S65A)‐mutant Parkin. Whole‐cell lysates (0.25 mg) were immunoprecipitated with anti‐HA agarose and immunoblotted with indicated phospho‐Ser111 antibodies. Part of the immunoprecipitates was used to immunoblot for HA antibody as loading controls. For the lower panel, whole‐cell lysates (30 μg) were immunoblotted with indicated antibodies.


Time ‐ course comparison of endogenous PINK1 ‐ mediated Rab8A , Rab8B and Rab13 Ser111 phosphorylation vs . Parkin Ser65 phosphorylation . HeLa cells were transfected with either WT or S111A ‐ mutant HA ‐ Rab8A , HA ‐ Rab8B or HA ‐ Rab13 for at least 24 h before CCCP treatment for the indicated time . Whole ‐ cell lysates ( 1 mg ) were immunoprecipitated with anti ‐ HA agarose and immunoblotted with indicated phospho ‐ Ser111 antibodies . In parallel , HeLa cells were transfected with either WT or S65A ‐ mutant Parkin and whole ‐ cell lysates ( 30 μg ) were immunoblotted with indicated antibodies .

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

Time‐course comparison of endogenous PINK1‐mediated Rab8A, Rab8B and Rab13 Ser111 phosphorylation vs. Parkin Ser65 phosphorylation. HeLa cells were transfected with either WT or S111A‐mutant HA‐Rab8A, HA‐Rab8B or HA‐Rab13 for at least 24 h before CCCP treatment for the indicated time. Whole‐cell lysates (1 mg) were immunoprecipitated with anti‐HA agarose and immunoblotted with indicated phospho‐Ser111 antibodies. In parallel, HeLa cells were transfected with either WT or S65A‐mutant Parkin and whole‐cell lysates (30 μg) were immunoblotted with indicated antibodies.


Rabin8 ‐ catalysed mant ‐ GDP release from mant ‐ GDP loaded WT , S111A and S111E mutants of Rab8A . The Rab proteins ( 1 μM ) were incubated with 100 μM GDP in buffer ( 20 mM HEPES pH 7 . 5 , 50 mM NaCl , 1 mM MgCl2 , 2 mM DTE ) , and the reaction was started by the addition of 0 . 5 μM Rabin8 . The decrease in mant fluorescence was used as a measure of mantGDP release .

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

Rabin8‐catalysed mant‐GDP release from mant‐GDP loaded WT, S111A and S111E mutants of Rab8A. The Rab proteins (1 μM) were incubated with 100 μM GDP in buffer (20 mM HEPES pH 7.5, 50 mM NaCl, 1 mM MgCl2, 2 mM DTE), and the reaction was started by the addition of 0.5 μM Rabin8. The decrease in mant fluorescence was used as a measure of mantGDP release.


Rab8A Ser111 phosphorylation impairs Rabin8 interaction in cells . Rab8A KO HeLa cells were transfected with wild ‐ type ( WT ) , S111E or S111A HA ‐ Rab8A . Whole ‐ cell lysates ( 1 mg ) were immunoprecipitated with anti ‐ HA agarose and immunoblotted with Rabin8 or anti ‐ HA antibody . Lysates were immunoblotted with Rabin8 or anti ‐ HA antibody to confirm equivalent expression of Rabin8 and WT and mutant HA ‐ Rab8A in extracts .

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

Rab8A Ser111 phosphorylation impairs Rabin8 interaction in cells. Rab8A KO HeLa cells were transfected with wild‐type (WT), S111E or S111A HA‐Rab8A. Whole‐cell lysates (1 mg) were immunoprecipitated with anti‐HA agarose and immunoblotted with Rabin8 or anti‐HA antibody. Lysates were immunoblotted with Rabin8 or anti‐HA antibody to confirm equivalent expression of Rabin8 and WT and mutant HA‐Rab8A in extracts.


VGLL4 protein expression levels in 6 murine tumor cell lines . Total cell lysates were analyzed by western

VGLL4 protein expression levels in 6 murine tumor cell lines. Total cell lysates were analyzed by western blot


VGLL4 protein levels were reduced in Vgll4 depleted cells . Total cell lysates of Vgll4 - knockdown LLC and MB49 cells were analyzed by western

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

VGLL4 protein levels were reduced in Vgll4 depleted cells. Total cell lysates of Vgll4-knockdown LLC and MB49 cells were analyzed by western blot


Control or shVgll4 LLC cells were transplanted into C57BL / 6 mice , and tumor - growth kinetics was measured at the indicated times . n = 12 tumors for each

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

Control or shVgll4 LLC cells were transplanted into C57BL/6 mice, and tumor-growth kinetics was measured at the indicated times. n=12 tumors for each group


Quantification of tumor weights 21 days after transplantation of control or shVgll4 LLC cells into C57BL / 6 mice from ( C )

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 tumor weights 21 days after transplantation of control or shVgll4 LLC cells into C57BL/6 mice from (C)


Control or shVgll4 LLC cells were transplanted into nude mice , and tumor - growth kinetics was measured at the indicated times . n = 8 tumors for each group . ( F ) Quantification of tumor weights 15 days after transplantation of control or shVgll4 LLC cells into nude mice from ( E ) . n = 8 tumors for each group . n . s . p > 0 . 05 , two - tailed Student " s t - test . The solid line represents the average

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

Control or shVgll4 LLC cells were transplanted into nude mice, and tumor-growth kinetics was measured at the indicated times. n=8 tumors for each group. (F) Quantification of tumor weights 15 days after transplantation of control or shVgll4 LLC cells into nude mice from (E). n=8 tumors for each group. n.s. p>0.05, two-tailed Student"s t-test. The solid line represents the average weight


Control or shVgll4 MB49 cells were transplanted into C57BL / 6 mice , and tumor - growth kinetics was measured at the indicated times . n = 9 tumors for each group Quantification of tumor weights 30 days after transplantation of control or shVgll4 MB49 cells into C57BL / 6

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

Control or shVgll4 MB49 cells were transplanted into C57BL/6 mice, and tumor-growth kinetics was measured at the indicated times. n=9 tumors for each group Quantification of tumor weights 30 days after transplantation of control or shVgll4 MB49 cells into C57BL/6 mice


Control or shVgll4 MB49 cells were transplanted into nude mice , and tumor - growth kinetics was measured at the indicated times . n = 8 tumors for each group Quantification of tumor weights 15 days after transplantation of control or shVgll4 MB49 cells into nude mice from ( I ) . n = 8 tumors for each

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

Control or shVgll4 MB49 cells were transplanted into nude mice, and tumor-growth kinetics was measured at the indicated times. n=8 tumors for each group Quantification of tumor weights 15 days after transplantation of control or shVgll4 MB49 cells into nude mice from (I). n=8 tumors for each group


Immunofluorescent staining of CD3 , CD8 and CD45 ( red ) counterstained with DAPI for DNA ( blue ) using control or shVgll4 LLC tumors from C57BL / 6 mice . Statistical analysis of the percentage of CD3 , CD8 and CD45 positive cells in the tumors is shown in the right panel respectively . n = 3 tumors for each

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

Immunofluorescent staining of CD3, CD8 and CD45 (red) counterstained with DAPI for DNA (blue) using control or shVgll4 LLC tumors from C57BL/6 mice. Statistical analysis of the percentage of CD3, CD8 and CD45 positive cells in the tumors is shown in the right panel respectively. n=3 tumors for each group


Quantification of tumor weights 30 days after transplantation of shVgll4 MB49 cells into C57BL / 6 mice receiving anti - CD4 alone , anti - CD8 alone , or both anti - CD4 and anti - CD8 blocking antibodies . n = 20 tumors for each

O O O O O O O O O O O O O O O O O O O O O O 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 tumor weights 30 days after transplantation of shVgll4 MB49 cells into C57BL/6 mice receiving anti-CD4 alone, anti-CD8 alone, or both anti-CD4 and anti-CD8 blocking antibodies. n=20 tumors for each group


Relative mRNA levels of immune related genes in Vgll4 - knockdown LLC or MB49 cells by qRT - PCR

Relative mRNA levels of immune related genes in Vgll4-knockdown LLC or MB49 cells by qRT-PCR analysis


Knockdown of Vgll4 in MB49 cells decreased the surface expression of PD - L1 protein on control or shVgll4 MB49 cells measured by flow cytometry analysis with anti - mPD - L1

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

Knockdown of Vgll4 in MB49 cells decreased the surface expression of PD-L1 protein on control or shVgll4 MB49 cells measured by flow cytometry analysis with anti-mPD-L1 antibody


Relative mRNA levels of immune related genes in Vgll4 - knockdown A549 cells by qRT - PCR

Relative mRNA levels of immune related genes in Vgll4-knockdown A549 cells by qRT-PCR analysis


Immunoblot analysis revealed the reduced expression of PD - L1 protein in VGLL4 knockdown A549 , 16HBE and H358 cells compared with control

Immunoblot analysis revealed the reduced expression of PD-L1 protein in VGLL4 knockdown A549, 16HBE and H358 cells compared with control cells


qRT - PCR analysis revealed a reduction of PD - L1 mRNA levels in VGLL4 - knockdown 16HBE and H358 cells by

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

qRT-PCR analysis revealed a reduction of PD-L1 mRNA levels in VGLL4-knockdown 16HBE and H358 cells by siRNA


Flow cytometry analysis of surface PD - L1 levels in control and VGLL4 knockdown A549 cells treated with IFNγ for the indicated

Flow cytometry analysis of surface PD-L1 levels in control and VGLL4 knockdown A549 cells treated with IFNγ for the indicated times


Immunoblot analysis revealed the reduced IFNγ - inducible PD - L1 expression in VGLL4 knockdown A549 and 16HBE

Immunoblot analysis revealed the reduced IFNγ-inducible PD-L1 expression in VGLL4 knockdown A549 and 16HBE cells


Immunoblot analysis of total cell lysates with the indicated antibodies revealed the attenuated IFNγ - inducible PD - L1 expression in VGLL4 knockout ( KO ) A549 cells generated by CRISPR /

Immunoblot analysis of total cell lysates with the indicated antibodies revealed the attenuated IFNγ-inducible PD-L1 expression in VGLL4 knockout (KO) A549 cells generated by CRISPR/Cas9


Loss of VGLL4 dampened the activity of PD - L1 promoter reporter in a luciferase assay . A549 cells were transfected with human PD - L1 - promoter luciferase plasmid and indicated siRNA , treated with IFNγ for 12 h , and subjected to a luciferase

Loss of VGLL4 dampened the activity of PD-L1 promoter reporter in a luciferase assay. A549 cells were transfected with human PD-L1-promoter luciferase plasmid and indicated siRNA, treated with IFNγ for 12 h, and subjected to a luciferase assay


Overexpression of mouse PD - L1 in Vgll4 knockdown LLC cells restored the tumor growth in C57BL / 6 mice . Tumor weights of shVgll4 or shVgll4 - lenti - mPD - L1 LLC cells were determined 21 days after tumor cell transplantation into C57BL / 6 mice ( left panel ) . Expression of mPD - L1 was shown in the right panel by immunoblot analysis with anti - mPD - L1 antibody . n = 10 tumors for each

Overexpression of mouse PD-L1 in Vgll4 knockdown LLC cells restored the tumor growth in C57BL/6 mice. Tumor weights of shVgll4 or shVgll4-lenti-mPD-L1 LLC cells were determined 21 days after tumor cell transplantation into C57BL/6 mice (left panel). Expression of mPD-L1 was shown in the right panel by immunoblot analysis with anti-mPD-L1 antibody. n=10 tumors for each group


Knockdown of VGLL4 in A549 cells enhances T cell - meditated tumor cell killing . Activated T cells and A549 cells were co - cultured in 24 - well plates for 4 days and then surviving tumor cells were visualized by crystal violet staining . Relative fold ratios of surviving cell intensities are shown in right

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

Knockdown of VGLL4 in A549 cells enhances T cell-meditated tumor cell killing. Activated T cells and A549 cells were co-cultured in 24-well plates for 4 days and then surviving tumor cells were visualized by crystal violet staining. Relative fold ratios of surviving cell intensities are shown in right panel


The endogenous complex of VGLL4 and IRF2BP2 was detected by immunoprecipitation using anti - VGLL4 antibody and visualized by anti - IRF2BP2 antibody in A549

The endogenous complex of VGLL4 and IRF2BP2 was detected by immunoprecipitation using anti-VGLL4 antibody and visualized by anti-IRF2BP2 antibody in A549 cells


The endogenous IRF2BP2 was immunoprecipitated by VGLL4 - FLAG proteins in HEK293T

The endogenous IRF2BP2 was immunoprecipitated by VGLL4-FLAG proteins in HEK293T cells


Interaction between VGLL4 and IRF2BP1 or IRF2BP2 was revealed by co - immunoprecipitation of differentially tagged proteins in HEK293T

Interaction between VGLL4 and IRF2BP1 or IRF2BP2 was revealed by co-immunoprecipitation of differentially tagged proteins in HEK293T cells


C - terminus of IRF2BP2 interacted with VGLL4 by co - immunoprecipitation assay in HEK293T

C-terminus of IRF2BP2 interacted with VGLL4 by co-immunoprecipitation assay in HEK293T cells


Interaction of IRF2BP2 with VGLL1 - 4 was revealed by co - immunoprecipitation of differentially tagged proteins in HEK293T

Interaction of IRF2BP2 with VGLL1-4 was revealed by co-immunoprecipitation of differentially tagged proteins in HEK293T cells


TDU domains in VGLL4 are required for the interaction with TEADs by co - immunoprecipitation assay in HEK293T

TDU domains in VGLL4 are required for the interaction with TEADs by co-immunoprecipitation assay in HEK293T cells


TDU - deleted or HF4A VGLL4 mutations interacted with IRF2BP by co - immunoprecipitation analysis in HEK293T

TDU-deleted or HF4A VGLL4 mutations interacted with IRF2BP by co-immunoprecipitation analysis in HEK293T cells


TDU - deleted or HF4A VGLL4 mutations interacted wit IRF2BP by co - immunoprecipitation analysis in HEK293T

TDU-deleted or HF4A VGLL4 mutations interacted wit IRF2BP by co-immunoprecipitation analysis in HEK293T cells


Expression of VGLL4 - WT or VGLL4 - HF4A enhances PD - L1 expression in A549 cells by immunoblot analysis . A549 cells were transfected with indicated plasmids and subjected to immunoblot with indicated

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

Expression of VGLL4-WT or VGLL4-HF4A enhances PD-L1 expression in A549 cells by immunoblot analysis. A549 cells were transfected with indicated plasmids and subjected to immunoblot with indicated antibodies


VGLL4 - HF4A rescues PD - L1 expression in VGLL4 - knockdown A549 cells . A549 cells were transfected with siRNA targeting to the 3 " UTR of VGLL4 mRNA and followed by transduction with lenti - VGLL4 - HF4A virus . Cell lysates were analyzed by western blot with indicated

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

VGLL4-HF4A rescues PD-L1 expression in VGLL4-knockdown A549 cells. A549 cells were transfected with siRNA targeting to the 3"UTR of VGLL4 mRNA and followed by transduction with lenti-VGLL4-HF4A virus. Cell lysates were analyzed by western blot with indicated antib


Expression of VGLL4 - HF4A rescues tumor growth of VGLL4 - knockdown LLC cells in C57BL / 6 mice . Control , Vgll4 knockdown or Vgll4 knockdown together with VGLL4 - HF4A overexpressing LLC cells were transplanted into nude mice or C57BL6 mice . Tumor volumes were measured 15 days for nude mice and 21 days for C57BL / 6 mice after tumor cell

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

Expression of VGLL4-HF4A rescues tumor growth of VGLL4-knockdown LLC cells in C57BL/6 mice. Control, Vgll4 knockdown or Vgll4 knockdown together with VGLL4-HF4A overexpressing LLC cells were transplanted into nude mice or C57BL6 mice. Tumor volumes were measured 15 days for nude mice and 21 days for C57BL/6 mice after tumor cell inoculation


Expression of VGLL4 - HF4A attenuates the T cell - meditated tumor cell killing . Activated T cells and A549 cells were co - cultured in 24 - well plates for 4 days and Relative fold ratios of surviving cells are shown by measuring the intensities of surviving cells stained with crystal

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

Expression of VGLL4-HF4A attenuates the T cell-meditated tumor cell killing. Activated T cells and A549 cells were co-cultured in 24-well plates for 4 days and Relative fold ratios of surviving cells are shown by measuring the intensities of surviving cells stained with crystal violet


Correlation between protein expression of VGLL4 and IRF2BP2 in 6 murine tumor cell line with the indicated antibodies


Correlation between protein expression of VGLL4 and IRF2BP2 i human cell line with the indicated antibodies


Immunoblot analysis of A549 cells transfected with VGLL4 - FLAG or VGLL4 - HF4A plasmids revealed an increased IRF2BP2 protein levels compared with 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

Immunoblot analysis of A549 cells transfected with VGLL4-FLAG or VGLL4-HF4A plasmids revealed an increased IRF2BP2 protein levels compared with control cells


Deficiency of VGLL4 reduces IRF2BP2 protein levels . Immunoblot analysis of A549 and LLC cell lysates transfected with siRNA for control and

Deficiency of VGLL4 reduces IRF2BP2 protein levels. Immunoblot analysis of A549 and LLC cell lysates transfected with siRNA for control and VGLL4


The indicated cell lines were transfected with control or VGLL4 siRNA and subjected to immunoblot analysis with the indicated antibodies


qRT - PCR analysis of IRF2BP2 mRNA levels in control and siVGLL4 A549

qRT-PCR analysis of IRF2BP2 mRNA levels in control and siVGLL4 A549 cells


A549 or 16HBE cells were transfected with control or VGLL4 siRNA and treated with MG132 for the indicated times and subjected to immunoblot analysis with the indicated antibodies . Relative IRF2BP2 expression levels were quantified in the right

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

A549 or 16HBE cells were transfected with control or VGLL4 siRNA and treated with MG132 for the indicated times and subjected to immunoblot analysis with the indicated antibodies. Relative IRF2BP2 expression levels were quantified in the right panel


Lys48 - linked ubiquitylation of IRF2BP2 is strongly inhibited when VGLL4 was overexpressed in HEK293T cells . HEK293T cells were transfected with indicated plasmids , treated with MG132 for 3 h before harvest , and subjected to immunoprecipitation and immunoblot analysis as

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

Lys48-linked ubiquitylation of IRF2BP2 is strongly inhibited when VGLL4 was overexpressed in HEK293T cells. HEK293T cells were transfected with indicated plasmids, treated with MG132 for 3 h before harvest, and subjected to immunoprecipitation and immunoblot analysis as indicated


Lys48 - linked ubiquitylation of IRF2BP2 is increased in the absence of VGLL4 in HEK293T cells . HEK293T cells were transfected with indicated siRNA and plasmids , and subjected to immunoprecipitation and immunoblot analysis as

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

Lys48-linked ubiquitylation of IRF2BP2 is increased in the absence of VGLL4 in HEK293T cells. HEK293T cells were transfected with indicated siRNA and plasmids, and subjected to immunoprecipitation and immunoblot analysis as indicated


IRF2 inhibits IRF1 transcriptional activity . A549 cells transfected with human PD - L1 promoter luciferase reporter and indicated plasmids were analyzed using a luciferase

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

IRF2 inhibits IRF1 transcriptional activity. A549 cells transfected with human PD-L1 promoter luciferase reporter and indicated plasmids were analyzed using a luciferase assay


Knockdown IRF2 restores PD - L1 expression in VGLL4 - knockdown A549 cells . Gene expression levels were analyzed by qRT - PCR in A549 cells transfected or transduced as

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

Knockdown IRF2 restores PD-L1 expression in VGLL4-knockdown A549 cells. Gene expression levels were analyzed by qRT-PCR in A549 cells transfected or transduced as indicated


Overexpression of IRF1 increases PD - L1 expression in VGLL4 - knockdown A549 cells . A549 cells were transfected with VGLL4 siRNA or together with HA - IRF1 plasmid and total cell lysates were analyzed by western

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

Overexpression of IRF1 increases PD-L1 expression in VGLL4-knockdown A549 cells. A549 cells were transfected with VGLL4 siRNA or together with HA-IRF1 plasmid and total cell lysates were analyzed by western blot


IRF2 is released from PD - L1 promoter upon IFNγ stimulation . IRF2 - 3 × HA knockin A549 cells were stimulated with IFNγ and subjected to ChIP - qPCR analysis in the PD - L1 promoter using control and HA antibodies . Normal mouse IgG was used as

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

IRF2 is released from PD-L1 promoter upon IFNγ stimulation. IRF2-3×HA knockin A549 cells were stimulated with IFNγ and subjected to ChIP-qPCR analysis in the PD-L1 promoter using control and HA antibodies. Normal mouse IgG was used as control


Increased association of IRF2 with PD - L1 promoter in IRF2BP2 knockout ( KO ) A549 cells revealed by ChIP - qPCR analysis . Immunoblot showed the loss of IRF2BP2 in IRF2BP2 KO A549 cells in the lower

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

Increased association of IRF2 with PD-L1 promoter in IRF2BP2 knockout (KO) A549 cells revealed by ChIP-qPCR analysis. Immunoblot showed the loss of IRF2BP2 in IRF2BP2 KO A549 cells in the lower panel


Dynamic association of IRF2 with IRF2BP2 during IFNγ stimulation by immunoprecipitation analysis using anti - HA antibody in IRF2 - 3 × HA knockin A549

Dynamic association of IRF2 with IRF2BP2 during IFNγ stimulation by immunoprecipitation analysis using anti-HA antibody in IRF2-3×HA knockin A549 cells


Loss of IRF2BP2 attenuated IFNγ - inducible PD - L1 protein expression revealed by immunoblot in A549 control and IRF2BP2 knockout cells Quantification of PD - L1 protein

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

Loss of IRF2BP2 attenuated IFNγ-inducible PD-L1 protein expression revealed by immunoblot in A549 control and IRF2BP2 knockout cells Quantification of PD-L1 protein level


Loss of IRF2BP2 attenuated IFNγ - inducible PD - L1 mRNA expression revealed by qRT - PCR analysis in A549 control and IRF2BP2 knockout

Loss of IRF2BP2 attenuated IFNγ-inducible PD-L1 mRNA expression revealed by qRT-PCR analysis in A549 control and IRF2BP2 knockout cells


miR - 130a represses VGLL4 and PD - L1 protein levels . A549 cells were transfected with control and miR - 130a mimic and subjected to immunoblot

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

miR-130a represses VGLL4 and PD-L1 protein levels. A549 cells were transfected with control and miR-130a mimic and subjected to immunoblot analysis


Inhibition of miR - 130a by its sponge enhances IFNγ - inducible PD - L1 expression . A549 cells were transduced with control or miR - 130a sponge lentivirus and subjected to

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

Inhibition of miR-130a by its sponge enhances IFNγ-inducible PD-L1 expression. A549 cells were transduced with control or miR-130a sponge lentivirus and subjected to immunoblot


Inhibition of miR - 130a by its sponge enhances IFNγ - inducible PD - L1 expression . A549 cells were transduced with control or miR - 130a sponge lentivirus and subjected t qRT - PCR

Inhibition of miR-130a by its sponge enhances IFNγ-inducible PD-L1 expression. A549 cells were transduced with control or miR-130a sponge lentivirus and subjected t qRT-PCR analysis


Decreased YAP target gene expression by inhibition of miR - 130a using its sponge in A549

Decreased YAP target gene expression by inhibition of miR-130a using its sponge in A549 cells


YAP5SA expression attenuates IFNγ - inducible PD - L1 expression . A549 cells were transduced with control , YAP - 5SA or YAP - S94A lentivirus and treated with IFNγ for the indicated times and subjected to immunoblot

YAP5SA expression attenuates IFNγ-inducible PD-L1 expression. A549 cells were transduced with control, YAP-5SA or YAP-S94A lentivirus and treated with IFNγ for the indicated times and subjected to immunoblot analysis


qRT - PCR analysis of cell lysates from control , YAP - 5SA or YAP - S94A expressing A549 cells treated with IFNγ for the indicated

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

qRT-PCR analysis of cell lysates from control, YAP-5SA or YAP-S94A expressing A549 cells treated with IFNγ for the indicated times


Control or YAP - overexpressing LLC cells were transplanted into nude mice ( upper panel ) or C57BL / 6 ( lower panel ) mice , and tumor - growth kinetics was measured at the indicated times . Tumor volumes at the end time points were analyzed and shown at the right

Control or YAP-overexpressing LLC cells were transplanted into nude mice (upper panel) or C57BL/6 (lower panel) mice, and tumor-growth kinetics was measured at the indicated times. Tumor volumes at the end time points were analyzed and shown at the right panel


miR - 130a regulates IRF1 3 ′ UTR sensor activity . A549 cells were transfected with WT or mutant IRF1 3 ′ UTR sensor plasmids together with miR - 130a mimic . Sensor activities were determined by dual luciferase

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

miR-130a regulates IRF1 3′UTR sensor activity. A549 cells were transfected with WT or mutant IRF1 3′UTR sensor plasmids together with miR-130a mimic. Sensor activities were determined by dual luciferase assay


YAP5SA induces miR - 130a expression and represses IRF1 expression . A549 cells were transduced with control or YAP - 5SA lentivirus and subjected to qRT - PCR

YAP5SA induces miR-130a expression and represses IRF1 expression. A549 cells were transduced with control or YAP-5SA lentivirus and subjected to qRT-PCR analysis


Inhibition of miR - 130a by its sponge enhances IFNγ - inducible IRF1 expression . A549 cells were transduced with control or miR - 130a sponge lentivirus and subjected to qRT - PCR

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

Inhibition of miR-130a by its sponge enhances IFNγ-inducible IRF1 expression. A549 cells were transduced with control or miR-130a sponge lentivirus and subjected to qRT-PCR analysis


Suppression of IFNγ - inducible PD - L1 expression by YAP5SA is compromised in miR - 130a KO A549 cells . PD - L1 expression levels were analyzed by qRT - PCR in IFNγ - treated control or miR - 130a KO A549 cells pre - transduced with lenti - YAP5SA

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

Suppression of IFNγ-inducible PD-L1 expression by YAP5SA is compromised in miR-130a KO A549 cells. PD-L1 expression levels were analyzed by qRT-PCR in IFNγ-treated control or miR-130a KO A549 cells pre-transduced with lenti-YAP5SA virus


Representative immunohistochemical staining for VGLL4 and IRF2BP2 in human non - small cell lung cancer tissues . IHC score is indicated . Scale bar : 50

Representative immunohistochemical staining for VGLL4 and IRF2BP2 in human non-small cell lung cancer tissues. IHC score is indicated. Scale bar: 50 μm


Staining of VGLL4 and IRF2BP2 on human non - small cell lung cancer tissue arrays containing 71 samples was quantified as described in the Materials and Methods section . Correlation of VGLL4 and IRF2BP2 expressions was significant ( Pearson correlation test ; R = 0 . 6341 , P < 0 . 001 ) Correlation analysis of VGLL4 and PD - L1 expression on human non - small cell lung cancer tissue arrays containing 71

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

Staining of VGLL4 and IRF2BP2 on human non-small cell lung cancer tissue arrays containing 71 samples was quantified as described in the Materials and Methods section. Correlation of VGLL4 and IRF2BP2 expressions was significant (Pearson correlation test; R = 0.6341, P < 0.001) Correlation analysis of VGLL4 and PD-L1 expression on human non-small cell lung cancer tissue arrays containing 71 samples


Kaplan - Meier survival analysis of patients with high ( n = 74 ) or low ( n = 130 ) VGLL4 mRNA levels from GSE31210 , a lung cancer data set . Survival curves are calculated according to the Kaplan - Meier

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

Kaplan-Meier survival analysis of patients with high (n=74) or low (n=130) VGLL4 mRNA levels from GSE31210, a lung cancer data set. Survival curves are calculated according to the Kaplan-Meier method


( A , B ) Effect of MPC inhibition on cellular lipid droplet content . ( A ) Live - cell super - resolution confocal imaging of primary brown adipocytes pre - stained overnight with the fatty acid tracer BODIPY C12 558 / 568 ( Bodipy C12 , red ) . Cells were stained with mitotracker green ( MTG , green ) prior to imaging . Cells were imaged before and 120 minutes after treatment with either vehicle ( DMSO ) or 100 nM UK5099 . As a positive control , cells were imaged 30 min after treatment with 1 µM norepinephrine . LD , lipid droplet ; N , nucleus . Scale bar = 10 µm . ( B ) Quantification of changes in lipid droplet cross - sectional area with indicated treatments from images shown in ( A ) . Data are represented as percentage change in LD area at time = 120 min compared to time = 0 min .

O O O O O O O O O O O O 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-MEASURED_VAR I-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 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 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

(A,B) Effect of MPC inhibition on cellular lipid droplet content. (A) Live-cell super-resolution confocal imaging of primary brown adipocytes pre-stained overnight with the fatty acid tracer BODIPY C12 558/568 (Bodipy C12, red). Cells were stained with mitotracker green (MTG, green) prior to imaging. Cells were imaged before and 120 minutes after treatment with either vehicle (DMSO) or 100 nM UK5099. As a positive control, cells were imaged 30 min after treatment with 1 µM norepinephrine. LD, lipid droplet; N, nucleus. Scale bar=10 µm. (B) Quantification of changes in lipid droplet cross-sectional area with indicated treatments from images shown in (A). Data are represented as percentage change in LD area at time=120 min compared to time=0 min.


( C , D ) Effect of MPC inhibition on fatty acid uptake and incorporation into triacylglycerides ( TAGs ) ( C ) Representative thin - layer chromatography ( TLC ) plate of lipids extracted from primary brown adipocytes and cell culture media . Cells were incubated with Bodipy C12 in presence of vehicle ( V ) , 100 nM UK5099 ( UK ) or 1 µM norepinephrine ( NE ) for 24 h and triacylglyceride ( TAG ) synthesis ( Lipids in cells ) and fatty acid uptake ( Lipids in media ) were detected . The relative polarity of the lipid species determines the motility , with nonpolar TAG migrating the furthest . ( D ) Quantification of C12 uptake from media , TAG in cells and TAG per amount of free fatty acid uptake shown in ( C ) , n = 5 individual experiments . UK5099 treatment decreases the amount of synthesized TAG per free fatty acid uptake , similarly to NE .

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

(C,D) Effect of MPC inhibition on fatty acid uptake and incorporation into triacylglycerides (TAGs) (C) Representative thin-layer chromatography (TLC) plate of lipids extracted from primary brown adipocytes and cell culture media. Cells were incubated with Bodipy C12 in presence of vehicle (V), 100 nM UK5099 (UK) or 1 µM norepinephrine (NE) for 24 h and triacylglyceride (TAG) synthesis (Lipids in cells) and fatty acid uptake (Lipids in media) were detected. The relative polarity of the lipid species determines the motility, with nonpolar TAG migrating the furthest. (D) Quantification of C12 uptake from media, TAG in cells and TAG per amount of free fatty acid uptake shown in (C), n=5 individual experiments. UK5099 treatment decreases the amount of synthesized TAG per free fatty acid uptake, similarly to NE.


( E - G ) Effect of UK5099 treatment on cellular energy expenditure . Fully differentiated primary brown adipocytes were pre - treated with vehicle ( DMSO ) or UK5099 at indicated concentrations for 2 h . Oxygen consumption rates ( OCR ) were measured in respirometry media supplemented with 5 mM glucose and 3 mM glutamine in the presence of vehicle or UK - 5099 . ( E ) Quantification of non - stimulated OCR from n = 3 - 7 individual experiments . Data were normalized to vehicle for each individual experiment . UK5099 increases OCR in non - stimulated brown adipocytes . vehicle . ( F ) Brown adipocytes were treated with 100 nM UK5099 or vehicle . Etomoxir ( Eto ; 40 µM ) and antimycin A ( AA ; 4 µM ) were injected where indicated . Representative OCR traces averaging 6 technical replicates . ( G ) Quantification of non - stimulated OCR , etomoxir sensitive , and etomoxir insensitive OCR from n = 4 individual experiments . Data were normalized to vehicle for each individual experiment . *

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

(E-G) Effect of UK5099 treatment on cellular energy expenditure. Fully differentiated primary brown adipocytes were pre-treated with vehicle (DMSO) or UK5099 at indicated concentrations for 2 h. Oxygen consumption rates (OCR) were measured in respirometry media supplemented with 5 mM glucose and 3 mM glutamine in the presence of vehicle or UK-5099. (E) Quantification of non-stimulated OCR from n=3-7 individual experiments. Data were normalized to vehicle for each individual experiment. UK5099 increases OCR in non-stimulated brown adipocytes. vehicle. (F) Brown adipocytes were treated with 100 nM UK5099 or vehicle. Etomoxir (Eto; 40 µM) and antimycin A (AA; 4 µM) were injected where indicated. Representative OCR traces averaging 6 technical replicates. (G) Quantification of non-stimulated OCR, etomoxir sensitive, and etomoxir insensitive OCR from n=4 individual experiments. Data were normalized to vehicle for each individual experiment. *


( H , I ) Effect of MPC inhibition on adipocyte fuel preference towards fatty acid . ( H ) Schematic representation of metabolite tracing using [ U - 13C16 ] palmitate . ( I ) [ U - 13C16 ] palmitate tracing in fully differentiated primary brown adipocytes treated with 5 µM UK5099 or vehicle for 24 h . Data shows mole percent enrichment ( MPE ) of isotope labeled substrate into respective metabolite .

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

(H,I) Effect of MPC inhibition on adipocyte fuel preference towards fatty acid. (H) Schematic representation of metabolite tracing using [U-13C16] palmitate. (I) [U-13C16] palmitate tracing in fully differentiated primary brown adipocytes treated with 5 µM UK5099 or vehicle for 24 h. Data shows mole percent enrichment (MPE) of isotope labeled substrate into respective metabolite.


( A , B ) Effect of UK5099 treatment on norepinephrine - stimulated energy expenditure . Fully differentiated primary brown adipocytes were pre - treated with vehicle ( DMSO ) or 100 nM UK5099 for 2 h . OCR were measured in respirometry media supplemented with 5 mM glucose and 3 mM glutamine in the presence of vehicle or UK5099 . Norepinephrine ( NE ; 1 µM ) , oligomycin A ( Oligo ; 4 µM ) , etomoxir ( Eto ; 40 µM ) and antimycin A ( AA ; 4 µM ) were injected where indicated . ( A ) Representative OCR traces averaging 4 technical replicates . ( B ) Quantification of non - stimulated OCR , NE - stimulated OCR , ATP - linked OCR , mitochondrial proton leak and Eto - sensitive OCR as shown in ( A ) from n = 9 individual experiments . The effects of UK5099 treatment were normalized to vehicle for each experiment .

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

(A,B) Effect of UK5099 treatment on norepinephrine-stimulated energy expenditure. Fully differentiated primary brown adipocytes were pre-treated with vehicle (DMSO) or 100 nM UK5099 for 2 h. OCR were measured in respirometry media supplemented with 5 mM glucose and 3 mM glutamine in the presence of vehicle or UK5099. Norepinephrine (NE; 1 µM), oligomycin A (Oligo; 4 µM), etomoxir (Eto; 40 µM) and antimycin A (AA; 4 µM) were injected where indicated. (A) Representative OCR traces averaging 4 technical replicates. (B) Quantification of non-stimulated OCR, NE-stimulated OCR, ATP-linked OCR, mitochondrial proton leak and Eto-sensitive OCR as shown in (A) from n=9 individual experiments. The effects of UK5099 treatment were normalized to vehicle for each experiment.