( H ) ECAR assay of naive human neurons , VPA - pretreated human neurons with or without XAV939 treatment . Notice the higher levels of glycolysis in VPA - pretreated human neurons and the inhibitory effects of XAV939 . , 4 - 5 batches of cells in per group .

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(H) ECAR assay of naive human neurons, VPA-pretreated human neurons with or without XAV939 treatment. Notice the higher levels of glycolysis in VPA-pretreated human neurons and the inhibitory effects of XAV939. , 4-5 batches of cells in per group.


The syntheses of the ligands PR - 924 , 9 , 14 , 16 , 17 and 18 have been described previously ( de Bruin et al , 2014 ) . All ligands share the peptide scaffold shown in the upper left corner with P1 , P2 and P3 side chains and an N - cap ; they are equipped with a l - Ala ( stereochemistry ( S ) ) or d - Ala ( stereochemistry ( R ) ) residue at P3 ( red ) and either an N - acylmorpholine or a 3 - methyl - 1H - indene group ( green ) . Furthermore , compounds differ in their P2 residue ( either methoxytyrosine or tryptophane ; blue ) . Inhibitor 9 , also termed LU - 015i , is endowed with an exceptional cyclohexyl residue ( gray ) , which is associated with increased selectivity for human β5i .

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The syntheses of the ligands PR-924, 9, 14, 16, 17 and 18 have been described previously (de Bruin et al, 2014). All ligands share the peptide scaffold shown in the upper left corner with P1, P2 and P3 side chains and an N-cap; they are equipped with a l-Ala (stereochemistry (S)) or d-Ala (stereochemistry (R)) residue at P3 (red) and either an N-acylmorpholine or a 3-methyl-1H-indene group (green). Furthermore, compounds differ in their P2 residue (either methoxytyrosine or tryptophane; blue). Inhibitor 9, also termed LU-015i, is endowed with an exceptional cyclohexyl residue (gray), which is associated with increased selectivity for human β5i.


A . Inhibition of human β5c and β5i by ONX 0914 and PR - 924 . IC50 values are deduced from the fitted data .

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A. Inhibition of human β5c and β5i by ONX 0914 and PR-924. IC50 values are deduced from the fitted data.


B . IC50 values [ µM ] for all compounds towards the yeast , human and mouse β5 subunits .

B. IC50 values [µM] for all compounds towards the yeast, human and mouse β5 subunits.


E . Serial dilutions of yeast cells were spotted on YPD plates and grown for 2 days either at 30 ° C or 37 ° C . Mutant yeasts are significantly retarded in growth and show increased temperature sensitivity .

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E. Serial dilutions of yeast cells were spotted on YPD plates and grown for 2 days either at 30 °C or 37 °C. Mutant yeasts are significantly retarded in growth and show increased temperature sensitivity.


F . Surface illustration of the yCP . The β5 and β6 subunits are depicted in dark gray , with the residue ranges that correspond to mammalian β5 and β6 subunits in the chimeric proteasomes highlighted in gold and blue , respectively . The β5 ' and β6 ' subunits of the second β - ring are hidden on the back of 20S proteasome .

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F. Surface illustration of the yCP. The β5 and β6 subunits are depicted in dark gray, with the residue ranges that correspond to mammalian β5 and β6 subunits in the chimeric proteasomes highlighted in gold and blue, respectively. The β5' and β6' subunits of the second β-ring are hidden on the back of 20S proteasome.


A . Superposition of the β5i / β6 active sites of genuine mouse iCP and the hβ5i / hβ6 human / yeast chimera illustrates that Met45 adopts the same ' open ' conformation in both structures . Amino acid side chains important for substrate binding are depicted as sticks . The catalytic Thr1 is colored in black . Met31 , the single amino acid difference between mouse and human β5i active sites , is highlighted in green .

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A. Superposition of the β5i/β6 active sites of genuine mouse iCP and the hβ5i/hβ6 human/yeast chimera illustrates that Met45 adopts the same 'open' conformation in both structures. Amino acid side chains important for substrate binding are depicted as sticks. The catalytic Thr1 is colored in black. Met31, the single amino acid difference between mouse and human β5i active sites, is highlighted in green.


B . Superposition of the genuine mouse and the human chimeric β5c / β6 active sites according to panel A proves the proper orientation of Met45 in the ' closed ' conformation .

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B. Superposition of the genuine mouse and the human chimeric β5c/β6 active sites according to panel A proves the proper orientation of Met45 in the 'closed' conformation.


C . Superposition of the β5c / β6 and β5i / β6 active sites of the natural mouse cCP and iCP according to panel A . Note the distinct conformations of Met45 ( Huber et al , 2012 ) .

C. Superposition of the β5c/β6 and β5i/β6 active sites of the natural mouse cCP and iCP according to panel A. Note the distinct conformations of Met45 (Huber et al, 2012).


D . Superposition of the hβ5c / hβ6 and the hβ5i / hβ6 chimeric proteasomes confirm the distinct positions of Met45 analogously to the natural mouse subunits shown in panel C .

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D. Superposition of the hβ5c/hβ6 and the hβ5i/hβ6 chimeric proteasomes confirm the distinct positions of Met45 analogously to the natural mouse subunits shown in panel C.


A . Complex structures of the human β5c / β6 ( left ) and β5i / β6 ( right ) chimeric proteasomes with PR - 924 . The ligand binds in a linear ( l ) manner to the β5c / β6 substrate binding channel , but in a kinked ( k ) conformation to the β5i / β6 active site . The 2FO - FC electron density maps for the ligands bound to Thr1 of the chimeric β5 active sites are shown as blue meshes contoured at 1σ . The inhibitor and Thr1 have been omitted for phasing .

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A. Complex structures of the human β5c/β6 (left) and β5i/β6 (right) chimeric proteasomes with PR-924. The ligand binds in a linear (l) manner to the β5c/β6 substrate binding channel, but in a kinked (k) conformation to the β5i/β6 active site. The 2FO-FC electron density maps for the ligands bound to Thr1 of the chimeric β5 active sites are shown as blue meshes contoured at 1σ. The inhibitor and Thr1 have been omitted for phasing.


B . Comparison of the conformations of ONX 0914 , PR - 924 , 14 , 16 , 17 and 18 bound to the chimeric hβ5 / hβ6 substrate binding channels . The l amino acid compounds ONX 0914 and 14 display a linear ( l ) binding mode to the hβ5c / hβ6 ( left ) and the hβ5i / hβ6 ( right ) chimeras , whereas the ligands 16 and 17 ( P3 - d - Ala and morpholine cap ) adopt a kinked ( k ) conformation in both β5 substrate binding channels . Remarkably , the orientation of PR - 924 and its analogue 18 ( P3 - d - Ala and 3 - methyl - 1H - indene cap ) in the hβ5c / hβ6 chimera ( and in WT yβ5 ; see Appendix Figure S5 ) ) differs from that observed with the hβ5i / hβ6 chimera . Note that the inhibitors are rotated by about 45 ° compared to panel A .

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B. Comparison of the conformations of ONX 0914, PR-924, 14, 16, 17 and 18 bound to the chimeric hβ5/hβ6 substrate binding channels. The l amino acid compounds ONX 0914 and 14 display a linear (l) binding mode to the hβ5c/hβ6 (left) and the hβ5i/hβ6 (right) chimeras, whereas the ligands 16 and 17 (P3-d-Ala and morpholine cap) adopt a kinked (k) conformation in both β5 substrate binding channels. Remarkably, the orientation of PR-924 and its analogue 18 (P3-d-Ala and 3-methyl-1H-indene cap) in the hβ5c/hβ6 chimera (and in WT yβ5; see Appendix Figure S5)) differs from that observed with the hβ5i/hβ6 chimera. Note that the inhibitors are rotated by about 45 ° compared to panel A.


A . Complex structures of the mouse chimeric proteasome ( left ) and the hβ5i - V31M / hβ6 mutant ( right ) with PR - 924 visualize the kinked ( k ) orientation . The 2FO - FC electron density maps for the ligands bound to Thr1 of the chimeric β5 active sites are depicted according to Fig 5A . Steric hindrance of Met31 ( green ) with the bulky 3 - methyl - 1H - indene cap is indicated by black double arrows .

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A. Complex structures of the mouse chimeric proteasome (left) and the hβ5i-V31M/hβ6 mutant (right) with PR-924 visualize the kinked (k) orientation. The 2FO-FC electron density maps for the ligands bound to Thr1 of the chimeric β5 active sites are depicted according to Fig 5A. Steric hindrance of Met31 (green) with the bulky 3-methyl-1H-indene cap is indicated by black double arrows.


B . Superposition of inhibitors bound to the mouse chimeric proteasome ( left ) and the hβ5i - V31M / hβ6 mutant ( right ) . Compounds display the same binding modes ( kinked ( k ) or linear ( l ) ) to the mβ5i / mβ6 active site as to the corresponding human β5i / β6 subunit ( Fig . 5 ) and to the hβ5i - V31M / hβ6 mutant . Note that the inhibitors are rotated by about 45 ° compared to panel A .

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B. Superposition of inhibitors bound to the mouse chimeric proteasome (left) and the hβ5i-V31M/hβ6 mutant (right). Compounds display the same binding modes (kinked (k) or linear (l)) to the mβ5i/mβ6 active site as to the corresponding human β5i/β6 subunit (Fig. 5) and to the hβ5i-V31M/hβ6 mutant. Note that the inhibitors are rotated by about 45° compared to panel A.


C . Superposition of chimeric mammalian and WT yeast β5 active sites bound to the P3 - d - Ala compounds PR - 924 and 18 . The nucleophilic Thr1 is depicted as well as the S1 pocket forming residues 31 and 45 . Residue 31 impacts on the position of the 3 - methyl - 1H - indene cap of PR - 924 and 18 . Due to steric hindrance , the N - cap is shifted in the mouse and in the hβ5i - V31M / hβ6 chimera compared to the hβ5i / hβ6 mutant proteasome crystal structures ( black double arrow ) . Note that the structures are rotated by 90 ° compared to panel B .

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C. Superposition of chimeric mammalian and WT yeast β5 active sites bound to the P3-d-Ala compounds PR-924 and 18. The nucleophilic Thr1 is depicted as well as the S1 pocket forming residues 31 and 45. Residue 31 impacts on the position of the 3-methyl-1H-indene cap of PR-924 and 18. Due to steric hindrance, the N-cap is shifted in the mouse and in the hβ5i-V31M/hβ6 chimera compared to the hβ5i/hβ6 mutant proteasome crystal structures (black double arrow). Note that the structures are rotated by 90° compared to panel B.


ONX 0914 binds in a linear mode to all active sites . Changing the stereochemistry at the P3 site from l - Ala ( S ) to d - Ala ( R ) leads to a kinked conformation in the β5 substrate binding channels ( compound 16 ) . Substitution of the morpholine cap of 16 by a larger 3 - metyhl - 1H - indene moiety yields PR - 924 and results in different inhibitor conformations at the yβ5 / hβ5c and the mammalian β5i active sites . Hereby , the selectivity for hβ5i over hβ5c is drastically increased ( IC50 ratio hβ5c / hβ5i : 255 ) compared to ONX 0914 ( IC50 ratio hβ5c / hβ5i : 20 ) and compound 16 ( IC50 ratio hβ5c / hβ5i : 5 . 2 , Fig 2 ) . Residue Met31 of mβ5i sterically hinders the kinked binding mode of PR - 924 , thereby creating 7 - fold selectivity for human β5i .

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ONX 0914 binds in a linear mode to all active sites. Changing the stereochemistry at the P3 site from l-Ala (S) to d-Ala (R) leads to a kinked conformation in the β5 substrate binding channels (compound 16). Substitution of the morpholine cap of 16 by a larger 3-metyhl-1H-indene moiety yields PR-924 and results in different inhibitor conformations at the yβ5/hβ5c and the mammalian β5i active sites. Hereby, the selectivity for hβ5i over hβ5c is drastically increased (IC50 ratio hβ5c/hβ5i: 255) compared to ONX 0914 (IC50 ratio hβ5c/hβ5i: 20) and compound 16 (IC50 ratio hβ5c/hβ5i: 5.2, Fig 2). Residue Met31 of mβ5i sterically hinders the kinked binding mode of PR-924, thereby creating 7-fold selectivity for human β5i.


( a - c ) Low - magnification cryo - EM projection images of pig ( a ) , horse ( b ) , and mouse ( c ) sperm . Different regions of the flagellum are annotated as follows : green - neck , yellow - midpiece , coral - principal piece , pink - endpiece . ( d - f ) Tomographic slices through cryo - FIB milled lamellae of pig ( d ) , horse ( e ) , and mouse ( f ) sperm . Transverse slices ( d ' - f ' ) show complete triplets in the pig ( d ' ) and the horse ( e ' ) , but not in the mouse ( f ' ) . Complete triplets are indicated by green arrowheads with black outlines , while degenerated triplets are indicated by white arrowheads with green outlines . Note the electron - dense material in the lumen of the pig sperm proximal centriole that is continuous with the connecting piece ( asterisks in d and d ' ) .

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(a-c) Low-magnification cryo-EM projection images of pig (a), horse (b), and mouse (c) sperm. Different regions of the flagellum are annotated as follows: green - neck, yellow - midpiece, coral - principal piece, pink - endpiece. (d-f) Tomographic slices through cryo-FIB milled lamellae of pig (d), horse (e), and mouse (f) sperm. Transverse slices (d'-f') show complete triplets in the pig (d') and the horse (e'), but not in the mouse (f'). Complete triplets are indicated by green arrowheads with black outlines, while degenerated triplets are indicated by white arrowheads with green outlines. Note the electron-dense material in the lumen of the pig sperm proximal centriole that is continuous with the connecting piece (asterisks in d and d').


( g - i ) Tomographic slices through cryo - FIB milled lamellae of the DC - to - axoneme transition in pig sperm show how the change in geometry ( g , white arrows and white dashed lines ) coincides with the appearance of axoneme accessory structures ( h , white arrows ) and with density in the A - tubule ( i , compare insets in white and black boxes ) . In ( i ) , the white and black arrows indicate where the cross - sections in white and black boxes were taken from .

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(g-i) Tomographic slices through cryo-FIB milled lamellae of the DC-to-axoneme transition in pig sperm show how the change in geometry (g, white arrows and white dashed lines) coincides with the appearance of axoneme accessory structures (h, white arrows) and with density in the A-tubule (i, compare insets in white and black boxes). In (i), the white and black arrows indicate where the cross-sections in white and black boxes were taken from.


( a - c ) Slices through defocus cryo - tomograms of the endpiece in pig ( a ) , horse ( b ) , and mouse ( c ) sperm . Insets show digital zooms ( black boxes ) and subtomogram averages ( white boxes ) of microtubule tips . Note the presence of a plug ( arrowhead ) extending into the lumen .

(a-c) Slices through defocus cryo-tomograms of the endpiece in pig (a), horse (b), and mouse (c) sperm. Insets show digital zooms (black boxes) and subtomogram averages (white boxes) of microtubule tips. Note the presence of a plug (arrowhead) extending into the lumen.


( a ) Identification of Beclin1 as a substrate of ROCK1 via affinity purification . Left panel : HeLa cells growing in control ( C : nutrient - rich media ) or starved in HBSS ( H ) for 4 h were lysed and subjected to IP with ROCK1 cross - linked beads . Proteins bound were eluted and detected by SDS - PAGE , followed by silver staining that presents precipitated proteins identified by LC - MS / MS . Right panel : HeLa cells grown in control media or HBSS ( 4 h ) were collected , whole cell lysates prepared and immunoprecipitated with ROCK1 cross - linked beads . The resulting immune complexes were divided into two , and were subjected to SDS - PAGE and analysed for Beclin1 ( MES buffer ) and ROCK1 ( MOPS buffer ) . Inputs and IP were analysed by western blotting . HBSS - treated cell extracts were used for IgG IP controls . Whole cell lysates ( 10 % ) were run for inputs . Total cell lysates were rerun and analysed for LC3 and β - actin .

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(a) Identification of Beclin1 as a substrate of ROCK1 via affinity purification. Left panel: HeLa cells growing in control (C: nutrient-rich media) or starved in HBSS (H) for 4 h were lysed and subjected to IP with ROCK1 cross-linked beads. Proteins bound were eluted and detected by SDS-PAGE, followed by silver staining that presents precipitated proteins identified by LC-MS/MS. Right panel: HeLa cells grown in control media or HBSS (4 h) were collected, whole cell lysates prepared and immunoprecipitated with ROCK1 cross-linked beads. The resulting immune complexes were divided into two, and were subjected to SDS-PAGE and analysed for Beclin1 (MES buffer) and ROCK1 (MOPS buffer). Inputs and IP were analysed by western blotting. HBSS-treated cell extracts were used for IgG IP controls. Whole cell lysates (10%) were run for inputs. Total cell lysates were rerun and analysed for LC3 and β-actin.


( b ) Co - IP was performed with 293T whole cell extracts by using ROCK1 - crosslinked agarose , and the resulting IP was blotted with indicated antibodies . 293T cells transfected with Flag - Beclin1 for 36 h , cultured in control media or starved ( 4 h ) , were immunoprecipitated with anti - Flag beads . Inputs and immune complexes were resolved and detected . Total cell lysates were rerun and examined for LC3 and β - actin expression .

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(b) Co-IP was performed with 293T whole cell extracts by using ROCK1-crosslinked agarose, and the resulting IP was blotted with indicated antibodies. 293T cells transfected with Flag-Beclin1 for 36 h, cultured in control media or starved (4 h), were immunoprecipitated with anti-Flag beads. Inputs and immune complexes were resolved and detected. Total cell lysates were rerun and examined for LC3 and β-actin expression.


( c ) Interaction of YFP - ROCK1 and CFP - Beclin1 was examined by using live cell spinning disk microscopy and FRET in live cells . Graph represents FRETAverage ratio changes ( YFP - ROCK1 / CFP - Beclin1 ) upon starvation . Data represents mean ± s . e . m . ; n = 6 . Three different experiments were performed . For the control , the right top graph represents CFP intensity over time ( corrected for photobleach ) , and the right bottom right represents YFP intensity ( corrected for photobleach ) . In both control FRET experiments , no significant change was observed in fluorescence , in the presence or absence of HBSS medium . P values were calculated using Student ' s t - test .

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(c) Interaction of YFP-ROCK1 and CFP-Beclin1 was examined by using live cell spinning disk microscopy and FRET in live cells. Graph represents FRETAverage ratio changes (YFP-ROCK1/CFP-Beclin1) upon starvation. Data represents mean±s.e.m.; n=6. Three different experiments were performed. For the control, the right top graph represents CFP intensity over time (corrected for photobleach), and the right bottom right represents YFP intensity (corrected for photobleach). In both control FRET experiments, no significant change was observed in fluorescence, in the presence or absence of HBSS medium. P values were calculated using Student's t-test.


( a ) Inhibition of ROCK1 activity ablates the interaction between Beclin1 and ROCK1 . HeLa cells were untreated ( H2O ) or treated with 10 μM ROCK1 inhibitor , Y27632 , for 8 h and then cultured in control media ( + ) or HBSS ( − ) for 4 h . Cells were collected , lysed and immunoprecipitated with Beclin1 ( left panel ) or ROCK1 ( right panel ) antibody . Left panel : immune complexes were divided into two ( ROCK1 - MOPS buffer and Beclin1 - MES buffer ) and probed for ROCK1 and Beclin1 . Relative values of ROCK1 versus Beclin1 are shown .

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(a) Inhibition of ROCK1 activity ablates the interaction between Beclin1 and ROCK1. HeLa cells were untreated (H2O) or treated with 10 μM ROCK1 inhibitor, Y27632, for 8 h and then cultured in control media (+) or HBSS (−) for 4 h. Cells were collected, lysed and immunoprecipitated with Beclin1 (left panel) or ROCK1 (right panel) antibody. Left panel: immune complexes were divided into two (ROCK1-MOPS buffer and Beclin1-MES buffer) and probed for ROCK1 and Beclin1. Relative values of ROCK1 versus Beclin1 are shown.


( b ) An increase in ROCK1 - mediated phosphorylation of MYPT1 upon starvation . HeLa and EJ cells were starved in HBSS for indicated time points and endogenous ROCK1 was immunoprecipitated . An in vitro kinase assay was performed using MYPT1 as substrate and immune - complexed ROCK1 . Proteins were resolved and visualized with Coomassie staining ( top ) , western blot analysis ( middle ) and autoradiography ( bottom ) . Inputs were examined for ROCK1 and β - actin .

(b) An increase in ROCK1-mediated phosphorylation of MYPT1 upon starvation. HeLa and EJ cells were starved in HBSS for indicated time points and endogenous ROCK1 was immunoprecipitated. An in vitro kinase assay was performed using MYPT1 as substrate and immune-complexed ROCK1. Proteins were resolved and visualized with Coomassie staining (top), western blot analysis (middle) and autoradiography (bottom). Inputs were examined for ROCK1 and β-actin.


( c ) HeLa cells incubated in HBSS were collected and endogenous phosphorylation of MYPT1 was determined by western blotting . The same blot was then washed in TBST for 30 min , blocked in 5 % milk and was analysed for total MYPT1 as a control . Extracts from this experiment were rerun on 7 . 5 % gel and analysed for ROCK1 and β - actin .

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(c) HeLa cells incubated in HBSS were collected and endogenous phosphorylation of MYPT1 was determined by western blotting. The same blot was then washed in TBST for 30 min, blocked in 5% milk and was analysed for total MYPT1 as a control. Extracts from this experiment were rerun on 7.5% gel and analysed for ROCK1 and β-actin.


( d ) ROCK activity is increased upon starvation . Cell lines ( HeLa and EJ ) grown in control ( + Glu ) or starved with HBSS for 1 and 3 h were lysed , extracts normalized and ROCK kinase activity was measured by performing enzyme - linked immunosorbent assay . Graphs represent ROCK activity in starved cells plotted as percent of ROCK1 activity in control ( glucose - rich ) cells . Data were obtained from triplicates performed at the same time ( mean ± s . d . ) . P - value was calculated using Student ' s t - test .

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(d) ROCK activity is increased upon starvation. Cell lines (HeLa and EJ) grown in control (+Glu) or starved with HBSS for 1 and 3 h were lysed, extracts normalized and ROCK kinase activity was measured by performing enzyme-linked immunosorbent assay. Graphs represent ROCK activity in starved cells plotted as percent of ROCK1 activity in control (glucose-rich) cells. Data were obtained from triplicates performed at the same time (mean±s.d.). P-value was calculated using Student's t-test.


( e ) ROCK1 activity is not dependent on Beclin1 . siControl ( siCont . ) and siBeclin1 knockdown cells were incubated in starvation media ( HBSS ) for indicated time . Resulting cell extracts were used to confirm Beclin1 knockdown and ROCK1 expression ( left panel ) . Cell extracts from the same experiment were used for enzyme - linked immunosorbent assay measuring ROCK activity ( right panel ) . Graph represents mean ± s . d . of duplicate samples read at the same time ( Student ' s t - test ; NS , not significant ) .

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(e) ROCK1 activity is not dependent on Beclin1. siControl (siCont.) and siBeclin1 knockdown cells were incubated in starvation media (HBSS) for indicated time. Resulting cell extracts were used to confirm Beclin1 knockdown and ROCK1 expression (left panel). Cell extracts from the same experiment were used for enzyme-linked immunosorbent assay measuring ROCK activity (right panel). Graph represents mean±s.d. of duplicate samples read at the same time (Student's t-test; NS, not significant).


( a ) The effect of ROCK1 depletion on autophagy marker , LC3II . MEF ROCK1flox / flox cells were generated and treated with Con or Ad - Cre for 72 h . Cells were then incubated in nutrient - rich ( + Glucose ) or HBSS media for 2 h . Cell extracts were resolved by SDS - PAGE and ROCK1 KO was confirmed by western blotting ( left panel ) . The same blot was used for LC3 and β - actin expression . Cells from the same experiment were fixed with chilled acetone and immunofluroscence was performed against endogenous LC3 ( red ) . Blue represents nuclear DAPI staining . Scale bar , 20 μm .

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

(a) The effect of ROCK1 depletion on autophagy marker, LC3II. MEF ROCK1flox/flox cells were generated and treated with Con or Ad-Cre for 72 h. Cells were then incubated in nutrient-rich (+Glucose) or HBSS media for 2 h. Cell extracts were resolved by SDS-PAGE and ROCK1 KO was confirmed by western blotting (left panel). The same blot was used for LC3 and β-actin expression. Cells from the same experiment were fixed with chilled acetone and immunofluroscence was performed against endogenous LC3 (red). Blue represents nuclear DAPI staining. Scale bar, 20 μm.


( b ) EJ cells ( left ) with a stable knockdown using shCont . or shROCK1 # 1 were incubated in control ( + glucose ) or nutrient - free ( HBSS ) medium ( − glucose ) for 24 h and lysed . Lysates were subjected to western blotting with antibodies indicated . HeLa cells ( right ) with a transient knockdown using siCont . and siROCK1 # 2 for 30 h were further incubated in control or HBSS media for 8 h . Whole cell extracts were prepared , resolved by SDS - PAGE and analysed for ROCK1 , LC3 and β - actin . Relative ratios of LC3II / LC3I are shown .

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(b) EJ cells (left) with a stable knockdown using shCont. or shROCK1#1 were incubated in control (+glucose) or nutrient-free (HBSS) medium (−glucose) for 24 h and lysed. Lysates were subjected to western blotting with antibodies indicated. HeLa cells (right) with a transient knockdown using siCont. and siROCK1#2 for 30 h were further incubated in control or HBSS media for 8 h. Whole cell extracts were prepared, resolved by SDS-PAGE and analysed for ROCK1, LC3 and β-actin. Relative ratios of LC3II/LC3I are shown.


( c ) HeLa cells were treated with H2O or 10 μM ROCK1 inhibitor , Y27632 , for 8 h . Cells were then cultured in high glucose ( 0 h ) or starved for in HBSS nutrient - free media for 6 h . One hour before collection , cells were incubated in dimethylsulphoxide ( control ) or 0 . 1 μM bafilomycin - A1 . Whole cell lysates were resolved by SDS - PAGE and were analysed by western blotting with indicated antibodies .

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(c) HeLa cells were treated with H2O or 10 μM ROCK1 inhibitor, Y27632, for 8 h. Cells were then cultured in high glucose (0 h) or starved for in HBSS nutrient-free media for 6 h. One hour before collection, cells were incubated in dimethylsulphoxide (control) or 0.1 μM bafilomycin-A1. Whole cell lysates were resolved by SDS-PAGE and were analysed by western blotting with indicated antibodies.


( d ) HeLa cells incubated with H2O or 10 μM Y27632 for 8 h were washed with PBS and cultured in control ( + Glu ) or HBSS media ( − Glu ) for an additional 8 h . Cells were fixed in acetone and immunofluorescence was performed against endogenous LC3 antibody ( green ) . Scale bar , 20 μm . Graph represents % of LC3 punctae / cell ( mean ± s . d . ) , n = 25 cells ; ( Student ' s t - test ) * * P = 0 . 00041 .

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

(d) HeLa cells incubated with H2O or 10 μM Y27632 for 8 h were washed with PBS and cultured in control (+Glu) or HBSS media (−Glu) for an additional 8 h. Cells were fixed in acetone and immunofluorescence was performed against endogenous LC3 antibody (green). Scale bar, 20 μm. Graph represents % of LC3 punctae/cell (mean±s.d.), n=25 cells; (Student's t-test) **P=0.00041.


( e ) Transmission electron micrographs of EJ cells with stable shCont . or shROCK1 # 1 knockdown , cultured in control or HBSS medium for 16 h . Arrow bars indicate autophagic vacuoles . Scale bar , 500 nm .

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(e) Transmission electron micrographs of EJ cells with stable shCont. or shROCK1#1 knockdown, cultured in control or HBSS medium for 16 h. Arrow bars indicate autophagic vacuoles. Scale bar, 500 nm.


( a ) Inhibition of ROCK1 kinase activity prevents metabolic stress - mediated phosphorylation of Beclin1 . HeLa cells left untreated or treated with a ROCK1 inhibitor , Y27632 ( 10 μM ) , were starved of glucose ( 4 h ) as indicated , and endogenous Beclin1 was immunoprecipitated and blotted against phospho - Ser / Thr antibody . Whole cell lysates were run as inputs for Beclin1 and β - actin .

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

(a) Inhibition of ROCK1 kinase activity prevents metabolic stress-mediated phosphorylation of Beclin1. HeLa cells left untreated or treated with a ROCK1 inhibitor, Y27632 (10 μM), were starved of glucose (4 h) as indicated, and endogenous Beclin1 was immunoprecipitated and blotted against phospho-Ser/Thr antibody. Whole cell lysates were run as inputs for Beclin1 and β-actin.


( b ) Phosphorylation of Beclin1 by ROCK1 . Recombinant ROCK1 was used for in vitro kinase assay in the presence or absence of Y27632 , using recombinant His - Beclin1 ( left ) or Flag - Beclin1 immunopurified from transfected HeLa cells ( right ) . Proteins were resolved by SDS - PAGE ; phosphorylated protein was visualized with autoradiography , and Beclin1 by Coomassie staining .

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

(b) Phosphorylation of Beclin1 by ROCK1. Recombinant ROCK1 was used for in vitro kinase assay in the presence or absence of Y27632, using recombinant His-Beclin1 (left) or Flag-Beclin1 immunopurified from transfected HeLa cells (right). Proteins were resolved by SDS-PAGE; phosphorylated protein was visualized with autoradiography, and Beclin1 by Coomassie staining.


( c ) Identification of ROCK1 - mediated phosphorylation domains in Beclin1 . The indicated Flag - Beclin1 fragments were purified from HeLa cells and used as substrates for in vitro ROCK1 kinase assay . 32P - autoradiogram ( centre ) analysed phosphorylation and western blotting ( WB ; right ) determined protein levels . Schematic representation of Beclin1 domain structure and deletion constructs are shown ( left ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ROCK1-mediated phosphorylation domains in Beclin1. The indicated Flag-Beclin1 fragments were purified from HeLa cells and used as substrates for in vitro ROCK1 kinase assay. 32P-autoradiogram (centre) analysed phosphorylation and western blotting (WB; right) determined protein levels. Schematic representation of Beclin1 domain structure and deletion constructs are shown (left).


( d ) Identification of the phosphorylation site on Beclin1 . Representation of point mutations in the different domains of Beclin1 are shown ( left ) . In vitro kinase assay using Flag - Beclin1 WT and mutants ( T38A and T119A ) , immunoprecipitated from transfected HeLa cells , as substrate and recombinant ROCK1 was performed . Phosphorylation was detected by 32P - autoradiogram , and Flag - Beclin1 levels were examined by WB .

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(d) Identification of the phosphorylation site on Beclin1. Representation of point mutations in the different domains of Beclin1 are shown (left). In vitro kinase assay using Flag-Beclin1 WT and mutants (T38A and T119A), immunoprecipitated from transfected HeLa cells, as substrate and recombinant ROCK1 was performed. Phosphorylation was detected by 32P-autoradiogram, and Flag-Beclin1 levels were examined by WB.


( e ) Flag - Beclin1 Wt or T119A transfected HeLa cells were incubated in glucose - rich or nutrient - free ( HBSS ) media , in the presence or absence of ROCK1 inhibitor Y27632 . Total cell extracts were used for IP using Flag agarose . Eluted protein was analysed by WB against phospho - T119 ( Beclin ) antibody and Beclin1 . Input for Beclin1 was run on 7 . 5 % gel and immunoblotted .

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(e) Flag-Beclin1 Wt or T119A transfected HeLa cells were incubated in glucose-rich or nutrient-free (HBSS) media, in the presence or absence of ROCK1 inhibitor Y27632. Total cell extracts were used for IP using Flag agarose. Eluted protein was analysed by WB against phospho-T119 (Beclin) antibody and Beclin1. Input for Beclin1 was run on 7.5% gel and immunoblotted.


( a ) Inhibiting ROCK1 activity increases the association between Beclin1 and Bcl - 2 . HeLa cells , untreated or treated with 10 μM Y27632 ( top panel ) , were cultured in control or glucose - free ( 4 h ) medium , lysed and cell extracts prepared . Endogenous Bcl - 2 was immunoprecipitated with cross - linked Bcl - 2 agarose and resulting eluted immune complexes were resolved by SDS - PAGE and blotted against indicated antibodies . EJ shCont . and shROCK1 # 1 cells ( bottom panel ) were grown in control or starvation media for 4 h and Bcl - 2 IP was performed as above . Inputs were rerun to confirm ROCK1 knockdown .

O O 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 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

(a) Inhibiting ROCK1 activity increases the association between Beclin1 and Bcl-2. HeLa cells, untreated or treated with 10 μM Y27632 (top panel), were cultured in control or glucose-free (4 h) medium, lysed and cell extracts prepared. Endogenous Bcl-2 was immunoprecipitated with cross-linked Bcl-2 agarose and resulting eluted immune complexes were resolved by SDS-PAGE and blotted against indicated antibodies. EJ shCont. and shROCK1#1 cells (bottom panel) were grown in control or starvation media for 4 h and Bcl-2 IP was performed as above. Inputs were rerun to confirm ROCK1 knockdown.


( b ) HeLa cells were transfected with full - length constructs of Flag - Beclin WT , Flag - Beclin T119A or Flag - Beclin T119E . Flag - Beclin WT cells were then treated with Y27632 for 8 h . Post transfection and treatment with Y27632 , cells were starved in HBSS medium for 4 h , after which cells were collected and whole cell lysates prepared . IP with Flag - agarose was performed for 2 h and proteins bound to beads were eluted and used for western blotting .

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(b) HeLa cells were transfected with full-length constructs of Flag-Beclin WT, Flag-Beclin T119A or Flag-Beclin T119E. Flag-Beclin WT cells were then treated with Y27632 for 8 h. Post transfection and treatment with Y27632, cells were starved in HBSS medium for 4 h, after which cells were collected and whole cell lysates prepared. IP with Flag-agarose was performed for 2 h and proteins bound to beads were eluted and used for western blotting.


( c ) Flag - Beclin1 Wt or T119A transfected HeLa cells were incubated in glucose - rich or nutrient - free ( HBSS ) media , in the presence or absence of ROCK1 inhibitor Y27632 . Total cell extracts were used for IP using Flag agarose . Eluted protein was analysed by western blotting against Bcl - 2 and Beclin1 . Inputs and IP were run on separate gels and same exposure is shown .

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(c) Flag-Beclin1 Wt or T119A transfected HeLa cells were incubated in glucose-rich or nutrient-free (HBSS) media, in the presence or absence of ROCK1 inhibitor Y27632. Total cell extracts were used for IP using Flag agarose. Eluted protein was analysed by western blotting against Bcl-2 and Beclin1. Inputs and IP were run on separate gels and same exposure is shown.


( d ) HeLa cells with a transient knockdown using siCont . and siBeclin1 - 3 ′ - untranslated region for 30 h were further left untreated or treated with 10 μM Y27632 . Cells were then incubated in control or HBSS media for 8 h . Whole cell extracts were prepared , resolved by SDS - PAGE and were analysed for Beclin1 , LC3 and β - actin .

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(d) HeLa cells with a transient knockdown using siCont. and siBeclin1-3′-untranslated region for 30 h were further left untreated or treated with 10 μM Y27632. Cells were then incubated in control or HBSS media for 8 h. Whole cell extracts were prepared, resolved by SDS-PAGE and were analysed for Beclin1, LC3 and β-actin.


( e ) Endogenous LC3 staining in WT - or T119A - Beclin1 - transfected cells . HeLa cells transfected with Flag - Beclin WT ( ± Y27632 ) and T119A were cultured in HBSS media for 6 h , fixed with cold acetone and endogenous LC3 immunofluorescence was performed . Representative images are shown ; scale bar , 20 μm . Arrows indicate punctate LC3 staining . Graph represents mean ± s . d . , n = 25 , LC3 puncta / cell .

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(e) Endogenous LC3 staining in WT- or T119A-Beclin1-transfected cells. HeLa cells transfected with Flag-Beclin WT (±Y27632) and T119A were cultured in HBSS media for 6 h, fixed with cold acetone and endogenous LC3 immunofluorescence was performed. Representative images are shown; scale bar, 20 μm. Arrows indicate punctate LC3 staining. Graph represents mean±s.d., n=25, LC3 puncta/cell.


( b ) PCR assay using tail genomic DNA from three different genotypes , separated on 0 . 7 % agarose gel ( top ) . Western blot analysis of ROCK1 expression in the heart tissue from three different genotypes using anti - ROCK1 antibody ( bottom ) . β - Actin was used for protein loading control in each lane .

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(b) PCR assay using tail genomic DNA from three different genotypes, separated on 0.7% agarose gel (top). Western blot analysis of ROCK1 expression in the heart tissue from three different genotypes using anti-ROCK1 antibody (bottom). β-Actin was used for protein loading control in each lane.


( c ) Mice were fed or starved for 16 h , and heart samples were prepared in lysis buffer . Resulting proteins were resolved by SDS - PAGE and blotted against ROCK1 , LC3 , autophagy marker p62 and β - actin .

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(c) Mice were fed or starved for 16 h, and heart samples were prepared in lysis buffer. Resulting proteins were resolved by SDS-PAGE and blotted against ROCK1, LC3, autophagy marker p62 and β-actin.


( d ) ROCK1 - deficient hearts or control hearts from animals fed or starved for 16 h were subjected to electron microscopy to identify autophagosome structures . Arrows indicate autophagic vacuoles . Scale bar , 500 nm .

(d) ROCK1-deficient hearts or control hearts from animals fed or starved for 16 h were subjected to electron microscopy to identify autophagosome structures. Arrows indicate autophagic vacuoles. Scale bar, 500 nm.


( e ) Hearts from fed and starved ROCK1 WT and KO mice were sectioned , fixed in acetone and immunofluorescence assay was performed against endogenous LC3 . Data are representative of two independent experiments from each genotype . Scale bar , 50 μm .

(e) Hearts from fed and starved ROCK1 WT and KO mice were sectioned, fixed in acetone and immunofluorescence assay was performed against endogenous LC3. Data are representative of two independent experiments from each genotype. Scale bar, 50 μm.


MCF10A cells were treated with 5uM RSL3 for times that ranged from 0 to 120 minutes . mRNA was isolated from each time point and PROM2 expression was quantified by qPCR . Shown is an average of three independent experiments with standard deviation ( n = 3 experiments per time point ) . P ‑ values were obtained by unpaired Student ' s t ‑ test with * P < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 005 . Exact p ‑ values are reported in the Appendix Table S1 . MCF10A cells were pretreated for 15 minutes with either DMSO or 2uM fer - 1 , followed by 60 minutes with either DMSO , 5uM RSL3 or RSL3 and fer - 1 . mRNA was isolated and PROM2 expression was quantified by qPCR . Shown are three independent experiments with standard deviation ( n = 3 experiments per group ) . P ‑ values were obtained by unpaired Student ' s t ‑ test with * P < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 005 . Exact p ‑ values are reported in the Appendix Table S1 . MCF10A cells were treated with 25uM 4HNE for times that ranged from 0 to 120 minutes . mRNA was isolated from each time point and PROM2 expression was quantified by qPCR . Shown is an average of three independent experiments with standard deviation ( n = 3 experiments per time point ) . P ‑ values were obtained by unpaired Student ' s t ‑ test with * P < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 005 . Exact p ‑ values are reported in the Appendix Table S1 . MCF10A cells were pretreated for 15 minutes with either DMSO or 2uM fer - 1 , followed by 60 minutes with either EtOH , 25uM 4HNE or RSL3 and fer - 1 . mRNA was isolated and PROM2 expression was quantified by qPCR . Shown are three independent experiments with standard deviation ( n = 3 experiments per group ) . P ‑ values were obtained by unpaired Student ' s t ‑ test with * P < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 005 . n = 3 experiments per group . Exact p ‑ values are reported in the Appendix Table S1 .

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

MCF10A cells were treated with 5uM RSL3 for times that ranged from 0 to 120 minutes. mRNA was isolated from each time point and PROM2 expression was quantified by qPCR. Shown is an average of three independent experiments with standard deviation (n=3 experiments per time point). P‑values were obtained by unpaired Student's t‑test with *P<0.05, **p<0.01, ***p<0.005. Exact p‑values are reported in the Appendix Table S1. MCF10A cells were pretreated for 15 minutes with either DMSO or 2uM fer-1, followed by 60 minutes with either DMSO, 5uM RSL3 or RSL3 and fer-1. mRNA was isolated and PROM2 expression was quantified by qPCR. Shown are three independent experiments with standard deviation (n=3 experiments per group). P‑values were obtained by unpaired Student's t‑test with *P<0.05, **p<0.01, ***p<0.005. Exact p‑values are reported in the Appendix Table S1. MCF10A cells were treated with 25uM 4HNE for times that ranged from 0 to 120 minutes. mRNA was isolated from each time point and PROM2 expression was quantified by qPCR. Shown is an average of three independent experiments with standard deviation (n=3 experiments per time point). P‑values were obtained by unpaired Student's t‑test with *P<0.05, **p<0.01, ***p<0.005. Exact p‑values are reported in the Appendix Table S1. MCF10A cells were pretreated for 15 minutes with either DMSO or 2uM fer-1, followed by 60 minutes with either EtOH, 25uM 4HNE or RSL3 and fer-1. mRNA was isolated and PROM2 expression was quantified by qPCR. Shown are three independent experiments with standard deviation (n=3 experiments per group). P‑values were obtained by unpaired Student's t‑test with *P<0.05, **p<0.01, ***p<0.005. n=3 experiments per group. Exact p‑values are reported in the Appendix Table S1.


MCF10A cells were treated with either EtOH or 25uM 4HNE for 60 minutes . Isolated protein was assessed by immunoprecipitating phospho - p38 ( Thr180 / Tyr182 ) and immunoblotting for 4HNE and phospho - p38 ( Thr180 / Tyr182 ) . Shown is one replicate of three independent experiments ( n = 3 ) .

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MCF10A cells were treated with either EtOH or 25uM 4HNE for 60 minutes. Isolated protein was assessed by immunoprecipitating phospho-p38 (Thr180/Tyr182) and immunoblotting for 4HNE and phospho-p38 (Thr180/Tyr182). Shown is one replicate of three independent experiments (n=3).


MCF10A cells were pretreated for 15 minutes with either DMSO , EtOH , or 10uM BIRB , followed by 60 minutes with either DMSO , EtOH , 25uM 4HNE , 5uM RSL3 , 4HNE and BIRB or RSL3 and BIRB . Isolated protein was assessed by immunoblotting for prominin2 , phospho - p38 ( Thr180 / Tyr182 ) and total p38 . Shown is one replicate of three independent experiments ( n = 3 ) .

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MCF10A cells were pretreated for 15 minutes with either DMSO, EtOH, or 10uM BIRB, followed by 60 minutes with either DMSO, EtOH, 25uM 4HNE, 5uM RSL3, 4HNE and BIRB or RSL3 and BIRB. Isolated protein was assessed by immunoblotting for prominin2, phospho-p38 (Thr180/Tyr182) and total p38. Shown is one replicate of three independent experiments (n=3).


HSF1 expression was diminished in MCF10A cells for 48 hours using siRNA prior to treatment with either DMSO , EtOH , DMSO , 25uM 4HNE or 5uM RSL3 . Isolated protein was assessed by immunoblotting for prominin2 , phospho - HSF1 ( Ser326 ) , total HSF1 , phospho - p38 ( Thr180 / Tyr182 ) , total p38 , and β - actin expression . Shown is one replicate of three independent experiments ( n = 3 ) .

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

HSF1 expression was diminished in MCF10A cells for 48 hours using siRNA prior to treatment with either DMSO, EtOH, DMSO, 25uM 4HNE or 5uM RSL3. Isolated protein was assessed by immunoblotting for prominin2, phospho-HSF1 (Ser326), total HSF1, phospho-p38 (Thr180/Tyr182), total p38, and β-actin expression. Shown is one replicate of three independent experiments (n=3).


MCF10A cells were treated for 60 minutes with either DMSO , 25uM 4HNE , 5uM RSL3 , 4HNE and 10uM KRIBB11 or RSL3 and KRIBB11 . mRNA was isolated and PROM2 expression was quantified by qPCR . Shown are three independent experiments with standard deviation ( n = 3 experiments per group ) . P ‑ values were obtained by unpaired Student ' s t ‑ test with * P < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 005 . Exact p ‑ values are reported in the Appendix Table S1 . MCF10A cells were treated for 60 minutes with either DMSO , 25uM 4HNE , 5uM RSL3 , 4HNE and 10uM KRIBB11 or RSL3 and KRIBB11 . mRNA was isolated and HSF1 expression was quantified by qPCR . Shown are three independent experiments with standard deviation ( n = 3 experiments per group ) . P ‑ values were obtained by unpaired Student ' s t ‑ test with * P < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 005 . Exact p ‑ values are reported in the Appendix Table S1 .

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MCF10A cells were treated for 60 minutes with either DMSO, 25uM 4HNE, 5uM RSL3, 4HNE and 10uM KRIBB11 or RSL3 and KRIBB11. mRNA was isolated and PROM2 expression was quantified by qPCR. Shown are three independent experiments with standard deviation (n=3 experiments per group). P‑values were obtained by unpaired Student's t‑test with *P<0.05, **p<0.01, ***p<0.005. Exact p‑values are reported in the Appendix Table S1. MCF10A cells were treated for 60 minutes with either DMSO, 25uM 4HNE, 5uM RSL3, 4HNE and 10uM KRIBB11 or RSL3 and KRIBB11. mRNA was isolated and HSF1 expression was quantified by qPCR. Shown are three independent experiments with standard deviation (n=3 experiments per group). P‑values were obtained by unpaired Student's t‑test with *P<0.05, **p<0.01, ***p<0.005. Exact p‑values are reported in the Appendix Table S1.


MCF10A cells were treated for 60 minutes with either EtOH , DMSO , 25uM 4HNE , 5uM RSL3 , 4HNE and 10uM KRIBB11 or RSL3 and KRIBB11 . Isolated protein was assessed by immunoblotting for prominin2 , phospho ‑ HSF1 ( Ser326 ) , total HSF1 , phospho - p38 ( Thr180 / Tyr182 ) , total p38 , and β - actin expression . Shown is one replicate of three independent experiments ( n = 3 ) .

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MCF10A cells were treated for 60 minutes with either EtOH, DMSO, 25uM 4HNE, 5uM RSL3, 4HNE and 10uM KRIBB11 or RSL3 and KRIBB11. Isolated protein was assessed by immunoblotting for prominin2, phospho‑HSF1 (Ser326), total HSF1, phospho-p38 (Thr180/Tyr182), total p38, and β-actin expression. Shown is one replicate of three independent experiments (n=3).


Hs578t cells were treated with either RSL3 or IKE at the range of concentrations shown in combination with either DMSO or 10uM KRIBB11 . Cells were assessed for viability after 24 hours . Absorbance was normalized to DMSO control . Shown are three independent replicates with standard deviation ( n = 3 experiments per group ) . P ‑ values were obtained by unpaired Student ' s t ‑ test with * P < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 005 . Exact p ‑ values are reported in the Appendix Table S1 . SF295 cells were treated with either RSL3 or IKE at the range of concentrations shown in combination with either DMSO or 10uM KRIBB11 . Cells were assessed for viability after 24 hours . Absorbance was normalized to DMSO control . Shown are three independent replicates with standard deviation ( n = 3 experiments per group ) . P ‑ values were obtained by unpaired Student ' s t ‑ test with * P < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 005 . Exact p ‑ values are reported in the Appendix Table S1 . NCI H1975 cells were treated with either RSL3 or IKE at the range of concentrations shown in combination with either DMSO or 10uM KRIBB11 . Cells were assessed for viability after 24 hours . Absorbance was normalized to DMSO control . Shown are three independent replicates with standard deviation ( n = 3 experiments per group ) . P ‑ values were obtained by unpaired Student ' s t ‑ test with * P < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 005 . Exact p ‑ values are reported in the Appendix Table S1 .

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Hs578t cells were treated with either RSL3 or IKE at the range of concentrations shown in combination with either DMSO or 10uM KRIBB11. Cells were assessed for viability after 24 hours. Absorbance was normalized to DMSO control. Shown are three independent replicates with standard deviation (n=3 experiments per group). P‑values were obtained by unpaired Student's t‑test with *P<0.05, **p<0.01, ***p<0.005. Exact p‑values are reported in the Appendix Table S1. SF295 cells were treated with either RSL3 or IKE at the range of concentrations shown in combination with either DMSO or 10uM KRIBB11. Cells were assessed for viability after 24 hours. Absorbance was normalized to DMSO control. Shown are three independent replicates with standard deviation (n=3 experiments per group). P‑values were obtained by unpaired Student's t‑test with *P<0.05, **p<0.01, ***p<0.005. Exact p‑values are reported in the Appendix Table S1. NCI H1975 cells were treated with either RSL3 or IKE at the range of concentrations shown in combination with either DMSO or 10uM KRIBB11. Cells were assessed for viability after 24 hours. Absorbance was normalized to DMSO control. Shown are three independent replicates with standard deviation (n=3 experiments per group). P‑values were obtained by unpaired Student's t‑test with *P<0.05, **p<0.01, ***p<0.005. Exact p‑values are reported in the Appendix Table S1.


Twenty - four hours after the last treatment , tumors were excised , frozen and sectioned . Each tumor was stained with Abs specific for either 4HNE , prominin2 or HSF1 . Images were taken at 20x magnification . Higher ‑ magnification images ( 1 . 5x ) are shown to the right of its corresponding inset ( white box ) . Scale bar is 50 microns . Shown is one representative image for each condition .

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Twenty-four hours after the last treatment, tumors were excised, frozen and sectioned. Each tumor was stained with Abs specific for either 4HNE, prominin2 or HSF1. Images were taken at 20x magnification. Higher‑magnification images (1.5x) are shown to the right of its corresponding inset (white box). Scale bar is 50 microns. Shown is one representative image for each condition.


A . Lysates from 293T cells transfected with the indicated DNA constructs were incubated with 1 mM Mg + + plus 0 . 1 mM GTPγS , GDP or 2 mM EDTA and immunoprecipitated with anti - Myc antibody . Rab18 - HA co - immunoprecipitated with Myc - TRAPPC9 when it was nucleotide free ( + EDTA ) .

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A. Lysates from 293T cells transfected with the indicated DNA constructs were incubated with 1 mM Mg++ plus 0.1 mM GTPγS, GDP or 2 mM EDTA and immunoprecipitated with anti-Myc antibody. Rab18-HA co-immunoprecipitated with Myc-TRAPPC9 when it was nucleotide free (+EDTA).


B . A mutant of Rab18 unable to bind nucleotide , N122I , was co - immunoprecipitated with Myc - TRAPPC9 by anti - Myc antibody .

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B. A mutant of Rab18 unable to bind nucleotide, N122I, was co-immunoprecipitated with Myc-TRAPPC9 by anti-Myc antibody.


C . HA - tagged Rab1 and Rab18 , but not Rab2 , could co - immunoprecipitate endogenous TRAPPII complex but not TRAPPIII complex .

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C. HA-tagged Rab1 and Rab18, but not Rab2, could co-immunoprecipitate endogenous TRAPPII complex but not TRAPPIII complex.


D . GST - Rab18 could pull down endogenous TRAPPII complex in an in vitro binding assay . GST - Rab18 was pre - loaded with GTPγS or GDP , or stripped off nucleotide by EDTA . Lysates from 293T cells were subjected to GST - Rab18 pull - down . The presence of various TRAPP subunits was detected by immunoblotting . TRAPPII , but not TRAPPIII complex was co - isolated with GST - Rab18 , most strongly with Rab18 in nucleotide - free state .

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D. GST-Rab18 could pull down endogenous TRAPPII complex in an in vitro binding assay. GST-Rab18 was pre-loaded with GTPγS or GDP, or stripped off nucleotide by EDTA. Lysates from 293T cells were subjected to GST-Rab18 pull-down. The presence of various TRAPP subunits was detected by immunoblotting. TRAPPII, but not TRAPPIII complex was co-isolated with GST-Rab18, most strongly with Rab18 in nucleotide-free state.


A . TRAPPII and TRAPPIII complexes were immunoprecipitated by antibodies specifically recognizing TRAPPC9 or TRAPPC12 , and the presence of various co - precipitating TRAPP subunits was detected by immunoblotting .

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A. TRAPPII and TRAPPIII complexes were immunoprecipitated by antibodies specifically recognizing TRAPPC9 or TRAPPC12, and the presence of various co-precipitating TRAPP subunits was detected by immunoblotting.


B - D . TRAPP stimulated guanine nucleotide exchange on purified Rab18 , Rab1 and Rab2 GST fusion proteins measured by release of 3H - GDP that was pre - loaded onto the Rab proteins .

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B-D. TRAPP stimulated guanine nucleotide exchange on purified Rab18, Rab1 and Rab2 GST fusion proteins measured by release of 3H-GDP that was pre-loaded onto the Rab proteins.


E - G . Nucleotide exchange reactions measured by time - dependent binding of [ 35S ] GTPγS . Immunoprecipitates using the indicated antibodies were included in the assay as the GEF to be tested . In the 3H - GDP release experiments , the reactions were incubated for 30 minutes at 37 ° C before the Rab proteins were subjected to nitrocellulose filter binding . In the [ 35S ] GTPγS binding experiments , the reactions were incubated for the indicated amount of time before filter binding .

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E-G. Nucleotide exchange reactions measured by time-dependent binding of [35S]GTPγS. Immunoprecipitates using the indicated antibodies were included in the assay as the GEF to be tested. In the 3H-GDP release experiments, the reactions were incubated for 30 minutes at 37°C before the Rab proteins were subjected to nitrocellulose filter binding. In the [35S]GTPγS binding experiments, the reactions were incubated for the indicated amount of time before filter binding.


H - I . Anti - Myc antibody immunoprecipitates from lysates of HEK293T cells transfected Myc - TRAPPC9 were used as the GEF for GST - Rab1 or GST - Rab18 in 30 min incubations at 37 ° C with [ 35S ] GTPγS .

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H-I. Anti-Myc antibody immunoprecipitates from lysates of HEK293T cells transfected Myc-TRAPPC9 were used as the GEF for GST-Rab1 or GST-Rab18 in 30 min incubations at 37°C with [35S]GTPγS.


A . Depleting TRAPPC9 , but not TRAPPC8 , increased lipid droplet size in HEK293T cells . The efficiency of siRNA depletion of TRAPPC8 and TRAPPC9 in Hela cells is shown by immunoblotting in the top left panel .

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A. Depleting TRAPPC9, but not TRAPPC8, increased lipid droplet size in HEK293T cells. The efficiency of siRNA depletion of TRAPPC8 and TRAPPC9 in Hela cells is shown by immunoblotting in the top left panel.


A . Depleting TRAPPC9 , but not TRAPPC8 , increased lipid droplet size in HEK293T cells . Representative fluorescence images show the status of lipid droplets in these siRNA treated cells after oleic acid incubation . Bar = 10 μm .

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A. Depleting TRAPPC9, but not TRAPPC8, increased lipid droplet size in HEK293T cells. Representative fluorescence images show the status of lipid droplets in these siRNA treated cells after oleic acid incubation. Bar =10 μm.


B . After loading with oleic acid , human skin fibroblasts containing a R475 * mutation in TRAPPC9 accumulated lipid droplets of greater sizes than skin fibroblasts similarly isolated from a TRAPPC9 wildtype human individual . Bar = 10 μm .

B. After loading with oleic acid, human skin fibroblasts containing a R475* mutation in TRAPPC9 accumulated lipid droplets of greater sizes than skin fibroblasts similarly isolated from a TRAPPC9 wildtype human individual. Bar = 10 μm.


A . HEK293T cells with the indicated genotypes were loaded with 400 μM oleic acid for 24 h before staining with bodipy 493 / 503 . Bar = 10 μm .

A. HEK293T cells with the indicated genotypes were loaded with 400 μM oleic acid for 24 h before staining with bodipy 493/503. Bar = 10 μm.


B . Wildtype or TRAPPII deleted cells were loaded with oleic acid and stained with bodipy 493 / 503 ( green ) and endogenous Rab18 by immunofluorescence ( red ) . Bar = 10 μm .

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B. Wildtype or TRAPPII deleted cells were loaded with oleic acid and stained with bodipy 493/503 (green) and endogenous Rab18 by immunofluorescence (red). Bar = 10 μm.


C . DsRed - Rab18 was transfected into wildtype ( top panels and insets ) or TRAPPII deleted HEK293T cells ( bottom panels and insets ) that were loaded with oleic acid and stained with bodipy 493 / 503 ( green ) . Bar = 10 μm .

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C. DsRed-Rab18 was transfected into wildtype (top panels and insets) or TRAPPII deleted HEK293T cells (bottom panels and insets) that were loaded with oleic acid and stained with bodipy 493/503 (green). Bar = 10 μm.


D . Homogenates of wildtype or TRAPPII deleted cells were subjected to sucrose gradient fractionation . Fractions 1 and 2 were enriched with LDs . The presence of Rab18 , TRAPPC9 , TRAPPC2 , LD marker TIP47 , and ER marker Calnexin were detected by SDS - PAGE followed by immunoblotting .

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D. Homogenates of wildtype or TRAPPII deleted cells were subjected to sucrose gradient fractionation. Fractions 1 and 2 were enriched with LDs. The presence of Rab18, TRAPPC9, TRAPPC2, LD marker TIP47, and ER marker Calnexin were detected by SDS-PAGE followed by immunoblotting.


Control siRNA depletion ( siCtrl ) was conducted with siRNA oligoes specific to a firefly luciferase sequence ; siCOPB = depletion of γ - COP ; siRab18 = depletion of Rab18 . C9C10KO = HEK293T cells with TRAPPC9 and TRAPPC10 genes deleted . N = 3 , Error bars = S . D .

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Control siRNA depletion (siCtrl) was conducted with siRNA oligoes specific to a firefly luciferase sequence; siCOPB = depletion of γ-COP; siRab18 = depletion of Rab18. C9C10KO = HEK293T cells with TRAPPC9 and TRAPPC10 genes deleted. N= 3, Error bars = S.D.


A . Myc - TRAPPC9 directly interacted with FLAG - γ - COP . The presence of FLAG - γ - COP in immunoprecipitations of Myc - TRAPPC9 and Myc - TRAPPC10 from cell lysates of the indicated genetic backgrounds was investigated by immunoblotting .

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A. Myc-TRAPPC9 directly interacted with FLAG-γ-COP. The presence of FLAG-γ-COP in immunoprecipitations of Myc-TRAPPC9 and Myc-TRAPPC10 from cell lysates of the indicated genetic backgrounds was investigated by immunoblotting.


B , C . Co - immunoprecipitation between FLAG - γ - COP and HA - Rab18 in HEK293T cells with indicated genetic backgrounds . The presence of FLAG - γ - COP in immunoprecipitates of HA - Rab18 ( and reciprocal IP in C ) was investigated by immunoblotting .

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B,C. Co-immunoprecipitation between FLAG-γ-COP and HA-Rab18 in HEK293T cells with indicated genetic backgrounds. The presence of FLAG-γ-COP in immunoprecipitates of HA-Rab18 (and reciprocal IP in C) was investigated by immunoblotting.


D . The interaction between endogenous COPI and Rab18 was TRAPPII dependent . Endogenous Rab18 was immunoprecipitated by anti - Rab18 antibody from lysates of indicated genetic background . The presence of γ - COP and various TRAPP subunits were detected by their specific antibodies as indicated .

D. The interaction between endogenous COPI and Rab18 was TRAPPII dependent. Endogenous Rab18 was immunoprecipitated by anti-Rab18 antibody from lysates of indicated genetic background. The presence of γ-COP and various TRAPP subunits were detected by their specific antibodies as indicated.


E . Co - immunoprecipitation between TRAPPII and HA - Rab18 in control ( siFFL ) and γ - COP ( si - γ - COP ) siRNA depleted cells . TRAPPII was precipitated with transfected Myc - TRAPPC10 and the presence of HA - Rab18 was determined by immunoblotting .

E. Co-immunoprecipitation between TRAPPII and HA-Rab18 in control (siFFL) and γ-COP (si-γ-COP) siRNA depleted cells. TRAPPII was precipitated with transfected Myc-TRAPPC10 and the presence of HA-Rab18 was determined by immunoblotting.


F . Co - immunoprecipitation between FLAG - γ - COP and TRAPPII in control ( siFFL ) and Rab18 ( si - Rab18 ) siRNA depleted cells . TRAPPII was precipitated with Myc - TRAPPC10 and the presence of FLAG - γ - COP was determined by immunoblotting . The molecular weight of endogenous Rab18 overlaps with IgG light chain , and therefore , the amount of endogenous Rab18 precipitated with TRAPPII cannot be determined in this experiment .

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F. Co-immunoprecipitation between FLAG-γ-COP and TRAPPII in control (siFFL) and Rab18 (si-Rab18) siRNA depleted cells. TRAPPII was precipitated with Myc-TRAPPC10 and the presence of FLAG-γ-COP was determined by immunoblotting. The molecular weight of endogenous Rab18 overlaps with IgG light chain, and therefore, the amount of endogenous Rab18 precipitated with TRAPPII cannot be determined in this experiment.


A . Huh - 7 cells were depleted with control siRNA specific to firefly luciferase sequence ( top panels ) or with siRNA specific to γ - COP ( bottom panels ) . Endogenous Rab18 ( red ) and LD ( green ) were stained and visualized . Bar = 10 μm .

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A. Huh-7 cells were depleted with control siRNA specific to firefly luciferase sequence (top panels) or with siRNA specific to γ-COP (bottom panels). Endogenous Rab18 (red) and LD (green) were stained and visualized. Bar = 10 μm.


B . Huh - 7 cells were treated with or without BFA for 6 hours before staining for Rab18 ( red ) and LD ( green ) . Bar = 10 μm .

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B. Huh-7 cells were treated with or without BFA for 6 hours before staining for Rab18 (red) and LD (green). Bar = 10 μm.


C . TRAPPC9 were detected on Golgi but not LD surface . Huh - 7 cells were permeabilized with digitonin before fixation and staining . LDs were labeled with bodipy 493 / 503 and pseudocolored in white . TRAPPC9 ( red ) and LD surface marker ADRP ( green ) were detected by their respective antibodies . Bar = 10 μm .

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C. TRAPPC9 were detected on Golgi but not LD surface. Huh-7 cells were permeabilized with digitonin before fixation and staining. LDs were labeled with bodipy 493/503 and pseudocolored in white. TRAPPC9 (red) and LD surface marker ADRP (green) were detected by their respective antibodies. Bar = 10 μm.


Huh - 7 cells were first serum - starved and then incubated with oleic acid for the indicated time before staining with Bodipy 493 / 503 , TRAPPC9 , Rab18 and γ - COP . A . In the merged images , bodipy 493 / 503 was pseudocolored in green , TRAPPC9 in red , and ADRP in blue . In the colocalization between TRAPPC9 and ADRP on the right side , the ADRP signal was re - colored to green for easy visualization . Bar = 10μm .

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Huh-7 cells were first serum-starved and then incubated with oleic acid for the indicated time before staining with Bodipy 493/503, TRAPPC9, Rab18 and γ-COP.A. In the merged images, bodipy 493/503 was pseudocolored in green, TRAPPC9 in red, and ADRP in blue. In the colocalization between TRAPPC9 and ADRP on the right side, the ADRP signal was re-colored to green for easy visualization. Bar = 10μm.


Huh - 7 cells were first serum - starved and then incubated with oleic acid for the indicated time before staining with Bodipy 493 / 503 , TRAPPC9 , Rab18 and γ - COP . B . Bodipy 493 / 503 was pseudocolored in green , Rab18 in red , and γ - COP in blue . In the colocalization between Rab18 and γ - COP on the right side , the γ - COP signal was re - colored to green for easy visualization . Bar = 10μm .

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Huh-7 cells were first serum-starved and then incubated with oleic acid for the indicated time before staining with Bodipy 493/503, TRAPPC9, Rab18 and γ-COP.B. Bodipy 493/503 was pseudocolored in green, Rab18 in red, and γ-COP in blue. In the colocalization between Rab18 and γ-COP on the right side, the γ-COP signal was re-colored to green for easy visualization. Bar = 10μm.


Huh - 7 cells were first starved with serum free medium and then incubated with oleic acids for 12 hours . Condition 1 ( control ) , the cells were not treated with BFA at any time point ( left column ) . Condition 2 , the cells were incubated with both 5 μg / ml of BFA and oleic acid incubation for 6 h and then in oleic acid alone for 10 h ( middle column ) . Condition 3 , the cells were treated with 5 μg / ml of BFA starting at 12 hours after oleic acid incubation ( right column ) . Bar = 10μm .

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Huh-7 cells were first starved with serum free medium and then incubated with oleic acids for 12 hours. Condition 1 (control), the cells were not treated with BFA at any time point (left column). Condition 2, the cells were incubated with both 5 μg/ml of BFA and oleic acid incubation for 6 h and then in oleic acid alone for 10 h (middle column). Condition 3, the cells were treated with 5 μg/ml of BFA starting at 12 hours after oleic acid incubation (right column). Bar = 10μm.


Proteins and ( C ) peptides identified for platelets , erythrocytes , platelet - rich and platelet - free plasma .

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Proteins and (C) peptides identified for platelets, erythrocytes, platelet-rich and platelet-free plasma.


Selection of the most suitable quality marker proteins for platelet contamination ( blue dots ) and ( E ) erythrocyte contamination ( red dots ) based on their abundance , the platelet / erythrocyte to plasma ratio and the coefficient of variation . Proteins that were only detected in platelets or erythrocytes , but not in plasma are aligned on the right side of the graph .

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

Selection of the most suitable quality marker proteins for platelet contamination (blue dots) and (E) erythrocyte contamination (red dots) based on their abundance, the platelet/erythrocyte to plasma ratio and the coefficient of variation. Proteins that were only detected in platelets or erythrocytes, but not in plasma are aligned on the right side of the graph.


Protein intensities were Z - scored across the dilution series for the 29 quality markers of the erythrocyte panel and ( C ) for the 29 markers of the platelet panel as a function of their spike - in proportion to plasma . Whiskers indicate 10 - 90 percentiles and horizontal lines denote the mean . ( D ) Correlation of erythrocyte count to the ' contamination index ' for the erythrocyte marker panel . ( E ) Correlation of platelet count to contamination index for the platelet marker panel .

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

Protein intensities were Z-scored across the dilution series for the 29 quality markers of the erythrocyte panel and (C) for the 29 markers of the platelet panel as a function of their spike-in proportion to plasma. Whiskers indicate 10-90 percentiles and horizontal lines denote the mean. (D) Correlation of erythrocyte count to the 'contamination index' for the erythrocyte marker panel. (E) Correlation of platelet count to contamination index for the platelet marker panel.


Volcano plot comparing 72 plasma vs . 72 serum proteomes . Proteins highlighted in yellow were chosen according to their p - value as markers for coagulation . Only the plasma enriched proteins ( compared to serum ) were used in the calculation of the coagulation contamination index . Ratio of the summed intensities of all plasma or serum proteins to the sum of the plasma - enriched panel proteins are plotted for all samples . Whiskers indicate the 10 - 90 percentile .

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

Volcano plot comparing 72 plasma vs. 72 serum proteomes. Proteins highlighted in yellow were chosen according to their p-value as markers for coagulation. Only the plasma enriched proteins (compared to serum) were used in the calculation of the coagulation contamination index. Ratio of the summed intensities of all plasma or serum proteins to the sum of the plasma-enriched panel proteins are plotted for all samples. Whiskers indicate the 10-90 percentile.


Volcano plot of the proteome comparison of time point 1 vs 4 . Proteins of the platelet panel are highlighted in blue and two additional significantly regulated proteins in red .

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Volcano plot of the proteome comparison of time point 1 vs 4. Proteins of the platelet panel are highlighted in blue and two additional significantly regulated proteins in red.


Global correlation map on the left with an inset of the platelet cluster on the right . The two significant outliers of the volcano plot in ( B ) are marked in red . Platelet panel proteins are highlighted in blue in the inset . Red patches in the global correlation map indicate positive and blue patches negative correlations .

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

Global correlation map on the left with an inset of the platelet cluster on the right. The two significant outliers of the volcano plot in (B) are marked in red. Platelet panel proteins are highlighted in blue in the inset. Red patches in the global correlation map indicate positive and blue patches negative correlations.


B . Violin plots of the 3 ' READS signal on all the TNFα induced genes ( n = 307 ) or the set of TNFα induced genes longer than 40 kb ( n = 111 ) . * * * * p < 0 . 0001 . Statistical test : Friedman test with Dunn ' s multiple test correction . Violin plot : minimal to maximal value , dashed horizontal line : median , dotted horizontal lines : interquartile ( 25 % and 75 % ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Violin plots of the 3'READS signal on all the TNFα induced genes (n=307) or the set of TNFα induced genes longer than 40 kb (n=111). **** p < 0.0001. Statistical test: Friedman test with Dunn's multiple test correction. Violin plot: minimal to maximal value, dashed horizontal line: median, dotted horizontal lines: interquartile (25% and 75%).


C . Western blot of total pol II , Ser2P , Xrn2 , CPSF2 , CPSF73 , and histone H3 as a loading control , on the chromatin fraction . D . Western blot of Xrn2 , CPSF2 , and Nucleolin as a loading control , on the nucleoplasm fraction ( the CPSF73 antibody does not provide reliable results on the nucleoplasm fraction ) . E . Quantification of the western blots shown in C and D . n = 2 biological replicates , mean .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 of total pol II, Ser2P, Xrn2, CPSF2, CPSF73, and histone H3 as a loading control, on the chromatin fraction. D. Western blot of Xrn2, CPSF2, and Nucleolin as a loading control, on the nucleoplasm fraction (the CPSF73 antibody does not provide reliable results on the nucleoplasm fraction). E. Quantification of the western blots shown in C and D. n=2 biological replicates, mean.


F . Ratio of the different CPA and termination factors to total pol II . n = 3 biological replicates , mean ± SEM , p - value : * p < 0 . 05 , * * p < 0 . 01 . Statistical test : two - tailed unpaired t test .

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F. Ratio of the different CPA and termination factors to total pol II. n=3 biological replicates, mean ± SEM, p-value: * p < 0.05, ** p < 0.01. Statistical test: two-tailed unpaired t test.


A . Western blot of total pol II , Ser2P ( Abcam or Cell Signaling ) , Ser5P ( Abcam or Cell Signaling ) , and histone H3 as a loading control , on the chromatin fraction of CDK9as or wild - type HEK293 cells treated with DMSO or 15 µM 1 - NA - PP1 for 15 or 30 minutes . B . Quantification of the western blots shown in A . n = 2 biological replicates , except for Ser2P / pol II ( Abcam ) CDK9as NA 30 min , Ser5P / pol II ( Abcam ) CDK9as NA 15 min and NA 30 min , and Ser5P / pol II ( Cell Signaling ) CDK9as NA 30 min , where n = 3 biological replicates .

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

A. Western blot of total pol II, Ser2P (Abcam or Cell Signaling), Ser5P (Abcam or Cell Signaling), and histone H3 as a loading control, on the chromatin fraction of CDK9as or wild-type HEK293 cells treated with DMSO or 15 µM 1-NA-PP1 for 15 or 30 minutes. B. Quantification of the western blots shown in A. n=2 biological replicates, except for Ser2P / pol II (Abcam) CDK9as NA 30 min, Ser5P / pol II (Abcam) CDK9as NA 15 min and NA 30 min, and Ser5P / pol II (Cell Signaling) CDK9as NA 30 min, where n=3 biological replicates.


C . ChIP - qPCR of total pol II and of Ser2P ( Abcam or Cell Signaling ) , and Ser5P ( Abcam or Cell Signaling ) ratioed to total pol II on KPNB1 . n = 3 biological replicates , mean ± SEM , p - value : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . Statistical test : two - tailed unpaired t test .

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

C. ChIP-qPCR of total pol II and of Ser2P (Abcam or Cell Signaling), and Ser5P (Abcam or Cell Signaling) ratioed to total pol II on KPNB1. n=3 biological replicates, mean ± SEM, p-value: * p < 0.05, ** p < 0.01, *** p < 0.001. Statistical test: two-tailed unpaired t test.


B . Western blot of total SF3B1 , SF3B1 T142P , and histone H3 as a loading control , on the chromatin fraction of CDK9as cells treated for 30 minutes with DMSO , NA , tautomycetin ( TT ) , calyculin A ( CA ) , NA + TT , or NA + CA . The histone H3 loading control ( CA samples ) is the same as the loading control shown in Appendix Figure S3B as the same western blot experiment is shown in two different figures .

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

B. Western blot of total SF3B1, SF3B1 T142P, and histone H3 as a loading control, on the chromatin fraction of CDK9as cells treated for 30 minutes with DMSO, NA, tautomycetin (TT), calyculin A (CA), NA+TT, or NA+CA. The histone H3 loading control (CA samples) is the same as the loading control shown in Appendix Figure S3B as the same western blot experiment is shown in two different figures.


C . Co - immunoprecipitation of CPSF2 in the CDK9as cell treated for 30 minutes with DMSO or NA followed by western blot with total pol II , CPSF2 , CPSF3 , SF3B1 , SF3B3 and β - actin ( negative control ) antibodies .

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C. Co-immunoprecipitation of CPSF2 in the CDK9as cell treated for 30 minutes with DMSO or NA followed by western blot with total pol II, CPSF2, CPSF3, SF3B1, SF3B3 and β-actin (negative control) antibodies.


D . Co - immunoprecipitation of SF3B1 in the CDK9as cell treated for 30 minutes with DMSO , NA , or DRB followed by western blot with total pol II , SF3B1 , SF3B2 , and GAPDH ( negative control ) antibodies .

O O O O O O O O O O O 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

D. Co-immunoprecipitation of SF3B1 in the CDK9as cell treated for 30 minutes with DMSO, NA, or DRB followed by western blot with total pol II, SF3B1, SF3B2, and GAPDH (negative control) antibodies.


B . qRT - PCR of nuclear polyadenylated mRNAs of several TNFα induced or non - induced genes with a 30 minutes DMSO , DRB , CA , or DRB + CA treatment .

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 B-CONTROLLED_VAR O B-CONTROLLED_VAR O O

B. qRT-PCR of nuclear polyadenylated mRNAs of several TNFα induced or non-induced genes with a 30 minutes DMSO, DRB, CA, or DRB+CA treatment.


C . qRT - PCR of cytoplasmic polyadenylated mRNAs of several TNFα induced or non - induced genes with a 30 minutes DMSO , DRB , CA , or DRB + CA treatment .

C. qRT-PCR of cytoplasmic polyadenylated mRNAs of several TNFα induced or non-induced genes with a 30 minutes DMSO, DRB, CA, or DRB+CA treatment.


D . ChIP - qPCR of Ser2P , CPSF73 , or CPSF2 ratioed to total pol II after 30 minutes treatment with DMSO , DRB , CA , or DRB + CA on KPNB1 .

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 B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O

D. ChIP-qPCR of Ser2P, CPSF73, or CPSF2 ratioed to total pol II after 30 minutes treatment with DMSO, DRB, CA, or DRB+CA on KPNB1.


E . Co - immunoprecipitation of total pol II from HeLa cells treated for 30 minutes with DMSO , DRB , CA , or DRB + CA followed by western blot with total pol II , Ser2P , SF3B1 , CPSF2 , Xrn2 , and GAPDH antibodies .

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

E. Co-immunoprecipitation of total pol II from HeLa cells treated for 30 minutes with DMSO, DRB, CA, or DRB+CA followed by western blot with total pol II, Ser2P, SF3B1, CPSF2, Xrn2, and GAPDH antibodies.


( B - K ' ) The Drosophila posterior midguts containing Su ( H ) - Gal4 > UAS - CD8 : GFP ( Su ( H ) > GFP ) with sucrose ( Suc , control ) ( B - C ' ) , P . e for 36 h ( D - F ' ) , Ecc15 for 36 h ( G - I ' ) and DSS for 36 h ( J - K ' ) were immunostained for GFP ( green ) , PH3 ( red ) and DAPI ( blue ) . ( C - C ' , E - E ' , H - H ' and K - K ' ) Magnification of selected areas containing PH3 + GFP ─ cells from B , D , G and J . The EB cell close to the PH3 + cell ( white arrow ) in C - C ' is out of focus . ( F - F ' and I - I ' ) Magnification of selected areas containing PH3 + EBs ( GFP + ) from D and G . White arrowheads and red arrows indicate PH3 in GFP ─ and GFP + cells , respectively . ( L and M ) Quantification of PH3 GFP ─ ISCs ( L ) and PH3 + GFP + EBs ( M ) with given treatments . n = 21 guts for each treatment .

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

(B-K') The Drosophila posterior midguts containing Su(H)-Gal4>UAS-CD8:GFP (Su(H)>GFP) with sucrose (Suc, control) (B-C'), P.e for 36 h (D-F'), Ecc15 for 36 h (G-I') and DSS for 36 h (J-K') were immunostained for GFP (green), PH3 (red) and DAPI (blue). (C-C', E-E', H-H' and K-K') Magnification of selected areas containing PH3+GFP─ cells from B, D, G and J. The EB cell close to the PH3+ cell (white arrow) in C-C' is out of focus. (F-F' and I-I') Magnification of selected areas containing PH3+ EBs (GFP+) from D and G. White arrowheads and red arrows indicate PH3 in GFP─ and GFP+ cells, respectively. (L and M) Quantification of PH3 GFP─ ISCs (L) and PH3+GFP+ EBs (M) with given treatments. n = 21 guts for each treatment.


( D - F ' ' ) When the ISCs were marked by esgGal4 , su ( H ) - Gal80 > UAS - GFP , PH3 expression was found in GFP + ISCs with feeding of Suc ( control ) ( D - D ' ' ) , P . e ( E - E ' ' ) and Dss ( F - F ' ' ) . However , PH3 expression is present in EBs with GFP ─ Pros ─ and small nuclei upon P . e infection ( E - E ' ' , red arrows ) . The white arrows indicate GFP + cells with PH3 expression . ( G ) Quantification of PH3 + GFP ─ Pros ─ EBs with given treatments . n = 11 guts for each treatment . Data information : Three independent experiments were performed , and error bars are ± SEM . * * * , P < 0 . 001 ( Student ' s t - test ) ( G ) .

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

(D-F'') When the ISCs were marked by esgGal4, su(H)-Gal80>UAS-GFP, PH3 expression was found in GFP+ ISCs with feeding of Suc (control) (D-D''), P.e (E-E'') and Dss (F-F''). However, PH3 expression is present in EBs with GFP─Pros─ and small nuclei upon P.e infection (E-E'', red arrows). The white arrows indicate GFP+ cells with PH3 expression. (G) Quantification of PH3+GFP─Pros─ EBs with given treatments. n = 11 guts for each treatment. Data information: Three independent experiments were performed, and error bars are ± SEM. ***, P < 0.001 (Student's t-test) (G).


( D - N ' ) The Drosophila midguts expressing Su ( H ) ts > GFP with control ( D - E ' ) , UAS - RasV12 for 6 days ( F - H ' ) , EGFRA887T for 4 days ( I - K ' ) and UAS − λTop for 5 days ( L - N ' ) were immunostained for GFP ( green ) , PH3 ( red ) and DAPI ( blue ) . ( E - E ' , G - G ' , J - J ' and M - M ' ) Magnification of selected areas containing PH3 + GFP ─ cells from D , F , I and L . ( H - H ' , K - K ' and N - N ' ) Magnification of selected areas containing PH3 + GFP + cells from F , I and L . White arrowheads and red arrows indicate PH3 in GFP ─ ISCs and GFP + EBs , respectively . ( O , P ) Quantification of PH3 + GFP ─ ( P ) and PH3 + GFP + ( O ) in midguts with indicated genotypes . n = 13 guts for each genotype . ( Q ) Quantification of PH3 + EBs ( GFP + ) with the given genotypes in response to P . e infection . n = 11 guts for each genotype .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-N') The Drosophila midguts expressing Su(H)ts>GFP with control (D-E'), UAS-RasV12 for 6 days (F-H'), EGFRA887T for 4 days (I-K') and UAS−λTop for 5 days (L-N') were immunostained for GFP (green), PH3 (red) and DAPI (blue). (E-E', G-G', J-J' and M-M') Magnification of selected areas containing PH3+GFP─ cells from D, F, I and L. (H-H', K-K' and N-N') Magnification of selected areas containing PH3+GFP+ cells from F, I and L. White arrowheads and red arrows indicate PH3 in GFP─ ISCs and GFP+ EBs, respectively. (O, P) Quantification of PH3+GFP─ (P) and PH3+GFP+ (O) in midguts with indicated genotypes. n = 13 guts for each genotype. (Q) Quantification of PH3+ EBs (GFP+) with the given genotypes in response to P. e infection. n = 11 guts for each genotype.


( B - C ' ' ' ) Flies expressing UAS - Flp ; Su ( H ) ts UAS - CD8 : GFP ; ActP > Stop > LacZ were fed with Suc ( B - B ' ' ' ) or P . e ( C - C ' ' ' ) for 36h and their midguts were dissected and immunostained with antibodies against GFP , β - gal and Dl . Blue arrowheads indicate Dl + ISCs ; white arrows indicate GFP + EBs ; and red arrowheads indicate EB progeny cells ( LacZ + GFP ─ ) . ( D ) Quantification of Dl + ISC - like cells from EBs ( GFP ─ LacZ + Dl + ) in the control , P . e infected midguts , n = 50 . ( E ) Quantification of Dl + ISC - like cells from EBs ( GFP ─ LacZ + Dl + ) in the control with Suc or P . e , or knockdown of EGFR with Suc or P . e infection , n = 50 .

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

(B-C''') Flies expressing UAS-Flp; Su(H)ts UAS-CD8:GFP; ActP>Stop>LacZ were fed with Suc (B-B''') or P.e (C-C''') for 36h and their midguts were dissected and immunostained with antibodies against GFP, β-gal and Dl. Blue arrowheads indicate Dl+ ISCs; white arrows indicate GFP+ EBs; and red arrowheads indicate EB progeny cells (LacZ+GFP─). (D) Quantification of Dl+ ISC-like cells from EBs (GFP─LacZ+Dl+) in the control, P. e infected midguts, n=50. (E) Quantification of Dl+ ISC-like cells from EBs (GFP─LacZ+Dl+) in the control with Suc or P. e, or knockdown of EGFR with Suc or P. e infection, n=50.