Figure 3A . TRAPP and COPII associate more stably with SGs than with ERES . The membrane association of Sec24C was evaluated in non - permeabilized or permeabilized cells with or without SA treatment , as indicated . G3BP was used as an SG marker . Left panel insets , G3BP staining in non - permeabilized cells . Dashed white lines show the outline of permeabilized SA - untreated cells . Blue , DAPI . Data information ; Scale bars , 10 µm . B . Stabilizing TRAPP and COPII at the ERES prevents their relocalization to SGs . HeLa cells overexpressing GFP - Sar1H79G were treated with SA and immunostained for Sec24C , TRAPPC2 , and eIF3 , as indicated . Blue , DAPI . Graphs show quantification of Sec24C or TRAPPC2 at SGs in GFP - Sar1H79G - expressing cells . Data are the ratio between Sec24C or TRAPPC2 mean intensity in SG puncta and Sec24C or TRAPPC2 mean intensity at ERES , expressed as a percentage of the non - transfected ( NT ) cells . Mean ± s . e . m . n = 60 - 70 , three independent experiments . * * * * p < 0 . 0001 , Student ' s unpaired two - tailed t - test . Data information ; Scale bars , 10 µm . C . Effect of Sec31A depletion on TRAPPC2 recruitment to SGs . Cells were mock - treated or KD for Sec31 , treated with SA , and then immunostained for TRAPPC2 and eIF3 as indicated . Graphs , quantification of TRAPPC2 at ERES and SGs after SA treatment . Mean ± s . e . m . , one representative experiment ; n = 60 - 80 . * * p < 0 . 001 , * * * * p < 0 . 0001 , Student ' s unpaired two - tailed t - test . Data information ; Scale bars , 10 µm . D . Effect of Sec31A depletion on Sec24C recruitment to SGs . Cells were mock - treated or KD for Sec31 and then immunostained for Sec24C and cTAGE5 ( to stain ERES , top panels ) and for eIF3 ( bottom panels ) . Insets show eIF3 . Graphs , quantification of Sec24C at ERES and in SGs after SA treatment . n = 80 . * * * * p < 0 . 0001 , Student ' s unpaired two - tailed t - test . Data information ; Scale bars , 10 µm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 3A. TRAPP and COPII associate more stably with SGs than with ERES. The membrane association of Sec24C was evaluated in non-permeabilized or permeabilized cells with or without SA treatment, as indicated. G3BP was used as an SG marker. Left panel insets, G3BP staining in non-permeabilized cells. Dashed white lines show the outline of permeabilized SA-untreated cells. Blue, DAPI. Data information; Scale bars, 10 µm.B. Stabilizing TRAPP and COPII at the ERES prevents their relocalization to SGs. HeLa cells overexpressing GFP-Sar1H79G were treated with SA and immunostained for Sec24C, TRAPPC2, and eIF3, as indicated. Blue, DAPI. Graphs show quantification of Sec24C or TRAPPC2 at SGs in GFP-Sar1H79G-expressing cells. Data are the ratio between Sec24C or TRAPPC2 mean intensity in SG puncta and Sec24C or TRAPPC2 mean intensity at ERES, expressed as a percentage of the non-transfected (NT) cells. Mean ± s.e.m. n = 60-70, three independent experiments. ****p<0.0001, Student's unpaired two-tailed t-test. Data information; Scale bars, 10 µm.C. Effect of Sec31A depletion on TRAPPC2 recruitment to SGs. Cells were mock-treated or KD for Sec31, treated with SA, and then immunostained for TRAPPC2 and eIF3 as indicated. Graphs, quantification of TRAPPC2 at ERES and SGs after SA treatment. Mean ± s.e.m., one representative experiment; n=60-80. ** p<0.001, ****p<0.0001, Student's unpaired two-tailed t-test. Data information; Scale bars, 10 µm.D. Effect of Sec31A depletion on Sec24C recruitment to SGs. Cells were mock-treated or KD for Sec31 and then immunostained for Sec24C and cTAGE5 (to stain ERES, top panels) and for eIF3 (bottom panels). Insets show eIF3. Graphs, quantification of Sec24C at ERES and in SGs after SA treatment. n=80. ****p<0.0001, Student's unpaired two-tailed t-test. Data information; Scale bars, 10 µm.


Figure 4A . Cells were treated with 273 kinase inhibitors ( 10 µM ) from the Sellekchem library and with SA , and Sec24C localization in G3BP puncta ( % of total G3BP area ) was analyzed by automated microscopy under High content screening and is reported as a percentage of the control ( cells treated with SA alone ) . Gray dashed line , mean of control ; gray box , control standard deviation ( ± 18 . 2 % ) ; red line , threshold of positive hits ; red box : positive hits . B . Enrichment Analysis of the positive hits ; for each class of inhibitor , the enrichment ( in percentage ) of compounds falling into positive hits and the enrichment ( in percentage ) in the total number of compounds was calculated and expressed as a ratio . C . Evaluation of CDK kinase activity upon SA treatment . Western blot analysis of phosphoSer - CDK substrates in control , SA ( 300µM , 30 min ) , Dinaciclib ( 10 µM , 180 min ) and SA + Dinaciclib ( 150 min Dinaciclib and 30 min SA ) . CDKs are hyperactivated upon oxidative stress and this activation is partially prevented upon CDK inhibition . Left , Western blot , right : Ponceau was used as loading control . Image , one representative experiment out of 3 independent replicates . D . Drug specificity from high ( + + + + ) to low ( + ) of the different CDKs as described by Selleckchem , and Sec24C recruitment to SGs as a percentage of control . E . Analysis of Sec24C recruitment to SGs in control and CDK1 + 2 - KD HeLa cells . Different levels of CDK1 + 2 KD were achieved by transfecting cells with different amounts of siRNA for different times ( 2 nM 24 h ; 5 nM 24 h ; 5 nM 48 h ; 20 nM 48 h ) to give 50 , 30 , 20 and 10 % , respectively , of the CDK1 + 2 levels in the control . CDK1 + 2 levels measured by qRT - PCR and Sec24C recruitment to SGs were evaluated from parallel cultures . Sec24C recruitment to SGs was performed by automated microscopy as described in ( A ) and is expressed as a percentage of mock ( CTRL , set at 100 % ) ± s . d . N = 8 . F . Representative images of HeLa cells treated with the three best hits ( Flavopiridol , SNS - 032 , Dinaciclib ; 1µM 150 min ) and then exposed to SA ( 300 µM , 30 min ) , followed by immunostaining for Sec24C and G3BP . Scale bar , 10 µm . G . HeLa cells were treated with Flavopiridol , SNS - 032 , or Dinaciclib for 150 min at the indicated concentrations , subsequently treated with SA ( 300 µM , 30 min ) , and then immunostained for Sec24C and G3BP and imaged by OPERA . BS - 181 , a specific CDK7 inhibitor , was used as negative control . Sec24C localization to SGs is expressed as a percentage of the control ( cells treated with SA alone ) . Dashed red line , mean of control ; yellow box , standard deviation of control ( ± 9 . 8 % ) , N = 12 . H . Cells were treated as in ( G ) and immunostained for TRAPPC2 and eIF3 . Scale bar , 10 µm . The graph shows the quantification of TRAPPC2 localization in SG puncta ( mean intensity ) . Data are mean ± s . e . m . expressed as a percentage of TRAPPC2 signal in SGs after Flavopiridol , SNS - 032 or Dinaciclib treatment compared to the control ( cells treated with SA alone ) . N = 3 , three independent experiments , n = 60 - 80 cells per experiment . * * * * p < 0 . 0001 , One - way ANOVA with Dunnett ' s multiple comparison test .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 4A. Cells were treated with 273 kinase inhibitors (10 µM) from the Sellekchem library and with SA, and Sec24C localization in G3BP puncta (% of total G3BP area) was analyzed by automated microscopy under High content screening and is reported as a percentage of the control (cells treated with SA alone). Gray dashed line, mean of control; gray box, control standard deviation (± 18.2%); red line, threshold of positive hits; red box: positive hits. B. Enrichment Analysis of the positive hits; for each class of inhibitor, the enrichment (in percentage) of compounds falling into positive hits and the enrichment (in percentage) in the total number of compounds was calculated and expressed as a ratio. C. Evaluation of CDK kinase activity upon SA treatment. Western blot analysis of phosphoSer-CDK substrates in control, SA (300µM, 30 min), Dinaciclib (10 µM, 180 min) and SA+Dinaciclib (150 min Dinaciclib and 30 min SA). CDKs are hyperactivated upon oxidative stress and this activation is partially prevented upon CDK inhibition. Left, Western blot, right: Ponceau was used as loading control. Image, one representative experiment out of 3 independent replicates.D. Drug specificity from high (++++) to low (+) of the different CDKs as described by Selleckchem, and Sec24C recruitment to SGs as a percentage of control.E. Analysis of Sec24C recruitment to SGs in control and CDK1+2-KD HeLa cells. Different levels of CDK1+2 KD were achieved by transfecting cells with different amounts of siRNA for different times (2 nM 24 h; 5 nM 24 h; 5 nM 48 h; 20 nM 48 h) to give 50, 30, 20 and 10%, respectively, of the CDK1+2 levels in the control. CDK1+2 levels measured by qRT-PCR and Sec24C recruitment to SGs were evaluated from parallel cultures. Sec24C recruitment to SGs was performed by automated microscopy as described in (A) and is expressed as a percentage of mock (CTRL, set at 100%) ± s.d. N=8.F. Representative images of HeLa cells treated with the three best hits (Flavopiridol, SNS-032, Dinaciclib; 1µM 150 min) and then exposed to SA (300 µM, 30 min), followed by immunostaining for Sec24C and G3BP. Scale bar, 10 µm.G. HeLa cells were treated with Flavopiridol, SNS-032, or Dinaciclib for 150 min at the indicated concentrations, subsequently treated with SA (300 µM, 30 min), and then immunostained for Sec24C and G3BP and imaged by OPERA. BS-181, a specific CDK7 inhibitor, was used as negative control. Sec24C localization to SGs is expressed as a percentage of the control (cells treated with SA alone). Dashed red line, mean of control; yellow box, standard deviation of control (± 9.8%), N=12.H. Cells were treated as in (G) and immunostained for TRAPPC2 and eIF3. Scale bar, 10 µm. The graph shows the quantification of TRAPPC2 localization in SG puncta (mean intensity). Data are mean ± s.e.m. expressed as a percentage of TRAPPC2 signal in SGs after Flavopiridol, SNS-032 or Dinaciclib treatment compared to the control (cells treated with SA alone). N=3, three independent experiments, n=60-80 cells per experiment. ****p<0.0001, One-way ANOVA with Dunnett's multiple comparison test.


Figure 5A . Immunofluorescence images of Sec24C and TRAPPC2 in vehicle - treated and SNS - 032 - treated ( 1 µM , 3 h ) cells . Graphs show the quantification of Sec24C and TRAPPC2 at ERES ( mean intensity ) normalized in SNS - 032 - treated cells relative to vehicle - treated cells ( set as 100 % ) . Mean ± s . e . m . of three independent experiments . * * p < 0 . 05 , * * * * p < 0 . 0001 , Student ' s unpaired two - tailed t - test . Data information : Scale bars , 10 µm . B . HeLa cells were treated with Dinaciclib ( 1 µM , 3 h ) and permeabilized or not with digitonin A GM130 antibody was added to the buffer of living cells to monitor permeabilization efficiency . Upper panels , non - permeabilized ( NP ) control cells ; middle panels , permeabilized control cells ; lower panels , permeabilized Dinaciclib - treated cells . Data information : Scale bars , 10 µm . C . Quantification of Sec24C membrane association after CDK inhibitor treatment . Digitonin - permeabilized cells treated with vehicle ( CTRL ) or the indicated CDK inhibitor ( 1 µM , 3 h ) were immunostained for Sec24C . The mean intensity of Sec24C in the perinuclear area normalized for the cytosolic Sec24C signal in drug - treated cells is expressed as fold change compared to the control ; n = 60 - 80 ; mean ± s . e . m . of three independent experiments * * * * p < 0 . 0001 , One - way ANOVA with Dunnett ' s multiple comparison test . D . HeLa cells transfected with HA - hnRNPK were left untreated ( Steady state ) or treated with SA ( 300µM , 30 min ) or SA + Dinaciclib ( 10 µM , 150 min ) and then immunostained with anti - HA and anti - G3BP antibodies . Data information Scale bars , 10 µm . E . TRAPPC2 interacts with hnRNPK . ( Top ) SDS - PAGE and Western blot analysis of endogenous hnRNPK after immunoprecipitation with an anti - TRAPPC2 Ab or IgG , and ( bottom ) after immunoprecipitation with an anti - HA Ab from cells transfected ( + ) or not ( - ) with hnRNPK - HA detected with the indicated antibodies . F . Mock or hnRNPK KD cells treated with SA were immunostained for TRAPPC2 and eIF3 . Data information : Scale bars , 10 µm . G . Mock or hnRNPK KD cells treated with SA were immunostained for Sec24C and G3BP . Data information : Scale bars , 10 µm . H . Graphs show quantification of TRAPPC2 and Sec24C redistribution to SGs in mock treated ( CTRL ) and hnRNPK KD cells . n > 100 , N = 3 mean ± s . d . * * * p < 0 . 001 . * * * * p < 0 . 0001 , Student ' s unpaired two - tailed t - test .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 5A. Immunofluorescence images of Sec24C and TRAPPC2 in vehicle-treated and SNS-032-treated (1 µM, 3 h) cells. Graphs show the quantification of Sec24C and TRAPPC2 at ERES (mean intensity) normalized in SNS-032-treated cells relative to vehicle-treated cells (set as 100%). Mean ± s.e.m. of three independent experiments. **p<0.05, ****p<0.0001, Student's unpaired two-tailed t-test. Data information: Scale bars, 10 µm.B. HeLa cells were treated with Dinaciclib (1 µM, 3 h) and permeabilized or not with digitonin A GM130 antibody was added to the buffer of living cells to monitor permeabilization efficiency. Upper panels, non-permeabilized (NP) control cells; middle panels, permeabilized control cells; lower panels, permeabilized Dinaciclib-treated cells. Data information: Scale bars, 10 µm.C. Quantification of Sec24C membrane association after CDK inhibitor treatment. Digitonin-permeabilized cells treated with vehicle (CTRL) or the indicated CDK inhibitor (1 µM, 3 h) were immunostained for Sec24C. The mean intensity of Sec24C in the perinuclear area normalized for the cytosolic Sec24C signal in drug-treated cells is expressed as fold change compared to the control; n= 60-80; mean ± s.e.m. of three independent experiments ****p<0.0001, One-way ANOVA with Dunnett's multiple comparison test.D. HeLa cells transfected with HA-hnRNPK were left untreated (Steady state) or treated with SA (300µM, 30 min) or SA+Dinaciclib (10 µM, 150 min) and then immunostained with anti-HA and anti-G3BP antibodies. Data information Scale bars, 10 µm.E. TRAPPC2 interacts with hnRNPK. (Top) SDS-PAGE and Western blot analysis of endogenous hnRNPK after immunoprecipitation with an anti-TRAPPC2 Ab or IgG, and (bottom) after immunoprecipitation with an anti-HA Ab from cells transfected (+) or not (-) with hnRNPK-HA detected with the indicated antibodies.F. Mock or hnRNPK KD cells treated with SA were immunostained for TRAPPC2 and eIF3. Data information: Scale bars, 10 µm.G. Mock or hnRNPK KD cells treated with SA were immunostained for Sec24C and G3BP. Data information: Scale bars, 10 µm.H. Graphs show quantification of TRAPPC2 and Sec24C redistribution to SGs in mock treated (CTRL) and hnRNPK KD cells. n>100, N=3 mean ± s.d. ***p<0.001.****p<0.0001, Student's unpaired two-tailed t-test.


Figure 6A . HeLa cells were starved for 8 h with HBSS and subsequently exposed to SA ( 30 min , 300 µM ) , and immunostained for Sec24C or TRAPPC2 and eIF3 . Scale bar 10 µm . B . Analysis of the proliferation status of the cells after 8 h starvation in HBSS . Top , representative images of starved and non - starved ( CTRL ) cells using EdU incorporation ( see Materials and Methods ) . Scale bar 100 µm . Bottom , quantification of EdU incorporation in starved cells as a percentage of incorporation in control fed cells , N = 3 , mean ± s . d . C . HeLa cells were seeded at different confluency , treated with SA and stained for Sec24C and G3BP as an SG marker . Scale bar , 10 µm . Flow cytometry ( FACS ) analysis ( right panels ) was performed to evaluate the distribution of cell cycle phases in HeLa cell populations seeded at different confluency . The graph shows quantification of Sec24C mean intensity in SG puncta ( normalized for the cytosolic Sec24C ) at the indicated cell confluency . Mean ± s . e . m . of a representative experiment out of 5 biological replicates . n = 50 - 80 . ns : not significant ; * * * * p < 0 . 0001 , One - way ANOVA with Dunnett ' s multiple comparison test . D . Differentiation of podocytes . Western blot of growing and differentiated podocytes . Synaptopodin ( SYNPO ) was used as a differentiation marker , GAPDH as a loading control . E . Growing and differentiated podocytes were treated with SA ( 300 µM , 30 min ) and stained for TRAPPC2 or Sec24C and eIF3 . Scale bar , 10 µm . The graphs show quantification of TRAPPC2 and Sec24C ( mean intensity ) in SGs . Mean ± s . e . m . of three independents experiments . * * * * p < 0 . 0001 , Student ' s unpaired two - tailed t - test . F . Western blot analysis of total ( RB ) and phosphorylated ( p - RB ) retinoblastoma in growing ( non - differentiated ) versus differentiated podocytes . ß - actin was used as loading control . G . CDK kinase activity in growing and differentiated podocytes . Western blot using a specific antibody recognizing phosphoSer - CDK substrates was used on total cell lysates .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A. HeLa cells were starved for 8 h with HBSS and subsequently exposed to SA (30 min, 300 µM), and immunostained for Sec24C or TRAPPC2 and eIF3. Scale bar 10 µm.B. Analysis of the proliferation status of the cells after 8 h starvation in HBSS. Top, representative images of starved and non-starved (CTRL) cells using EdU incorporation (see Materials and Methods). Scale bar 100 µm. Bottom, quantification of EdU incorporation in starved cells as a percentage of incorporation in control fed cells, N=3, mean ± s.d.C. HeLa cells were seeded at different confluency, treated with SA and stained for Sec24C and G3BP as an SG marker. Scale bar, 10 µm. Flow cytometry (FACS) analysis (right panels) was performed to evaluate the distribution of cell cycle phases in HeLa cell populations seeded at different confluency. The graph shows quantification of Sec24C mean intensity in SG puncta (normalized for the cytosolic Sec24C) at the indicated cell confluency. Mean ± s.e.m. of a representative experiment out of 5 biological replicates. n= 50-80. ns: not significant; ****p<0.0001, One-way ANOVA with Dunnett's multiple comparison test.D. Differentiation of podocytes. Western blot of growing and differentiated podocytes. Synaptopodin (SYNPO) was used as a differentiation marker, GAPDH as a loading control.E. Growing and differentiated podocytes were treated with SA (300 µM, 30 min) and stained for TRAPPC2 or Sec24C and eIF3. Scale bar, 10 µm. The graphs show quantification of TRAPPC2 and Sec24C (mean intensity) in SGs. Mean ± s.e.m. of three independents experiments. ****p<0.0001, Student's unpaired two-tailed t-test.F. Western blot analysis of total (RB) and phosphorylated (p-RB) retinoblastoma in growing (non-differentiated) versus differentiated podocytes. ß-actin was used as loading control.G. CDK kinase activity in growing and differentiated podocytes. Western blot using a specific antibody recognizing phosphoSer-CDK substrates was used on total cell lysates.


Figure 7A . SG area in mock , TRAPPC2 TRAPPC3 , TRAPPC8 , and TRAPPC9 - depleted cells treated with SA . Data are shown in box - and - whisker plots : Box - plot central line , median ; box limits , upper and lower quartiles ; whiskers show the minimum to maximum . N = 3 . * * * p < 0 . 001 , * * * * p < 0 . 0001 , One - way ANOVA with Dunnett ' s multiple comparison test . B . HeLa cells microinjected with control IgG or a TRAPPC3 - specific antibody ( right panels in green ) were treated with SA . The TRAPPC3 Ab disrupts the Golgi ( Yu et al , 2006 ) , monitored using an anti - TGN46 Ab . Anti - G3BP was used to stain SGs . The graph shows quantification of the SG area . Mean ± s . e . m . three independent experiments ; n > 80 . * * * * p < 0 . 0001 , Student ' s unpaired two - tailed t - test . Scale bar , 10 µm . C . Structured Illumination Microscopy ( SIM ) - Super resolution ( SR ) images of endogenous TRAPPC2 and GFP - Sec23 localizing at SGs , stained for G3BP . Scale bar 5 µm . Right , magnification of boxed area . Scale bar , 0 . 5 µm . Localization of Raptor ( D ) in mock , TRAPPC3 - KD and TRAPPC2 - KD HeLa cells treated with SA . G3BP was used to stain SGs . Scale bar , 10 µm . Each graph shows the quantification ( mean intensity ) of the respective protein in SG spots expressed as a percentage of the mock ( TRL ) . Mean ± S . D . three independent replicates . * p < 0 . 02 ; * * p < 0 . 009 in ( D ) , One - way ANOVA with Dunnett ' s multiple comparison test . Localization of RACK1 ( E ) in mock , TRAPPC3 - KD and TRAPPC2 - KD HeLa cells treated with SA . G3BP was used to stain SGs . Scale bar , 10 µm . Each graph shows the quantification ( mean intensity ) of the respective protein in SG spots expressed as a percentage of the mock ( TRL ) . Mean ± S . D . independent replicates . , * p < 0 . 05 ; * * * * p < 0 . 0001 in ( E ) , One - way ANOVA with Dunnett ' s multiple comparison test . Immunoprecipitation ( IP ) with an anti - TRAPPC2 Ab or IgG from cells with or without SA treatment , as indicated , followed by SDS - PAGE and Western blot with ant - Raptor ( F ) Abs . Input : cell lysate ( 40 µg for TRAPPC2 , 10 µg for Raptor ) . Immunoprecipitation ( IP ) with an anti - TRAPPC2 Ab or IgG from cells with or without SA treatment , as indicated , followed by SDS - PAGE and Western blot with anti - RACK1 ( G ) Abs . Input : cell lysate ( 40 µg for TRAPPC2 , 10 µg for RACK1Localization of Raptor ( H ) untreated cells or cells pre - treated with the indicated CDK inhibitor ( 1 µM , 150 min ) and then with SA ( 300 µM , 30 min ) . G3BP was used to stain SGs . Scale bar , 10 µm . Graphs show quantification of the localization of the respective protein with SGs , expressed as a percentage of the control . Mean ± s . e . m . of one representative experiment out of three independent replicates , n = 60 - 80 . * * * * p < 0 . 0001 , One - way ANOVA with Dunnett ' s multiple comparison test . Localization of RACK1 ( I ) in untreated cells or cells pre - treated with the indicated CDK inhibitor ( 1 µM , 150 min ) and then with SA ( 300 µM , 30 min ) . G3BP was used to stain SGs . Scale bar , 10 µm . Graphs show quantification of the localization of the respective protein with SGs , expressed as a percentage of the control . Mean ± s . e . m . of one representative experiment out of three independent replicates , n = 60 - 80 . * * * * p < 0 . 0001 , One - way ANOVA with Dunnett ' s multiple comparison test . J . Analysis of cell death after overnight recovery of HeLa cells treated or untreated with CDKi ( SNS - 032 ; Flavopiridol or Dinaciclib , 1 µM , 150 min ) and then treated or not with SA ( 500 µM , 3 h ) . Images were acquired automatically by OPERETTA microscope . Values indicate the percentage of the total number of nuclei ( stained with DAPI ) positive for BoBo - 3 staining . Mean ± s . d . of one representative experiment out of three independent replicates . ns : not significant , * * * * p < 0 . 0001 , One - way ANOVA with Dunnett ' s multiple comparison test . K . Analysis of cell death in HeLa cells that were left untreated , treated with non - targeting siRNAs or siRNAs against TRAPPC2 , and in two cell clones knocked out for TRAPPC2 , with and without SA treatment . Mean ± s . d . of three independent replicates .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7A. SG area in mock, TRAPPC2 TRAPPC3, TRAPPC8, and TRAPPC9-depleted cells treated with SA. Data are shown in box-and-whisker plots: Box-plot central line, median; box limits, upper and lower quartiles; whiskers show the minimum to maximum. N=3. ***p<0.001, ****p<0.0001, One-way ANOVA with Dunnett's multiple comparison test.B. HeLa cells microinjected with control IgG or a TRAPPC3-specific antibody (right panels in green) were treated with SA. The TRAPPC3 Ab disrupts the Golgi (Yu et al, 2006), monitored using an anti-TGN46 Ab. Anti-G3BP was used to stain SGs. The graph shows quantification of the SG area. Mean ± s.e.m. three independent experiments; n>80. ****p<0.0001, Student's unpaired two-tailed t-test. Scale bar, 10 µm.C. Structured Illumination Microscopy (SIM)-Super resolution (SR) images of endogenous TRAPPC2 and GFP-Sec23 localizing at SGs, stained for G3BP. Scale bar 5 µm. Right, magnification of boxed area. Scale bar, 0.5 µm.Localization of Raptor (D) in mock, TRAPPC3-KD and TRAPPC2-KD HeLa cells treated with SA. G3BP was used to stain SGs. Scale bar, 10 µm. Each graph shows the quantification (mean intensity) of the respective protein in SG spots expressed as a percentage of the mock (TRL). Mean ± S.D. three independent replicates. *p<0.02; **p<0.009 in (D) , One-way ANOVA with Dunnett's multiple comparison test.Localization of RACK1 (E) in mock, TRAPPC3-KD and TRAPPC2-KD HeLa cells treated with SA. G3BP was used to stain SGs. Scale bar, 10 µm. Each graph shows the quantification (mean intensity) of the respective protein in SG spots expressed as a percentage of the mock (TRL). Mean ± S.D. independent replicates. , *p<0.05; ****p<0.0001 in (E), One-way ANOVA with Dunnett's multiple comparison test.Immunoprecipitation (IP) with an anti-TRAPPC2 Ab or IgG from cells with or without SA treatment, as indicated, followed by SDS-PAGE and Western blot with ant-Raptor (F) Abs. Input: cell lysate (40 µg for TRAPPC2, 10 µg for Raptor).Immunoprecipitation (IP) with an anti-TRAPPC2 Ab or IgG from cells with or without SA treatment, as indicated, followed by SDS-PAGE and Western blot with anti-RACK1 (G) Abs. Input: cell lysate (40 µg for TRAPPC2, 10 µg for RACK1Localization of Raptor (H) untreated cells or cells pre-treated with the indicated CDK inhibitor (1 µM, 150 min) and then with SA (300 µM, 30 min). G3BP was used to stain SGs. Scale bar, 10 µm. Graphs show quantification of the localization of the respective protein with SGs, expressed as a percentage of the control. Mean ± s.e.m. of one representative experiment out of three independent replicates, n=60-80. ****p<0.0001, One-way ANOVA with Dunnett's multiple comparison test.Localization of RACK1 (I) in untreated cells or cells pre-treated with the indicated CDK inhibitor (1 µM, 150 min) and then with SA (300 µM, 30 min). G3BP was used to stain SGs. Scale bar, 10 µm. Graphs show quantification of the localization of the respective protein with SGs, expressed as a percentage of the control. Mean ± s.e.m. of one representative experiment out of three independent replicates, n=60-80. ****p<0.0001, One-way ANOVA with Dunnett's multiple comparison test.J. Analysis of cell death after overnight recovery of HeLa cells treated or untreated with CDKi (SNS-032; Flavopiridol or Dinaciclib, 1 µM, 150 min) and then treated or not with SA (500 µM, 3 h). Images were acquired automatically by OPERETTA microscope. Values indicate the percentage of the total number of nuclei (stained with DAPI) positive for BoBo-3 staining. Mean ± s.d. of one representative experiment out of three independent replicates. ns: not significant, ****p<0.0001, One-way ANOVA with Dunnett's multiple comparison test.K. Analysis of cell death in HeLa cells that were left untreated, treated with non-targeting siRNAs or siRNAs against TRAPPC2, and in two cell clones knocked out for TRAPPC2, with and without SA treatment. Mean ± s.d. of three independent replicates.


Figure 8A . Left , control or SA - treated ( 500 µM , 120 min ) human fibroblasts ( HFs ) were incubated at 40 ° C for 180 min and stained for eIF3 , PCI , and Sec24C . Right , the cells were imaged 10 min after shifting the temperature from 40 ° C to 32 ° C and stained for eIF3 , PCI , and Giantin ( to label the Golgi ) . Data information Scale bars , 10 µm . B . HeLa cells were exposed to SA ( 200 µM ) for the indicated time , alone or in combination with ISRIB and SGs were analyzed . Quantification of SG area ( µm ) of three independent experiments . Mean ± s . e . m . n = 60 - 80 cells per experiment , N = 3 . C . Quantification of Sec24C localization at SGs over time ( the ratio between Sec24C ( mean fluorescence intensity ) in SG puncta and cytosolic Sec24C ) . Mean ± SD . n = 60 - 80 cells per experiment , N = 3 . D . HeLa cells expressing VSV - G - UVR8 mEOS were left untreated or treated with SA alone or in combination with ISRIB ( 1 µM ) ( 120 min ISRIB pre - treatment and 30 min SA + ISRIB ) and then pulsed with blue light . Images were taken 10 min after the UV pulse and processed for staining with the indicated markers . E . Cells were treated with SA for 30 min , the SA was washed out ( WO ) , and cells were left to recover for 120 min in the presence of cycloheximide , followed by a blue light pulse and processing for staining as described in ( D ) . F . Quantification of VSV - G ( mean intensity ) in the Golgi area to the total VSV - G per cell under the conditions described in ( D , E ) . Data are expressed as percentage of the control . Mean ± s . e . m . of three independent experiment . * * * * p < 0 . 0001 ; ns : not significant , One - way ANOVA with Dunnett ' s multiple comparison test . Data information : Scale bars , 10 µm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 8A. Left, control or SA-treated (500 µM, 120 min) human fibroblasts (HFs) were incubated at 40°C for 180 min and stained for eIF3, PCI, and Sec24C. Right, the cells were imaged 10 min after shifting the temperature from 40°C to 32°C and stained for eIF3, PCI, and Giantin (to label the Golgi). Data information Scale bars, 10 µm.B. HeLa cells were exposed to SA (200 µM) for the indicated time, alone or in combination with ISRIB and SGs were analyzed. Quantification of SG area (µm) of three independent experiments. Mean ± s.e.m. n = 60-80 cells per experiment, N = 3.C. Quantification of Sec24C localization at SGs over time (the ratio between Sec24C (mean fluorescence intensity) in SG puncta and cytosolic Sec24C). Mean ± SD. n = 60-80 cells per experiment, N = 3.D. HeLa cells expressing VSV-G-UVR8 mEOS were left untreated or treated with SA alone or in combination with ISRIB (1 µM) (120 min ISRIB pre-treatment and 30 min SA+ISRIB) and then pulsed with blue light. Images were taken 10 min after the UV pulse and processed for staining with the indicated markers. E. Cells were treated with SA for 30 min, the SA was washed out (WO), and cells were left to recover for 120 min in the presence of cycloheximide, followed by a blue light pulse and processing for staining as described in (D). F. Quantification of VSV-G (mean intensity) in the Golgi area to the total VSV-G per cell under the conditions described in (D,E). Data are expressed as percentage of the control. Mean ± s.e.m. of three independent experiment. ****p<0.0001; ns: not significant, One-way ANOVA with Dunnett's multiple comparison test. Data information: Scale bars, 10 µm.


Figure 9A . AiryScan images of HeLa cells treated with SA for the indicated times and stained with GM130 ( gray ) . Scale bar , 10 µm . The graph shows quantification of Golgi objects in SA - treated cells . Mean ± s . e . m . of three independent experiments . n = 90 - 100 . * * * * p < 0 . 0001 ; ns : not significant , Student ' s unpaired two - tailed t - test . B . Control cells and cells treated with Dinaciclib ( CDKi ) were treated with SA and stained for GM130 ( in gray ) . Insets , eIF3 . Blue , nuclear DAPI staining . The graph shows quantification of Golgi objects in HeLa cells untreated or treated with SNS - 032 , Flavopiridol , or Dinaciclib for 150 min and then treated with SA ( 300 µM , 30 min ) . Mean ± s . e . m . of three independent experiments . * * * * p < 0 . 0001 , One - way ANOVA with Dunnett ' s multiple comparison test . C . Quantification of Golgi objects in cells plated at different percentage of confluency and treated or untreated with SA . Mean ± s . e . m . of three independent experiments . * * p < 0 . 001 ; * * * p < 0 . 0002 ; ns : not significant , One - way ANOVA with Dunnett ' s multiple comparison test . D . Quantification of Golgi objects in cells pre - treated with ISRIB ( 1 µM ) for 3 h and then exposed to SA ( 30 min ) in the presence of ISRIB . * * * p < 0 . 002 , Student ' s unpaired two - tailed t - test . E . Electron microscopy images of cells treated ( 300 µM , 30 min ) or untreated with SA . The arrow indicates the Golgi complex . Scale bar , 200 nm . F . AiryScan images of HeLa cells at steady state or exposed to SA for the indicated times . A Rab1 - GTP specific antibody was used to monitor the pool of active Rab1 , and GM130 to stain the Golgi complex . Insets , eIF3 . Scale bar , 10 µm . The graph shows quantification of Rab1 - GTP at the Golgi complex expressed as a percentage of the control . Mean ± s . e . m . of three independent experiments . * p < 0 . 011 , * * p < 0 . 002 , Student ' s unpaired two - tailed t - test . G . HeLa cells transfected or not with WT GFP - Rab1B were left untreated or treated with SA and stained for GM130 and G3BP to monitor SGs . Dashed white line , WT GFP - Rab1B transfected cells . Scale bar , 10 µm . The graph shows quantification of Golgi objects in the different conditions . NT : non - transfected . Mean ± s . e . m . of three independent experiments . * * * * p < 0 . 0001 , Student ' s unpaired two - tailed t - test .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 9A. AiryScan images of HeLa cells treated with SA for the indicated times and stained with GM130 (gray). Scale bar, 10 µm. The graph shows quantification of Golgi objects in SA-treated cells. Mean ± s.e.m. of three independent experiments. n=90-100. ****p<0.0001; ns: not significant, Student's unpaired two-tailed t-test.B. Control cells and cells treated with Dinaciclib (CDKi) were treated with SA and stained for GM130 (in gray). Insets, eIF3. Blue, nuclear DAPI staining. The graph shows quantification of Golgi objects in HeLa cells untreated or treated with SNS-032, Flavopiridol, or Dinaciclib for 150 min and then treated with SA (300 µM, 30 min). Mean ± s.e.m. of three independent experiments. ****p<0.0001, One-way ANOVA with Dunnett's multiple comparison test.C. Quantification of Golgi objects in cells plated at different percentage of confluency and treated or untreated with SA. Mean ± s.e.m. of three independent experiments. **p<0.001; ***p<0.0002; ns: not significant, One-way ANOVA with Dunnett's multiple comparison test.D. Quantification of Golgi objects in cells pre-treated with ISRIB (1 µM) for 3 h and then exposed to SA (30 min) in the presence of ISRIB. ***p<0.002, Student's unpaired two-tailed t-test.E. Electron microscopy images of cells treated (300 µM, 30 min) or untreated with SA. The arrow indicates the Golgi complex. Scale bar, 200 nm.F. AiryScan images of HeLa cells at steady state or exposed to SA for the indicated times. A Rab1-GTP specific antibody was used to monitor the pool of active Rab1, and GM130 to stain the Golgi complex. Insets, eIF3. Scale bar, 10 µm. The graph shows quantification of Rab1-GTP at the Golgi complex expressed as a percentage of the control. Mean ± s.e.m. of three independent experiments. *p<0.011, **p<0.002, Student's unpaired two-tailed t-test.G. HeLa cells transfected or not with WT GFP-Rab1B were left untreated or treated with SA and stained for GM130 and G3BP to monitor SGs. Dashed white line, WT GFP-Rab1B transfected cells. Scale bar, 10 µm. The graph shows quantification of Golgi objects in the different conditions. NT: non-transfected. Mean ± s.e.m. of three independent experiments. ****p<0.0001, Student's unpaired two-tailed t-test.


Figure 1A Western blot analysis of the localization of endogenous PHGDH in PLC , HepG2 , and THLE3 cells . Lamin B1 , Tubulin , and Tom20 were used as markers for nuclear ( Nuc ) , cytosolic ( Cyto ) , and mitochondrial ( Mito ) proteins , respectively . B Western blot analysis of the localization of endogenous PHGDH in tumour tissues and adjacent normal tissues . Lamin B1 , GAPDH , and Tom20 were used as markers for Nuc , Cyto , and Mito proteins , respectively . C Western blot analysis of the localization of endogenous and exogenous Flag - tagged PHGDH in PLC cells . Lamin B1 , Tubulin , and Tom20 were used as markers for Nuc , Cyto , and Mito proteins , respectively . D Immunofluorescence images showing colocalization of Flag - PHGDH ( green ) and mitochondria ( red ) acquired with a laser confocal microscope system in PLC cells . Nuclei were stained with DAPI ( blue ) , and mitochondria were stained with MitoTracker Red . Scale bar , 10 μm ( left panel ) , 2 μm ( right panel ) . F Subcellular localization of PHGDH was determined by western blot in PLC cells overexpressing of Flag - tagged wild - type PHGDH , PHGDH ∆ SBD1 , or PHGDH ∆ RD mutants . WCL : whole - cell lysates , Mito : mitochondria . Tom20 was used as loading control . G Proteinase K protection assays were performed on purified mitochondria isolated from PLC and HepG2 cells . The outer mitochondrial membrane ( OMM ) protein Tom20 , the inner mitochondrial membrane ( IMM ) protein ANT2 , and the mitochondrial matrix ( MM ) protein TFAM were used as markers of the OMM , IMM , and MM , respectively . H Purified mitochondria of PLC or HepG2 cells were incubated with Na2CO3 ( pH 11 . 5 ) for 30 min at 4 ℃ . The soluble component ( Supernatant / Sup . ) and membrane component ( Pellet ) were separated by centrifugation prior to western blot analysis . COX2 , ATP8 , and ANT2 are markers for the membrane compotent ; CYTC and TFAM are markers for the soluble component .

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Figure 1A Western blot analysis of the localization of endogenous PHGDH in PLC, HepG2, and THLE3 cells. Lamin B1, Tubulin, and Tom20 were used as markers for nuclear (Nuc), cytosolic (Cyto), and mitochondrial (Mito) proteins, respectively.B Western blot analysis of the localization of endogenous PHGDH in tumour tissues and adjacent normal tissues. Lamin B1, GAPDH, and Tom20 were used as markers for Nuc, Cyto, and Mito proteins, respectively.C Western blot analysis of the localization of endogenous and exogenous Flag-tagged PHGDH in PLC cells. Lamin B1, Tubulin, and Tom20 were used as markers for Nuc, Cyto, and Mito proteins, respectively.D Immunofluorescence images showing colocalization of Flag-PHGDH (green) and mitochondria (red) acquired with a laser confocal microscope system in PLC cells. Nuclei were stained with DAPI (blue), and mitochondria were stained with MitoTracker Red. Scale bar, 10 μm (left panel), 2 μm (right panel).F Subcellular localization of PHGDH was determined by western blot in PLC cells overexpressing of Flag-tagged wild-type PHGDH, PHGDH∆SBD1, or PHGDH∆RD mutants. WCL: whole-cell lysates, Mito: mitochondria. Tom20 was used as loading control.G Proteinase K protection assays were performed on purified mitochondria isolated from PLC and HepG2 cells. The outer mitochondrial membrane (OMM) protein Tom20, the inner mitochondrial membrane (IMM) protein ANT2, and the mitochondrial matrix (MM) protein TFAM were used as markers of the OMM, IMM, and MM, respectively.H Purified mitochondria of PLC or HepG2 cells were incubated with Na2CO3 (pH 11.5) for 30 min at 4 ℃. The soluble component (Supernatant/Sup.) and membrane component (Pellet) were separated by centrifugation prior to western blot analysis. COX2, ATP8, and ANT2 are markers for the membrane compotent; CYTC and TFAM are markers for the soluble component.


Figure 2A The oxygen consumption rate ( OCR ) was measured by successive injections of oligomycin ( oligo ) , FCCP , and antimycin A / rotenone ( AA / Rot ) in PLC cells infected with viruses expressing nontargeting control ( NTC ) shRNA or shPHGDH . Basal respiration , ATP - linked respiration , and maximal respiration were analysed with the OCR curve . B Western blot analysis of mtDNA - encoded proteins in PLC cells stably expressing NTC or shPHGDH and empty vector ( EV ) or PLVX - PHGDH . β - Actin was used as the loading control . C PLC cells with endogenous PHGDH knockdown were further infected with viruses expressing EV or PLVX - PHGDH prior to western blot analysis of mtDNA - encoded proteins . β - Actin was used as the loading control . D PLC cells expressing NTC or shPHGDH were treated with 100 μg / ml cycloheximide ( CHX ) for 20 min prior to puromycin treatment for 15 min . Purified mitochondrial lysates were subjected to a SUnSET assay with an anti - puromycin antibody to measure the actively translated polypeptide chains . Western blot analysis of PHGDH confirmed the knockdown efficiency of the PHGDH shRNAs . β - Actin was used as the loading control . E SUnSET assays were performed in the indicated cell lines using whole - cell lysates . F PLC cells with or without PLVX - Flag - PHGDH overexpression were treated with DMSO or 50 μM chloramphenicol ( CAP ) in combination with 50 μM thiamphenicol ( TAP ) for 24 h prior to SUnSET assays . The assays were performed using whole - cell lysate . G PLC cells with or without PLVX - PHGDH overexpression were treated with DMSO or 50 μM CAP in combination with 50 μM TAP for 24 h prior to western blot analysis of mtDNA - encoded proteins . β - Actin was used as the loading control . H The translation efficiency of mtDNA - encoded proteins was determined by a SUnSET assay in PLC cells with endogenous PHGDH knockdown that were further infected with viruses expressing EV , wild - type PHGDH , or the catalytically inactive PHGDH mutant PHGDHR236E as indicated . β - Actin was used as the loading control . I mtDNA - encoded proteins were analysed by western blotting in PLC cells overexpressing EV , wild - type PHGDH , or PHGDHR236E . β - Actin was used as the loading control .

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Figure 2A The oxygen consumption rate (OCR) was measured by successive injections of oligomycin (oligo), FCCP, and antimycin A/rotenone (AA/Rot) in PLC cells infected with viruses expressing nontargeting control (NTC) shRNA or shPHGDH. Basal respiration, ATP-linked respiration, and maximal respiration were analysed with the OCR curve.B Western blot analysis of mtDNA-encoded proteins in PLC cells stably expressing NTC or shPHGDH and empty vector (EV) or PLVX-PHGDH. β-Actin was used as the loading control.C PLC cells with endogenous PHGDH knockdown were further infected with viruses expressing EV or PLVX-PHGDH prior to western blot analysis of mtDNA-encoded proteins. β-Actin was used as the loading control.D PLC cells expressing NTC or shPHGDH were treated with 100 μg/ml cycloheximide (CHX) for 20 min prior to puromycin treatment for 15 min. Purified mitochondrial lysates were subjected to a SUnSET assay with an anti-puromycin antibody to measure the actively translated polypeptide chains. Western blot analysis of PHGDH confirmed the knockdown efficiency of the PHGDH shRNAs. β-Actin was used as the loading control.E SUnSET assays were performed in the indicated cell lines using whole-cell lysates.F PLC cells with or without PLVX-Flag-PHGDH overexpression were treated with DMSO or 50 μM chloramphenicol (CAP) in combination with 50 μM thiamphenicol (TAP) for 24 h prior to SUnSET assays. The assays were performed using whole-cell lysate.G PLC cells with or without PLVX-PHGDH overexpression were treated with DMSO or 50 μM CAP in combination with 50 μM TAP for 24 h prior to western blot analysis of mtDNA-encoded proteins. β-Actin was used as the loading control.H The translation efficiency of mtDNA-encoded proteins was determined by a SUnSET assay in PLC cells with endogenous PHGDH knockdown that were further infected with viruses expressing EV, wild-type PHGDH, or the catalytically inactive PHGDH mutant PHGDHR236E as indicated. β-Actin was used as the loading control.I mtDNA-encoded proteins were analysed by western blotting in PLC cells overexpressing EV, wild-type PHGDH, or PHGDHR236E. β-Actin was used as the loading control.


Figure 3A HEK293T cells were transfected with Flag - tagged PHGDH in combination with HA - tagged EV , mtIF2 , mtIF3 , mtEFTu , mtEFG1 , mtEFG2 , or mtRRF . Co - immunoprecipitation ( Co - IP ) was performed with an anti - Flag antibody prior to western blot analysis . B HEK293T cells were transfected with HA - tagged PHGDHWT or PHGDHR236E in combination with Flag - EV or Flag - mtEFG2 , and Co - IP was performed with an anti - Flag antibody prior to western blot analysis . C HEK293T cells were transfected with HA - tagged PHGDHWT or PHGDHR236E in combination with Flag - EV or Flag - ANT2 , and Co - IP was performed with an anti - Flag antibody prior to western blot analysis . D HEK293T cells were transfected with HA - mtEFG2 in combination with Flag - EV or Flag - ANT2 , and Co - IP was performed with an anti - Flag antibody prior to western blot analysis . E GST pull - down of His - PHGDHWT and His - PHGDHR236E by GST - ANT2 using proteins purified in E . coli , followed by western blot analysis with anti - His and anti - GST antibodies . F GST pull - down of His - mtEFG2 by GST - ANT2 using proteins purified in E . coli , followed by western blot analysis with anti - His and anti - GST antibodies . G PLC cells stably expressing Flag - mtEFG2 and HA - PHGDH were further infected with viruses expressing NTC or shANT2 , and then cell lysates were immunoprecipitated with an anti - Flag antibody or IgG prior to western blot analysis . H PLC cells overexpressing PHGDH were further infected with viruses expressing NTC or shANT2 . The translation efficiency was measured by a SUnSET assay . I PLC cells overexpressing PLVX - PHGDH were further infected with viruses expressing NTC or shANT2 . The expression of mtDNA - encoded proteins was determined by western blotting . β - Actin was used as the loading control . PLC cells overexpressing PHGDH were further infected with viruses expressing NTC or shmtEFG2 . The translation efficiency was measured by a SUnSET assay . K PLC cells overexpressing pSin - 3xFlag - PHGDH were further infected with viruses expressing NTC or shmtEFG2 . The expression of mtDNA - encoded proteins was determined by western blotting . β - Actin was used as the loading control .

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Figure 3A HEK293T cells were transfected with Flag-tagged PHGDH in combination with HA-tagged EV, mtIF2, mtIF3, mtEFTu, mtEFG1, mtEFG2, or mtRRF. Co-immunoprecipitation (Co-IP) was performed with an anti-Flag antibody prior to western blot analysis.B HEK293T cells were transfected with HA-tagged PHGDHWT or PHGDHR236E in combination with Flag-EV or Flag-mtEFG2, and Co-IP was performed with an anti-Flag antibody prior to western blot analysis.C HEK293T cells were transfected with HA-tagged PHGDHWT or PHGDHR236E in combination with Flag-EV or Flag-ANT2, and Co-IP was performed with an anti-Flag antibody prior to western blot analysis.D HEK293T cells were transfected with HA-mtEFG2 in combination with Flag-EV or Flag-ANT2, and Co-IP was performed with an anti-Flag antibody prior to western blot analysis.E GST pull-down of His-PHGDHWT and His-PHGDHR236E by GST-ANT2 using proteins purified in E. coli, followed by western blot analysis with anti-His and anti-GST antibodies.F GST pull-down of His-mtEFG2 by GST-ANT2 using proteins purified in E. coli, followed by western blot analysis with anti-His and anti-GST antibodies.G PLC cells stably expressing Flag-mtEFG2 and HA-PHGDH were further infected with viruses expressing NTC or shANT2, and then cell lysates were immunoprecipitated with an anti-Flag antibody or IgG prior to western blot analysis.H PLC cells overexpressing PHGDH were further infected with viruses expressing NTC or shANT2. The translation efficiency was measured by a SUnSET assay.I PLC cells overexpressing PLVX-PHGDH were further infected with viruses expressing NTC or shANT2. The expression of mtDNA-encoded proteins was determined by western blotting. β-Actin was used as the loading control.PLC cells overexpressing PHGDH were further infected with viruses expressing NTC or shmtEFG2. The translation efficiency was measured by a SUnSET assay.K PLC cells overexpressing pSin-3xFlag-PHGDH were further infected with viruses expressing NTC or shmtEFG2. The expression of mtDNA-encoded proteins was determined by western blotting. β-Actin was used as the loading control.


Figure 4A HEK293T cells were transfected with HA - mtEFG2 in combination with Flag - EV or Flag - mtRRF , and Co - IP was performed with an anti - Flag antibody prior to western blot analysis . B PLC cells stably expressing Flag - mtEFG2 and HA - mtRRF were further infected with viruses expressing NTC or shPHGDH , and then cell lysates were immunoprecipitated with anti - Flag antibody or IgG prior to western blot analysis . C PLC cells stably overexpressing mtEFG2 were further infected with viruses expressing NTC or shPHGDH . The translation efficiency was measured by a SUnSET assay . D PLC cells stably overexpressing mtEFG2 were further infected with viruses expressing NTC or shPHGDH . mtDNA - encoded proteins were analysed by western blot . β - Actin was used as the loading control . E PLC cell lysates were immunoprecipitated with an anti - MRPL44 antibody or IgG prior to western blot analysis of MRPL44 , MRPL46 , MRPL48 , MRPS15 , MRPS16 , and MRPS35 . F Binding of mtDNA - encoded mRNA by endogenous MRPL44 ( representing mitochondrial ribosomes ) was evaluated by RNA immunoprecipitation ( RIP ) in PLC cells with PHGDH knockdown . G The same amount of mitochondria from PLC cells expressing NTC or shPHGDH treated with CAP and TAP were loaded onto the sucrose density gradient . After centrifugation , all fractions were subjected to western blot analysis of MRPS35 , MRPL44 , and MRPL48 proteins . RNA concentrations of ND6 were measured .

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Figure 4A HEK293T cells were transfected with HA-mtEFG2 in combination with Flag-EV or Flag-mtRRF, and Co-IP was performed with an anti-Flag antibody prior to western blot analysis.B PLC cells stably expressing Flag-mtEFG2 and HA-mtRRF were further infected with viruses expressing NTC or shPHGDH, and then cell lysates were immunoprecipitated with anti-Flag antibody or IgG prior to western blot analysis.C PLC cells stably overexpressing mtEFG2 were further infected with viruses expressing NTC or shPHGDH. The translation efficiency was measured by a SUnSET assay.D PLC cells stably overexpressing mtEFG2 were further infected with viruses expressing NTC or shPHGDH. mtDNA-encoded proteins were analysed by western blot. β-Actin was used as the loading control.E PLC cell lysates were immunoprecipitated with an anti-MRPL44 antibody or IgG prior to western blot analysis of MRPL44, MRPL46, MRPL48, MRPS15, MRPS16, and MRPS35.F Binding of mtDNA-encoded mRNA by endogenous MRPL44 (representing mitochondrial ribosomes) was evaluated by RNA immunoprecipitation (RIP) in PLC cells with PHGDH knockdown.G The same amount of mitochondria from PLC cells expressing NTC or shPHGDH treated with CAP and TAP were loaded onto the sucrose density gradient. After centrifugation, all fractions were subjected to western blot analysis of MRPS35, MRPL44, and MRPL48 proteins. RNA concentrations of ND6 were measured.


Figure 5A PLC cells overexpressing EV , PHGDHWT , or PHGDHR236E were further infected with viruses expressing NTC or shmtEFG2 . The OCR was measured after successive injections of oligomycin ( oligo ) , FCCP , and antimycin A / rotenone ( AA / Rot ) . Basal respiration , ATP - linked respiration , and maximal respiration were analysed with the OCR curve . B PLC cells overexpressing EV , PHGDHWT , or PHGDHR236E were further infected with viruses expressing NTC or shmtEFG2 prior to western blot analysis . β - Actin was used as the loading control ( left ) . Cell growth curves were constructed by cell counting with trypan blue exclusion ( right ) . C PLC cells overexpressing EV , PHGDHWT , or PHGDHR236E were further treated with H2O or tigecycline prior to western blot analysis . β - Actin was used as the loading control ( left ) . Cell growth curves were determined by cell counting with trypan blue exclusion ( right ) . D - F PLC cells overexpressing EV or PHGDH were further infected with viruses expressing NTC or shmtEFG2 . Then , these cells were subcutaneously injected into male nude mice ( n = 5 in each group ) . Tumour sizes were measured beginning 14 days after inoculation ( D ) . At the end of the experiment , the tumours were excised ( E ) , and PHGDH and mtEFG2 expression in tumour lysates was analysed by western blotting ( F ) . G - I PLC cells overexpressing EV or PHGDH were subcutaneously injected into male nude mice ( n = 5 in each group ) . Mice were intraperitoneally injected with 100 mg / kg tigecycline daily beginning 14 days after inoculation . Tumour sizes were measured beginning 14 days after inoculation ( G ) . At the end of the experiment , the tumours were excised ( H ) , and PHGDH expression in tumour lysates was analysed by western blotting ( I ) .

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Figure 5A PLC cells overexpressing EV, PHGDHWT, or PHGDHR236E were further infected with viruses expressing NTC or shmtEFG2. The OCR was measured after successive injections of oligomycin (oligo), FCCP, and antimycin A/rotenone (AA/Rot). Basal respiration, ATP-linked respiration, and maximal respiration were analysed with the OCR curve.B PLC cells overexpressing EV, PHGDHWT, or PHGDHR236E were further infected with viruses expressing NTC or shmtEFG2 prior to western blot analysis. β-Actin was used as the loading control (left). Cell growth curves were constructed by cell counting with trypan blue exclusion (right).C PLC cells overexpressing EV, PHGDHWT, or PHGDHR236E were further treated with H2O or tigecycline prior to western blot analysis. β-Actin was used as the loading control (left). Cell growth curves were determined by cell counting with trypan blue exclusion (right).D-F PLC cells overexpressing EV or PHGDH were further infected with viruses expressing NTC or shmtEFG2. Then, these cells were subcutaneously injected into male nude mice (n = 5 in each group). Tumour sizes were measured beginning 14 days after inoculation (D). At the end of the experiment, the tumours were excised (E), and PHGDH and mtEFG2 expression in tumour lysates was analysed by western blotting (F).G-I PLC cells overexpressing EV or PHGDH were subcutaneously injected into male nude mice (n = 5 in each group). Mice were intraperitoneally injected with 100 mg/kg tigecycline daily beginning 14 days after inoculation. Tumour sizes were measured beginning 14 days after inoculation (G). At the end of the experiment, the tumours were excised (H), and PHGDH expression in tumour lysates was analysed by western blotting (I).


Figure 1A HERC3 increases the protein levels of active YAP , p - YAP ( S381 ) , YAP and TAZ . active - YAP , p - YAP ( S381 ) , p - YAP ( S127 ) , YAP , p - TAZ ( S89 ) , TAZ , HERC3 and β - Actin were detected by Western blotting with appropriate antibodies as indicated in MDA - MB - 231 cells stably expressing HERC3 - WT or HERC3 - CA . Ctrl , Control ; WT , wildtype of HERC3 ; CA , HERC3 - CA , a catalytically inactive mutant of HERC3 . B HERC3 reverses the reduction in the active YAP and TAZ levels induced by serum starvation . MDA - MB - 231 cells stably expressing HERC3 or HERC3 - CA were cultured in medium with or without fetal bovine serum ( FBS ) for 1 h . Cells were harvested for Western blotting analysis with appropriate antibodies as indicated . Ctrl , Control ; WT , wildtype of HERC3 ; CA , HERC3 - CA , a catalytically inactive mutant of HERC3 . C Depletion of HERC3 decreases the steady - state levels of YAP / TAZ . Levels of active - YAP , p - YAP ( S381 ) , p - YAP ( S127 ) , YAP , p - TAZ ( S89 ) , TAZ , HERC3 and β - Actin were detected by Western blotting with appropriate antibodies as indicated in MDA - MB - 231 and SUM159 cells stably expressing shCtrl ( Control shRNA ) , sh - 1 and sh - 2 ( two shRNAs against HERC3 ) . E Depletion of HERC3 has no effects on mRNA levels of YAP / TAZ . Total mRNA levels of YAP / TAZ in control or HERC3 - deficient MDA - MB - 231 cells were analyzed by qRT - PCR using primers specific to YAP / TAZ . Data are shown as mean ± SEM ; n = 3 biological replicates . Statistical analysis was performed using two - tailed Student t test . n . s . , not significant . G Depletion of HERC3 attenuates YAP - or TAZ - induced 8xGTIIC - Luc reporter activity . HEK293T cells were transfected with expression plasmids for YAP or TAZ , shHERC3 or shCtrl , and reporter plasmids 8xGTIIC - Luc and Renilla - Luc as indicated . Relative luciferase activity was measured as described in the text . Western blots showing expression of indicated proteins were shown . H Depletion of HERC3 attenuates YAP - or TAZ - induced CTGF - Luc reporter activity . Cell transfection and luciferase assay were carried out as described in Panel G . Western blots showing expression of indicated proteins were shown . Data are shown as mean ± SEM ; n = 3 biological replicates . Statistical analysis was performed using two - tailed Student t test . * * P < 0 . 01 . I Depletion of HERC3 attenuates transcription of YAP / TAZ target genes . Total mRNA levels of CTGF and ANKRD1 in control or HERC3 - deficient MDA - MB - 231 cells were analyzed by qRT - PCR using primers specific to the indicated target gene . Data are shown as mean ± SEM ; n = 3 biological replicates . Statistical analysis was performed using two - tailed Student t test . * * * P < 0 . 001 .

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Figure 1A HERC3 increases the protein levels of active YAP, p-YAP (S381), YAP and TAZ. active-YAP, p-YAP (S381), p-YAP (S127), YAP, p-TAZ (S89), TAZ, HERC3 and β-Actin were detected by Western blotting with appropriate antibodies as indicated in MDA-MB-231 cells stably expressing HERC3-WT or HERC3-CA. Ctrl, Control; WT, wildtype of HERC3; CA, HERC3-CA, a catalytically inactive mutant of HERC3.B HERC3 reverses the reduction in the active YAP and TAZ levels induced by serum starvation. MDA-MB-231 cells stably expressing HERC3 or HERC3-CA were cultured in medium with or without fetal bovine serum (FBS) for 1 h. Cells were harvested for Western blotting analysis with appropriate antibodies as indicated. Ctrl, Control; WT, wildtype of HERC3; CA, HERC3-CA, a catalytically inactive mutant of HERC3.C Depletion of HERC3 decreases the steady-state levels of YAP/TAZ. Levels of active-YAP, p-YAP (S381), p-YAP (S127), YAP, p-TAZ (S89), TAZ, HERC3 and β-Actin were detected by Western blotting with appropriate antibodies as indicated in MDA-MB-231 and SUM159 cells stably expressing shCtrl (Control shRNA), sh-1 and sh-2 (two shRNAs against HERC3).E Depletion of HERC3 has no effects on mRNA levels of YAP/TAZ. Total mRNA levels of YAP/TAZ in control or HERC3-deficient MDA-MB-231 cells were analyzed by qRT-PCR using primers specific to YAP/TAZ. Data are shown as mean ±SEM; n = 3 biological replicates. Statistical analysis was performed using two-tailed Student t test. n.s., not significant.G Depletion of HERC3 attenuates YAP- or TAZ-induced 8xGTIIC-Luc reporter activity. HEK293T cells were transfected with expression plasmids for YAP or TAZ, shHERC3 or shCtrl, and reporter plasmids 8xGTIIC-Luc and Renilla-Luc as indicated. Relative luciferase activity was measured as described in the text. Western blots showing expression of indicated proteins were shown. H Depletion of HERC3 attenuates YAP- or TAZ-induced CTGF-Luc reporter activity. Cell transfection and luciferase assay were carried out as described in Panel G. Western blots showing expression of indicated proteins were shown. Data are shown as mean ±SEM; n = 3 biological replicates. Statistical analysis was performed using two-tailed Student t test. **P < 0.01.I Depletion of HERC3 attenuates transcription of YAP/TAZ target genes. Total mRNA levels of CTGF and ANKRD1 in control or HERC3-deficient MDA-MB-231 cells were analyzed by qRT-PCR using primers specific to the indicated target gene. Data are shown as mean ±SEM; n = 3 biological replicates. Statistical analysis was performed using two-tailed Student t test. ***P < 0.001.


Figure 2A HERC3 and HERC3 - CA block β - TrCP - mediated degradation of TAZ . HEK293T cells were transfected with expression plasmids for HA - β - TrCP , Myc - HERC3 or Myc - HERC3 - CA , FLAG - TAZ or FLAG - GFP as indicated . Cells were harvested 24 h post - transfection . Protein levels were detected by Western blotting with appropriate antibodies as indicated . D HERC3 and HERC3 - CA extend the half - life of TAZ . Control or HERC3 - deficient MDA - MB - 231 cells were treated with cycloheximide ( CHX , 20 µM ) for 0 , 4 , 8 or 12 h before harvested . Left , Western blotting with appropriate antibodies . Right , Image J quantitation of YAP intensity and normalized to β - Actin at the indicated time points ( n = 3 biological replicates ) . Data are represented as the mean ± SEM ( right panel ) . F Depletion of HERC3 decreases the half - life of YAP / TAZ . Control or HERC3 - deficient MDA - MB - 231 cells were treated with cycloheximide ( CHX , 20 µM ) for 0 , 2 , 4 or 6 h before harvested . Left , Western blotting with appropriate antibodies . Right , Image J quantitation of YAP intensity and normalized to β - Actin at the indicated time points ( n = 3 biological replicates ) . Data are represented as the mean ± SEM ( right panel ) . G Depletion of β - TrCP reverses shHERC3 - induced degradation of YAP / TAZ . Control or HERC3 - deficient MDA - MB - 231 were transfected with siCtrl or siβ - TrCP as indicated . Cells were harvested 48 h post - transfection . Protein levels were detected by Western blotting with appropriate antibodies as indicated .

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Figure 2A HERC3 and HERC3-CA block β-TrCP-mediated degradation of TAZ. HEK293T cells were transfected with expression plasmids for HA-β-TrCP, Myc-HERC3 or Myc-HERC3-CA, FLAG-TAZ or FLAG-GFP as indicated. Cells were harvested 24 h post-transfection. Protein levels were detected by Western blotting with appropriate antibodies as indicated.D HERC3 and HERC3-CA extend the half-life of TAZ. Control or HERC3-deficient MDA-MB-231 cells were treated with cycloheximide (CHX, 20 µM) for 0, 4, 8 or 12 h before harvested. Left, Western blotting with appropriate antibodies. Right, Image J quantitation of YAP intensity and normalized to β-Actin at the indicated time points (n = 3 biological replicates). Data are represented as the mean ± SEM (right panel).F Depletion of HERC3 decreases the half-life of YAP/TAZ. Control or HERC3-deficient MDA-MB-231 cells were treated with cycloheximide (CHX, 20 µM) for 0, 2, 4 or 6 h before harvested. Left, Western blotting with appropriate antibodies. Right, Image J quantitation of YAP intensity and normalized to β-Actin at the indicated time points (n = 3 biological replicates). Data are represented as the mean ± SEM (right panel).G Depletion of β-TrCP reverses shHERC3-induced degradation of YAP/TAZ. Control or HERC3-deficient MDA-MB-231 were transfected with siCtrl or siβ-TrCP as indicated. Cells were harvested 48 h post-transfection. Protein levels were detected by Western blotting with appropriate antibodies as indicated.


Figure 3A HERC3 interacts with β - TrCP , but not YAP or TAZ . HEK293T cells were transfected with expression plasmids for FLAG - HERC3 , HA - YAP or HA - TAZ or HA - β - TrCP . Cells were harvested 24 h post - transfection and subjected to IP with FLAG antibody - conjugated agarose beads . Protein levels were detected by Western blotting with HA and FLAG antibodies . B HERC3 interacts with endogenous β - TrCP . MDA - MB - 231 cells stably expressing HERC3 or HERC3 - CA were harvested and subjected to IP with IgG or HA antibodies . Protein levels were detected by Western blotting with β - TRCP , HA and β - Actin antibodies . CH HERC3 blocks the interaction between β - TrCP and YAP . HEK293T cells were transfected with expression plasmids for FLAG - YAP , HA - β - TRCP , Myc - HERC3 and Myc - HERC3 - CA as indicated . Cells were harvested 24 h post - transfection and subjected to IP with FLAG antibody - conjugated agarose beads . Protein levels were detected by Western blotting with HA , FLAG and Myc antibodies . L HERC3 displaces TAZ on β - TRCP . Increasing concentrations of purified HERC3 or HERC3 - CA proteins were added to the β - TrCP and TAZ complex , and followed by Western blotting analysis with indicated antibodies . The competitive binding assay in vitro was described in materials and methods .

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Figure 3A HERC3 interacts with β-TrCP, but not YAP or TAZ. HEK293T cells were transfected with expression plasmids for FLAG-HERC3, HA-YAP or HA-TAZ or HA-β-TrCP. Cells were harvested 24 h post-transfection and subjected to IP with FLAG antibody-conjugated agarose beads. Protein levels were detected by Western blotting with HA and FLAG antibodies. B HERC3 interacts with endogenous β-TrCP. MDA-MB-231 cells stably expressing HERC3 or HERC3-CA were harvested and subjected to IP with IgG or HA antibodies. Protein levels were detected by Western blotting with β-TRCP, HA and β-Actin antibodies. CH HERC3 blocks the interaction between β-TrCP and YAP. HEK293T cells were transfected with expression plasmids for FLAG-YAP, HA-β-TRCP, Myc-HERC3 and Myc-HERC3-CA as indicated. Cells were harvested 24 h post-transfection and subjected to IP with FLAG antibody-conjugated agarose beads. Protein levels were detected by Western blotting with HA, FLAG and Myc antibodies.L HERC3 displaces TAZ on β-TRCP. Increasing concentrations of purified HERC3 or HERC3-CA proteins were added to the β-TrCP and TAZ complex, and followed by Western blotting analysis with indicated antibodies. The competitive binding assay in vitro was described in materials and methods.


Figure 4A Depletion of HERC3 reduces cell proliferation . Control or HERC3 - deficient MDA - MB - 231 were analyzed for indicated days by CCK8 assay as described in the materials and methods . Data are shown as mean ± SEM ; n = 3 biological replicates . C Depletion of HERC3 inhibits colony formation in MDA - MB - 231 cells . Control or HERC3 - deficient MDA - MB - 231 were analyzed by colony formation assay . Quantification of the results in colony formation assays was analyzed by Image J . D Depletion of HERC3 inhibits colony formation in SUM159 cells . Control or HERC3 - deficient SUM159 were analyzed by colony formation assay . Quantification of the results in colony formation assays was analyzed by Image J . Depletion of HERC3 attenuates cell migration . Control or HERC3 - deficient MDA - MB - 231 cells ( upper panel ) and SUM159 cells ( lower panel ) were analyzed by transwell migration assay . E , migrated were fixed in 4 % PFA and stained with 0 . 1 % crystal violet and photographed . data quantification of migrated MDA - MB - 231 cells ( left panel ) and SUM159 ( right panel ) cells counts by Image J . H RNAi - resistant HERC3 and YAP / TAZ mutants reverse the inhibitory effect of HERC3 depletion on colony formation . Ectopic expression of RNAi - resistant HERC3 - WT , HERC3 - CA and YAP / TAZ mutants in HERC3 - deficient MDA - MB - 231 cells were subjected to colony formation assay as described in Panel C . I , J RNAi - resistant HERC3 and YAP / TAZ mutants reverse the inhibitory effect of HERC3 depletion on cell migration . I , ectopic expression of resistant variants of HERC3 - WT , HERC3 - CA and YAP / TAZ mutants in HERC3 - deficient MDA - MB - 231 cells were subjected to transwell migration assay . Scale bar , 100 μm . J , graphic representation of quantitated cell migration in Panel I .

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Figure 4A Depletion of HERC3 reduces cell proliferation. Control or HERC3-deficient MDA-MB-231 were analyzed for indicated days by CCK8 assay as described in the materials and methods. Data are shown as mean ± SEM; n = 3 biological replicates.C Depletion of HERC3 inhibits colony formation in MDA-MB-231 cells. Control or HERC3-deficient MDA-MB-231 were analyzed by colony formation assay. Quantification of the results in colony formation assays was analyzed by Image J. D Depletion of HERC3 inhibits colony formation in SUM159 cells. Control or HERC3-deficient SUM159 were analyzed by colony formation assay. Quantification of the results in colony formation assays was analyzed by Image J.Depletion of HERC3 attenuates cell migration. Control or HERC3-deficient MDA-MB-231 cells (upper panel) and SUM159 cells (lower panel) were analyzed by transwell migration assay. E, migrated were fixed in 4% PFA and stained with 0.1% crystal violet and photographed. data quantification of migrated MDA-MB-231 cells (left panel) and SUM159 (right panel) cells counts by Image J.H RNAi-resistant HERC3 and YAP/TAZ mutants reverse the inhibitory effect of HERC3 depletion on colony formation. Ectopic expression of RNAi-resistant HERC3-WT, HERC3-CA and YAP/TAZ mutants in HERC3-deficient MDA-MB-231 cells were subjected to colony formation assay as described in Panel C.I, J RNAi-resistant HERC3 and YAP/TAZ mutants reverse the inhibitory effect of HERC3 depletion on cell migration. I, ectopic expression of resistant variants of HERC3-WT, HERC3-CA and YAP/TAZ mutants in HERC3-deficient MDA-MB-231 cells were subjected to transwell migration assay. Scale bar, 100 μm. J, graphic representation of quantitated cell migration in Panel I.


Figure 5HERC3 knockdown inhibits breast tumorigenesis . Luciferase - harboring MDA - MB - 231 cells expressing shHERC3 or shCtrl were subcutaneously injected into 5 - week - old nude mice . Four weeks after injection , mice were monitored by bioluminescence using Xenogen IVIS imaging system . A , representative tumors expressing luciferase are indicated by radiance bar ( p / sec / cm2 / sr ) . B , quantitation of bioluminescence data . Data are shown as mean ± SEM ; n = 6 biological replicates . Statistical analysis was performed using two - tailed Student t test . * * P < 0 . 01 , * * * P < 0 . 001 . J , K shHERC3 attenuates breast cancer lung metastasis . Mice were injected with control or HERC3 - deficient MDA - MB - 231 cells . J , Bright - field images ( upper panel , scale bars , 2 mm ) and H & E staining ( lower panel , scale bars , 50 μm ) of the lungs from mice . K , number of lung metastasis nodules . Data are shown as mean ± SEM ; n = 5 biological replicates . Statistical analysis was performed using two - tailed Student t test . * * * P < 0 . 001 . RNAi - resistant HERC3 and unphosphorylatable YAP - S381A can counteract the tumor suppressive effect of HERC3 depletion . HERC3 - deficient cells with or without ectopic expression of YAP - S381A , RNAi - resistant variants of HERC3 - WT or HERC3 - CA and control MDA - MB - 231 cells were performed subcutaneously injection as described in Panel A . L , tumors were dissected and photographed at four weeks after cell implantation . M , volume from all tumors was recorded .

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Figure 5HERC3 knockdown inhibits breast tumorigenesis. Luciferase-harboring MDA-MB-231 cells expressing shHERC3 or shCtrl were subcutaneously injected into 5-week-old nude mice. Four weeks after injection, mice were monitored by bioluminescence using Xenogen IVIS imaging system. A, representative tumors expressing luciferase are indicated by radiance bar (p/sec/cm2/sr). B, quantitation of bioluminescence data. Data are shown as mean ±SEM; n = 6 biological replicates. Statistical analysis was performed using two-tailed Student t test. **P < 0.01, ***P < 0.001.J, K shHERC3 attenuates breast cancer lung metastasis. Mice were injected with control or HERC3-deficient MDA-MB-231 cells. J, Bright-field images (upper panel, scale bars, 2 mm) and H&E staining (lower panel, scale bars, 50 μm) of the lungs from mice. K, number of lung metastasis nodules. Data are shown as mean ±SEM; n = 5 biological replicates. Statistical analysis was performed using two-tailed Student t test. ***P < 0.001.RNAi-resistant HERC3 and unphosphorylatable YAP-S381A can counteract the tumor suppressive effect of HERC3 depletion. HERC3-deficient cells with or without ectopic expression of YAP-S381A, RNAi-resistant variants of HERC3-WT or HERC3-CA and control MDA-MB-231 cells were performed subcutaneously injection as described in Panel A. L, tumors were dissected and photographed at four weeks after cell implantation. M, volume from all tumors was recorded.


Figure 6HERC3 is highly expressed in breast cancer specimens . A , representative images of IHC staining of HERC3 in tumor samples of breast cancer specimens and their paired adjacent normal tissues . Scale bars , 50 µm . D HERC3 is correlated with poor prognosis in breast cancer patients . Kaplan - Meier curves for overall survival of breast cancer patients according to the HERC3 expression level ( upper left panel ) ( http : / / kmplot . com / analysis / index . php ? p = service & cancer = breast ) . HERC3 is negatively correlated with the overall survival ( upper right panel ) , the distant metastasis free survival ( lower left panel ) and the relapse free survival ( lower right panel ) in breast cancer patients . Microarray analysis was performed to examine the prognostic potential of HERC3 in breast cancer using the PrognoScan database with one cohort ( GSE7390 ) of 198 breast cancer specimens ( http : / / www . prognoscan . org ) . E HERC3 expression is positively correlated with TAZ or active YAP expression in breast cancer patients . Representative images of IHC staining of HERC3 , TAZ and active YAP in tumor samples of breast cancer

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Figure 6HERC3 is highly expressed in breast cancer specimens. A, representative images of IHC staining of HERC3 in tumor samples of breast cancer specimens and their paired adjacent normal tissues. Scale bars, 50 µm.D HERC3 is correlated with poor prognosis in breast cancer patients. Kaplan-Meier curves for overall survival of breast cancer patients according to the HERC3 expression level (upper left panel) (http://kmplot.com/analysis/index.php?p=service&cancer=breast). HERC3 is negatively correlated with the overall survival (upper right panel), the distant metastasis free survival (lower left panel) and the relapse free survival (lower right panel) in breast cancer patients. Microarray analysis was performed to examine the prognostic potential of HERC3 in breast cancer using the PrognoScan database with one cohort (GSE7390) of 198 breast cancer specimens (http://www.prognoscan.org). E HERC3 expression is positively correlated with TAZ or active YAP expression in breast cancer patients. Representative images of IHC staining of HERC3, TAZ and active YAP in tumor samples of breast cancer patients


Figure 1B . FPKM of the 7 LncRNAs identified in the RNA - seq with high expression in PGCs . The solid lines at each end of the box plot represent the maximum and minimum values , respectively . The three dotted lines in the middle of the violin diagram represent the 75 % percentile , the mean , and the 25 % percentile in turn .

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Figure 1B. FPKM of the 7 LncRNAs identified in the RNA-seq with high expression in PGCs. The solid lines at each end of the box plot represent the maximum and minimum values, respectively. The three dotted lines in the middle of the violin diagram represent the 75% percentile, the mean, and the 25% percentile in turn.


Figure 2A . Up : The schematic diagram of PGCs induced from chicken ESCs in vitro . Down : Morphological observation of the number of embryoid bodies in the in vitro PGCs induction model after LncBMP4overexpression and interference ; oeLncBMP4 indicates the overexpression of LncBMP4 ; shLncBMP4 indicates the interference of LncBMP4 ; BMP4 induction was regarded as the control . Scale bar : 60 µm ( n = 3 independent experiments ) . B . The expression of reproductive marker genes ( Cvh and C - kit ) , LncBMP4 , and BMP4 were detected by qRT - PCR after LncBMP4 overexpression and interference , in vitro ( Data are shown as mean ± SEM , n = 3 independent experiments , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , one - way ANOVA ) .

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Figure 2A. Up: The schematic diagram of PGCs induced from chicken ESCs in vitro. Down: Morphological observation of the number of embryoid bodies in the in vitro PGCs induction model after LncBMP4overexpression and interference; oeLncBMP4 indicates the overexpression of LncBMP4; shLncBMP4 indicates the interference of LncBMP4; BMP4 induction was regarded as the control. Scale bar: 60 µm (n = 3 independent experiments).B. The expression of reproductive marker genes (Cvh and C-kit), LncBMP4, and BMP4 were detected by qRT-PCR after LncBMP4 overexpression and interference, in vitro (Data are shown as mean ± SEM, n = 3 independent experiments, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, one-way ANOVA) .


Figure 3A . The results of IHC examining using anti - DDX4 antibody shows that the overexpression of LncBMP4 increases the number of PGCs in the genital ridge , and interference with LncBMP4 reduces the number of PGCs in the genital ridge . The number of PGCs was counted through the Segmentation module of Image J software . Scale bars : 200 μm ( top row ) , 40 μm ( bottom row ) ( Data are shown as mean ± SEM , n = 3 independent experiments , * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 , one - way ANOVA ) . B . The number of PGCs in the genital ridge , following the overexpression or interference of LncBMP4 , as determined by flow cytometry ( Data are shown as mean ± SEM , n = 3 independent experiments , * * * * p < 0 . 0001 , one - way ANOVA ) . C . The number of PGCs labeled with PkH26 ( red ) that migrated to the genital ridge in a frozen section , the number of PGCs labeled with PkH26 ( red ) was counted using Image J software . Scale bar : 70 µm ( Data are shown as mean ± SEM , n = 6 independent experiments , * * p < 0 . 01 , * * * * p < 0 . 0001 , one - way ANOVA . ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 3A. The results of IHC examining using anti-DDX4 antibody shows that the overexpression of LncBMP4 increases the number of PGCs in the genital ridge, and interference with LncBMP4 reduces the number of PGCs in the genital ridge. The number of PGCs was counted through the Segmentation module of Image J software. Scale bars:200 μm (top row),40 μm (bottom row) (Data are shown as mean ± SEM, n = 3 independent experiments, * p < 0.05, ** p < 0.01, **** p < 0.0001, one-way ANOVA).B. The number of PGCs in the genital ridge, following the overexpression or interference of LncBMP4, as determined by flow cytometry (Data are shown as mean ± SEM, n = 3 independent experiments, **** p < 0.0001, one-way ANOVA).C. The number of PGCs labeled with PkH26 (red) that migrated to the genital ridge in a frozen section, the number of PGCs labeled with PkH26 (red) was counted using Image J software. Scale bar: 70 µm (Data are shown as mean ± SEM, n = 6 independent experiments, ** p < 0.01, **** p < 0.0001, one-way ANOVA.).


Figure 4D . The expression of reproductive marker genes ( Cvh and C - kit ) , LncBMP4 , and BMP4 were detected by qRT - PCR in the genital ridge after gga - mir - 12211 overexpression and interference in vitro ( Data are shown as mean ± SEM , n = 3 independent experiments , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , one - way ANOVA ) . E . The efficiency of PGC differentiation was detected by IF , 4 d after induction , using C - KIT and CVH antibodies . Scale bar : 60 µm ( n = 3 independent experiments ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4D. The expression of reproductive marker genes (Cvh and C-kit), LncBMP4, and BMP4 were detected by qRT-PCR in the genital ridge after gga-mir-12211 overexpression and interference in vitro (Data are shown as mean ± SEM, n =3 independent experiments, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, one-way ANOVA).E. The efficiency of PGC differentiation was detected by IF, 4 d after induction, using C-KIT and CVH antibodies. Scale bar: 60 µm (n = 3 independent experiments).