Figure 7Immunofluorescence staining of M1AP ( green ) and TEX11 ( red ) on the spermatocyte spreads of WT mice . Ratios of M1AP - TEX11 co - localizing foci to the M1AP total foci or TEX11 total foci at the indicated meiotic prophase substages . Each dot represents the ratio calculated from one nucleus . Immunofluorescence staining of M1AP ( green ) and DMC1 ( red ) on the spermatocyte spreads of WT mice . Ratios of M1AP - DMC1 co - localizing foci to the M1AP total foci or DMC1 total foci at the indicated meiotic prophase substages . Each dot represents the ratio calculated from one nucleus . Immunofluorescence staining of M1AP ( green ) and RPA2 ( red ) on the spermatocyte spreads of WT mice . Ratios of M1AP - RPA2 co - localizing foci to the M1AP total foci or RPA2 total foci at the indicated meiotic prophase substages . Each dot represents the ratio calculated from one nucleus . Immunofluorescence staining of TEX11 ( green ) and SYCP3 ( red ) on the spermatocyte spreads of control and M1apKI / KI testes . The mean number of TEX11 foci per cell in control and M1apKI / KI spermatocytes . Frequencies of nuclei with TEX11 foci detected at the PAR in spread early pachytene spermatocytes with touching XY chromosomes .

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 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 O O O 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 7Immunofluorescence staining of M1AP (green) and TEX11 (red) on the spermatocyte spreads of WT mice.   Ratios of M1AP-TEX11 co-localizing foci to the M1AP total foci or TEX11 total foci at the indicated meiotic prophase substages. Each dot represents the ratio calculated from one nucleus.  Immunofluorescence staining of M1AP (green) and DMC1 (red) on the spermatocyte spreads of WT mice.   Ratios of M1AP-DMC1 co-localizing foci to the M1AP total foci or DMC1 total foci at the indicated meiotic prophase substages. Each dot represents the ratio calculated from one nucleus. Immunofluorescence staining of M1AP (green) and RPA2 (red) on the spermatocyte spreads of WT mice.   Ratios of M1AP-RPA2 co-localizing foci to the M1AP total foci or RPA2 total foci at the indicated meiotic prophase substages. Each dot represents the ratio calculated from one nucleus.  Immunofluorescence staining of TEX11 (green) and SYCP3 (red) on the spermatocyte spreads of control and M1apKI/KI testes.   The mean number of TEX11 foci per cell in control and M1apKI/KI spermatocytes.   Frequencies of nuclei with TEX11 foci detected at the PAR in spread early pachytene spermatocytes with touching XY chromosomes.


Figure 1A . Western blot analysis of mTORC1 activity in C2C12 - Vevtor ( VC ) and Aster - C KO ( KO ) cells in complete medium ( CM ) and in response to nutrient starvation or stimulation with amino acids for 30 min . B and C . Statistical analysis of phosphorylated level of S6K ( B ) and 4E - BP1 ( C ) in C2C12 - Vector and Aster - C KO cells in CM , under nutrient starvation ( - AA ) and in response to stimulation with amino acids ( + AA ) using ImageJ software . Data are represented as mean ± SD ( n = 3 biological replicates ) . * p < 0 . 05 , * * p < 0 . 01 by Student ' s t test . D . Immunostaining of endogenous mTOR and LAMP1 in C2C12 - Vector and Aster - C KO cells under nutrient starvation ( - AA ) and in response to AA stimulation ( + AA ) . LAMP1 was immunostained as a lysosome marker . Arrows highlight the co - localization of mTOR with LAMP1 . Scale bar , 40 μm ( Row 1 ) and 10 μm ( Rows 2 , 3 , 4 ) . E . Statistical analysis of the Pearson ' s correlation coefficient of mTOR and LAMP1 in Figure 1F ( n = 12 - 15 cells per group ) . Data are represented as mean ± SD . * * * p < 0 . 001 by one way ANOVA . F . Western blot analysis of mTORC1 activity in Aster - C KO cells stably re - expressing exogenous GFP - Aster - C under nutrient starvation and AA stimulation . Aster - C KO cells were transfected with GFP - Aster - C and selected with G418 for 2 weeks . G . Immunostaining of endogenous mTOR and LAMP1 in C2C12 - Vector and Aster - C KO cells under cholesterol depletion by treatment with 0 . 5 % MCD for 2 hrs ( - CHOL ) or cholesterol ( 50 µM ) re - stimulation for 1 hr ( + CHOL ) . Arrows highlight the co - localization of mTOR with LAMP1 . Scale bar , 40 μm ( Row 1 ) and 10 μm ( Rows 2 , 3 , 4 ) . H . Western blot analysis of mTORC1 activity in C2C12 - Vector and Aster - C KO cells in CM , under starvation , cholesterol depletion by treatment with 0 . 5 % MCD for 2 hrs or cholesterol ( 50 µM ) re - stimulation for the indicated time .

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 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 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 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

Figure 1A. Western blot analysis of mTORC1 activity in C2C12-Vevtor (VC) and Aster-C KO (KO) cells in complete medium (CM) and in response to nutrient starvation or stimulation with amino acids for 30 min. B and C. Statistical analysis of phosphorylated level of S6K (B) and 4E-BP1 (C) in C2C12-Vector and Aster-C KO cells in CM, under nutrient starvation (-AA) and in response to stimulation with amino acids (+AA) using ImageJ software. Data are represented as mean ± SD (n=3 biological replicates). *p < 0.05, **p<0.01 by Student's t test.D. Immunostaining of endogenous mTOR and LAMP1 in C2C12-Vector and Aster-C KO cells under nutrient starvation (-AA) and in response to AA stimulation (+AA). LAMP1 was immunostained as a lysosome marker. Arrows highlight the co-localization of mTOR with LAMP1. Scale bar, 40 μm (Row 1) and 10 μm (Rows 2, 3, 4).E. Statistical analysis of the Pearson's correlation coefficient of mTOR and LAMP1 in Figure 1F (n=12-15 cells per group). Data are represented as mean ± SD. ***p<0.001 by one way ANOVA.F. Western blot analysis of mTORC1 activity in Aster-C KO cells stably re-expressing exogenous GFP-Aster-C under nutrient starvation and AA stimulation. Aster-C KO cells were transfected with GFP-Aster-C and selected with G418 for 2 weeks.G. Immunostaining of endogenous mTOR and LAMP1 in C2C12-Vector and Aster-C KO cells under cholesterol depletion by treatment with 0.5% MCD for 2 hrs (-CHOL) or cholesterol (50 µM) re-stimulation for 1 hr (+CHOL). Arrows highlight the co-localization of mTOR with LAMP1. Scale bar, 40 μm (Row 1) and 10 μm (Rows 2, 3, 4).H. Western blot analysis of mTORC1 activity in C2C12-Vector and Aster-C KO cells in CM, under starvation, cholesterol depletion by treatment with 0.5% MCD for 2 hrs or cholesterol (50 µM) re-stimulation for the indicated time.


Figure 2A . Confocal image analysis depicting the co - localization of Aster - C with the ER in C2C12 cells transiently expressing GFP - Aster - C and DsRed - ER under starvation and in response to AA stimulation . Scale bar , 5 μm . B . Subcellular fractionation analysis of mTOR , GATOR2 complex and Aster - C localization in HEK293T cells transiently expressing FLAG - Aster - C under starvation and in response to AA stimulation . LAMP1 , Sec63 and GAPDH were used as lysosomal ( Lyso ) , rough ER ( RER ) and cytosol ( Cyto ) protein markers , respectively . Microsomal fraction ( Micro ) was also used in the blot . C . Subcellular fractionation analysis of mTOR and GATOR2 complex in C2C12 - Vector and Aster - C KO cells under starvation and in response to AA stimulation . LAMP1 , Sec63 and GAPDH were used as lysosomal ( Lyso ) , rough ER ( RER ) and cytosol ( Cyto ) protein markers , respectively . D . Co - IP analysis of the interaction of Aster - C with exogenous mTOR in response to starvation and AA or insulin stimulation in HEK293T cells transiently expressing the indicated proteins . Anti - Myc antibody was used for immunoprecipitation . E . Co - IP analysis of the interaction of Aster - C with endogenous mTOR and the GATOR2 complex using FLAG - Sestrin2 ( SESN2 ) as a positive control in HEK293T cells transiently expressing the indicated proteins . Anti - FLAG antibody was used for immunoprecipitation . F . Co - IP analysis of the interaction of Aster - C with mTOR , the GATOR2 complex , GATOR1 complex and the Ragulator - Rag complex using FLAG - DEPDC5 as a positive control in HEK293T cells transiently expressing the indicated proteins . Anti - FLAG antibody was used for immunoprecipitation . Black arrow indicates the RagC protein .

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 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 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 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 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

Figure 2A. Confocal image analysis depicting the co-localization of Aster-C with the ER in C2C12 cells transiently expressing GFP-Aster-C and DsRed-ER under starvation and in response to AA stimulation. Scale bar, 5 μm.B. Subcellular fractionation analysis of mTOR, GATOR2 complex and Aster-C localization in HEK293T cells transiently expressing FLAG-Aster-C under starvation and in response to AA stimulation. LAMP1, Sec63 and GAPDH were used as lysosomal (Lyso), rough ER (RER) and cytosol (Cyto) protein markers, respectively. Microsomal fraction (Micro) was also used in the blot.C. Subcellular fractionation analysis of mTOR and GATOR2 complex in C2C12-Vector and Aster-C KO cells under starvation and in response to AA stimulation. LAMP1, Sec63 and GAPDH were used as lysosomal (Lyso), rough ER (RER) and cytosol (Cyto) protein markers, respectively.D. Co-IP analysis of the interaction of Aster-C with exogenous mTOR in response to starvation and AA or insulin stimulation in HEK293T cells transiently expressing the indicated proteins. Anti-Myc antibody was used for immunoprecipitation.E. Co-IP analysis of the interaction of Aster-C with endogenous mTOR and the GATOR2 complex using FLAG-Sestrin2 (SESN2) as a positive control in HEK293T cells transiently expressing the indicated proteins. Anti-FLAG antibody was used for immunoprecipitation.F. Co-IP analysis of the interaction of Aster-C with mTOR, the GATOR2 complex, GATOR1 complex and the Ragulator-Rag complex using FLAG-DEPDC5 as a positive control in HEK293T cells transiently expressing the indicated proteins. Anti-FLAG antibody was used for immunoprecipitation. Black arrow indicates the RagC protein.


Figure 3A . Co - IP analysis of the interaction of Aster - C with COPA and COPG in response to starvation and AA stimulation using FLAG - DEPDC5 as a negative control in HEK293T cells transiently expressing the indicated proteins . Anti - FLAG antibody was used for immunoprecipitation . Black arrow indicates the COPG protein . B . Co - IP analysis of the interaction of COPA with mTOR in HEK293T cells transiently expressing GFP - COPA in response to starvation and AA stimulation . Anti - GFP antibody was used for immunoprecipitation . Black arrow indicates the COPG protein . C . Co - IP analysis of the interaction of COPA with mTOR in C2C12 - Vector and Aster - C KO cells in response to starvation and AA stimulation . Anti - COPA antibody was used for immunoprecipitation . D . Confocal imaging analysis depicting the co - localization of YFP - mTOR ( yellow ) with GFP - COPA ( green ) and lysosomes ( red ) in live C2C12 - Vector and Aster - C KO cells in response to nutrient starvation and re - stimulation by AA . Arrows highlight co - localization of mTOR , COPA and lysosomes . Scale bar , 20 μm . E and F . Statistical analysis of the Pearson ' s correlation coefficient of mTOR / Lysosome ( E ) and COPA / Lysosome ( F ) in Figure 3D ( n = 10 - 12 cells per group ) . Data are represented as mean ± SD . * * * p < 0 . 001 by 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 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 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

Figure 3A. Co-IP analysis of the interaction of Aster-C with COPA and COPG in response to starvation and AA stimulation using FLAG-DEPDC5 as a negative control in HEK293T cells transiently expressing the indicated proteins. Anti-FLAG antibody was used for immunoprecipitation. Black arrow indicates the COPG protein.B. Co-IP analysis of the interaction of COPA with mTOR in HEK293T cells transiently expressing GFP-COPA in response to starvation and AA stimulation. Anti-GFP antibody was used for immunoprecipitation. Black arrow indicates the COPG protein.C. Co-IP analysis of the interaction of COPA with mTOR in C2C12-Vector and Aster-C KO cells in response to starvation and AA stimulation. Anti-COPA antibody was used for immunoprecipitation.D. Confocal imaging analysis depicting the co-localization of YFP-mTOR (yellow) with GFP-COPA (green) and lysosomes (red) in live C2C12-Vector and Aster-C KO cells in response to nutrient starvation and re-stimulation by AA. Arrows highlight co-localization of mTOR, COPA and lysosomes. Scale bar, 20 μm. E and F. Statistical analysis of the Pearson's correlation coefficient of mTOR/Lysosome (E) and COPA/Lysosome (F) in Figure 3D (n=10-12 cells per group). Data are represented as mean ± SD. ***p<0.001 by one-way ANOVA.


Figure 4Time - lapse confocal imaging analysis of the co - localization of YFP - mTOR ( red ) with GFP - COPA ( green ) and lysosomes ( blue ) in live C2C12 cells under nutrient starvation ( 0 min ) , and in response to AA re - stimulation for indicated time ( 0 . 5 min , 2 min , 6 min and 8 min ) . Arrows highlight COPA puncta that were co - localized with mTOR or COPA puncta alone prior to their association with mTOR and lysosomes . . Scale bar , 5 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 O O O O O O O O O O O O O O O

Figure 4Time-lapse confocal imaging analysis of the co-localization of YFP-mTOR (red) with GFP-COPA (green) and lysosomes (blue) in live C2C12 cells under nutrient starvation (0 min), and in response to AA re-stimulation for indicated time (0.5 min, 2 min, 6 min and 8 min). Arrows highlight COPA puncta that were co-localized with mTOR or COPA puncta alone prior to their association with mTOR and lysosomes.. Scale bar, 5 μm.


Figure 5A . Confocal imaging analysis depicting the lysosomal association of mTOR in response to AA stimulation in the presence or absence of BFA ( 10 μM ) or Exo - 2 ( 10 μM ) in live C2C12 cells transiently expressing YFP - mTOR and stained with Lysotracker Red . Scale bar , 20 μm . Western blot analysis of mTORC1 activity in C2C12 cells in CM , in response to nutrient starvation and AA stimulation in the presence or absence of BFA ( 10 μM ) ( B ) C . Western blot analysis of mTORC1 activity in C2C12 cells in CM , in response to nutrient starvation and AA stimulation in the presence or absence of Exo - 2 ( 10 μM ) ( C ) . D . Western blot analysis of mTORC1 activity in C2C12 - Vector and ARF1 KO cells in CM , in response to nutrient starvation and AA stimulation . E . Confocal imaging analysis depicting COPA puncta ( green ) formation and co - localization with lysosomes ( red ) in live C2C12 cells transiently expressing GFP - COPA and stained with Lysotracker Red in response to nutrient starvation and AA stimulation in the presence or absence of BFA ( 10 μM ) or Exo - 2 ( 10 μM ) . Arrows highlight co - localization of COPA with lysosomes . Scale bar , 5 μm . F . Statistical analysis of the Pearson ' s correlation coefficient of COPA and lysosomes in Figure 5E ( n = 10 - 15 cells per group ) . Data are represented as mean ± SD . * * * p < 0 . 001 by 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 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 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 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 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 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

Figure 5A. Confocal imaging analysis depicting the lysosomal association of mTOR in response to AA stimulation in the presence or absence of BFA (10 μM) or Exo-2 (10 μM) in live C2C12 cells transiently expressing YFP-mTOR and stained with Lysotracker Red. Scale bar, 20 μm.Western blot analysis of mTORC1 activity in C2C12 cells in CM, in response to nutrient starvation and AA stimulation in the presence or absence of BFA (10 μM) (B)C. Western blot analysis of mTORC1 activity in C2C12 cells in CM, in response to nutrient starvation and AA stimulation in the presence or absence of Exo-2 (10 μM) (C).D. Western blot analysis of mTORC1 activity in C2C12-Vector and ARF1 KO cells in CM, in response to nutrient starvation and AA stimulation.E. Confocal imaging analysis depicting COPA puncta (green) formation and co-localization with lysosomes (red) in live C2C12 cells transiently expressing GFP-COPA and stained with Lysotracker Red in response to nutrient starvation and AA stimulation in the presence or absence of BFA (10 μM) or Exo-2 (10 μM). Arrows highlight co-localization of COPA with lysosomes. Scale bar, 5 μm.F. Statistical analysis of the Pearson's correlation coefficient of COPA and lysosomes in Figure 5E (n=10-15 cells per group). Data are represented as mean ± SD. ***p<0.001 by one-way ANOVA.


Figure 6A . Co - IP analysis of the interaction of Aster - C with COPA , MYH10 , WDR24 and Mios in HEK293T cells transiently expressing the indicated proteins in response to treatment with brefeldin A ( BFA , 10 μM ) and / or blebbistatin ( BBS , 10 μM ) , inhibitors of ARFs and MYH10 , respectively . Anti - FLAG antibody was used for immunoprecipitation . B . Western blot analysis of mTORC1 activity in wild type ( WT ) and MYH10 KO MEF cells cultured in CM , or in response to starvation and AA stimulation . C . Western blot analysis of mTORC1 activity in MYH10 KO MEF cells with stably re - expressed exogenous mCherry - MYH10 in response to starvation and AA stimulation . MYH10 KO MEF cells were transfected with mCherry - MYH10 and selected with G418 for 2 weeks . D . Confocal imaging analysis depicting COPA puncta ( green ) formation and co - localization with mTOR ( red ) in live C2C12 cells transiently expression GFP - COPA and YFP - mTOR in response to nutrient starvation in the presence or absence of BBS ( 10 µM ) . Arrows highlight the co - localization of mTOR with COPA . Scale bar , 20 μm . E . Statistical analysis of the Pearson ' s correlation coefficient of mTOR and COPA in Figure 6D ( n = 10 - 12 cells per group ) . Data are represented as mean ± SD . * * * p < 0 . 001 by student ' s 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 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 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 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

Figure 6A. Co-IP analysis of the interaction of Aster-C with COPA, MYH10, WDR24 and Mios in HEK293T cells transiently expressing the indicated proteins in response to treatment with brefeldin A (BFA, 10 μM) and/or blebbistatin (BBS, 10 μM), inhibitors of ARFs and MYH10, respectively. Anti-FLAG antibody was used for immunoprecipitation.B. Western blot analysis of mTORC1 activity in wild type (WT) and MYH10 KO MEF cells cultured in CM, or in response to starvation and AA stimulation.C. Western blot analysis of mTORC1 activity in MYH10 KO MEF cells with stably re-expressed exogenous mCherry-MYH10 in response to starvation and AA stimulation. MYH10 KO MEF cells were transfected with mCherry-MYH10 and selected with G418 for 2 weeks.D. Confocal imaging analysis depicting COPA puncta (green) formation and co-localization with mTOR (red) in live C2C12 cells transiently expression GFP-COPA and YFP-mTOR in response to nutrient starvation in the presence or absence of BBS (10 µM). Arrows highlight the co-localization of mTOR with COPA. Scale bar, 20 μm.E. Statistical analysis of the Pearson's correlation coefficient of mTOR and COPA in Figure 6D (n=10-12 cells per group). Data are represented as mean ± SD. ***p<0.001 by student's t test.


Figure 7A . Confocal imaging analysis depicting mTOR puncta ( green ) formation on actin filaments ( red ) in COS - 7 cells transiently expressing YFP - mTOR in response to AA stimulation in the presence or absence of latrunculin B ( LA - B , 100 nM ) . Actin filaments were stained using BODIPY ™ 558 / 568 - Phalloidin . Arrows indicated the association of mTOR with actin filaments . Scale bar , 5 μm . B . Immunostaining of endogenous mTOR and LAMP1 in C2C12 cells under amino acids starvation ( - AA ) , or in response to AA stimulation ( + AA ) in the presence or absence of LA - B ( 100 nM ) . Scale bar , 20 μm . C . Western blot analysis of mTORC1 activity in C2C12 cells in CM , in response to nutrient starvation and AA stimulation in the presence or absence of LA - B ( 100 nM ) . D . Co - IP analysis of the interaction of Aster - C with COPA and MYH10 in the presence or absence of LA - B ( 100 nM ) or cytochalasin D ( Cyto - D , 250 nM ) in HEK293T cells transiently expressing FLAG - Aster - C . Anti - FLAG antibody was used for immunoprecipitation .

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 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 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

Figure 7A. Confocal imaging analysis depicting mTOR puncta (green) formation on actin filaments (red) in COS-7 cells transiently expressing YFP-mTOR in response to AA stimulation in the presence or absence of latrunculin B (LA-B, 100 nM). Actin filaments were stained using BODIPY™ 558/568-Phalloidin. Arrows indicated the association of mTOR with actin filaments. Scale bar, 5 μm.B. Immunostaining of endogenous mTOR and LAMP1 in C2C12 cells under amino acids starvation (-AA), or in response to AA stimulation (+AA) in the presence or absence of LA-B (100 nM). Scale bar, 20 μm.C. Western blot analysis of mTORC1 activity in C2C12 cells in CM, in response to nutrient starvation and AA stimulation in the presence or absence of LA-B (100 nM).D. Co-IP analysis of the interaction of Aster-C with COPA and MYH10 in the presence or absence of LA-B (100 nM) or cytochalasin D (Cyto-D, 250 nM) in HEK293T cells transiently expressing FLAG-Aster-C. Anti-FLAG antibody was used for immunoprecipitation.


Figure 1A , Genome browser images of ChIP - seq for SAGA proteins before and after induction of ER stress at ER stress response genes CHOP , ERP70 , and GRP78 . ATF4 / Xbp1s binding sites are from publicly available datasets ( GEO accessions GSE69304 and GSE49952 ) . C , Mean ChIP - seq signal for USP22 , GCN5 , and ATXN7L3 surrounding gene promoters from B . Nonparametric Mann - Whitney analysis of 7 bins ( grey shade ) surrounding maximal peak in Thaps treated conditions are reported .

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 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

Figure 1A, Genome browser images of ChIP-seq for SAGA proteins before and after induction of ER stress at ER stress response genes CHOP, ERP70, and GRP78. ATF4/Xbp1s binding sites are from publicly available datasets (GEO accessions GSE69304 and GSE49952).C, Mean ChIP-seq signal for USP22, GCN5, and ATXN7L3 surrounding gene promoters from B. Nonparametric Mann-Whitney analysis of 7 bins (grey shade) surrounding maximal peak in Thaps treated conditions are reported.


Figure 2A , HCT116 cells were treated with 100 nM Thapsigargin for 24 hrs . Cells were stained with dye recognizing cleaved Caspases 3 and 7 and imaged every 4 hours . Scale bar = 400 µm . B , Quantification of apoptotic populations as indicated by Cas3 / 7 fluorescence from A . Three independent experiments are represented as mean ± SEM , with significance calculated by Two - way ANOVA between conditions over time ( f ( 3 ) = 50 . 530 , p < 0 . 001 ) ; significant pairwise comparisons by Tukey ' s HSD post - hoc assessment are indicated by * . C

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

Figure 2A, HCT116 cells were treated with 100 nM Thapsigargin for 24 hrs. Cells were stained with dye recognizing cleaved Caspases 3 and 7 and imaged every 4 hours. Scale bar = 400 µm. B, Quantification of apoptotic populations as indicated by Cas3/7 fluorescence from A. Three independent experiments are represented as mean ± SEM, with significance calculated by Two-way ANOVA between conditions over time (f(3)=50.530, p<0.001); significant pairwise comparisons by Tukey's HSD post-hoc assessment are indicated by *. C


Figure 3A , HCT116 cells were treated with 100 nM Thapsigargin for the indicated times in the presence or absence of shRNA targeting USP22 . Cells were harvested and whole - cell lysate was subjected to immunoblotting with the indicated antibodies .

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

Figure 3A, HCT116 cells were treated with 100 nM Thapsigargin for the indicated times in the presence or absence of shRNA targeting USP22. Cells were harvested and whole-cell lysate was subjected to immunoblotting with the indicated antibodies.


Figure 4A , Genome browser images of ChIP - seq for Pol II and Ub - H2B before and after induction of ER stress , with and without USP22 knockdown , at ER stress response genes CHOP , ERP70 , and GRP78 . ATF4 / Xbp1s binding sites are from publicly available datasets ( GEO accessions GSE69304 and GSE49952 ) . B , Metagene profiles for Pol II ( upper panel ) and Ub - H2B ( middle panel ) ChIP - seq for USP22 - dependent , USP22 - Bound genes . mRNA analyses of select Bound gene transcripts are presented ( lower panel ) . Three independent experiments are represented as mean ± SEM , with significance measured by Student ' s t - test , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 005 C , Metagene profiles for Pol II ( upper panel ) and Ub - H2B ( middle panel ) ChIP - seq for USP22 - dependent , SAGA - Unbound genes . Nonparametric Mann - Whitney analyses of 7 bins ( grey shade ) surrounding TSS ( TSS ) and the midpoint of the profiles ( Body ) in Thaps treated conditions are reported . mRNA analysis of select Unbound gene transcripts are presented ( lower panel ) . Three independent experiments are represented as mean ± SEM , with significance measured by Student ' s t - test , * * p < 0 . 01 , * * * p < 0 . 005 .

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 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

Figure 4A, Genome browser images of ChIP-seq for Pol II and Ub-H2B before and after induction of ER stress, with and without USP22 knockdown, at ER stress response genes CHOP, ERP70, and GRP78. ATF4/Xbp1s binding sites are from publicly available datasets (GEO accessions GSE69304 and GSE49952).B, Metagene profiles for Pol II (upper panel) and Ub-H2B (middle panel) ChIP-seq for USP22-dependent, USP22-Bound genes. mRNA analyses of select Bound gene transcripts are presented (lower panel). Three independent experiments are represented as mean ± SEM, with significance measured by Student's t-test, *p<0.05, **p<0.01, ***p<0.005 C, Metagene profiles for Pol II (upper panel) and Ub-H2B (middle panel) ChIP-seq for USP22-dependent, SAGA-Unbound genes. Nonparametric Mann-Whitney analyses of 7 bins (grey shade) surrounding TSS (TSS) and the midpoint of the profiles (Body) in Thaps treated conditions are reported. mRNA analysis of select Unbound gene transcripts are presented (lower panel). Three independent experiments are represented as mean ± SEM, with significance measured by Student's t-test, **p<0.01, ***p<0.005.


Figure 5C , ChIP - qPCR for total RNA Pol II , pSer5 Pol II , and pSer2 Pol II at ERP70 , GRP78 , and a gene desert ( Prom = gene promoter , Coding = downstream coding region - see Appendix Table S1 for primer sequences ) . Three independent experiments are represented as mean ± SEM . with significance measured by Student ' s t - test , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 005 . H , An aliquot of cells from D ‑ G were harvested for whole - cell lysate and subjected to immunoblotting with the indicated antibodies .

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 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

Figure 5C, ChIP-qPCR for total RNA Pol II, pSer5 Pol II, and pSer2 Pol II at ERP70, GRP78, and a gene desert (Prom = gene promoter, Coding = downstream coding region - see Appendix Table S1 for primer sequences). Three independent experiments are represented as mean ± SEM. with significance measured by Student's t-test, *p<0.05, **p<0.01, ***p<0.005.H, An aliquot of cells from D‑G were harvested for whole-cell lysate and subjected to immunoblotting with the indicated antibodies.


Figure 6B , HCT116 cells were treated for 2 hrs with 100 nM Thapsigargin in the presence or absence of shRNA targeting USP22 . Cells were harvested , fractionated , and fractions were subjected to immunoblotting with the indicated antibodies . C , In vitro deubiquitylation UbiTest of endogenously ubiquitylated MED24 . HCT116 cells were treated with MG132 in the presence or absence of shRNA targeting USP22 . Lysates were generated using buffer containing protease cocktail inhibitor , pan - DUB inhibitor PR619 , and the JAMM protease inhibitor o - phenanthroline . Ubiquitylated proteins were purified on ubiquitin binding resin and eluates were either undigested ( lanes 1 and 2 ) or digested with USP2 to strip polyubiquitin ( lanes 3 and 4 ) and reduce target proteins to unit length . Digestion reactions were subjected to immunoblotting with the indicated antibodies .

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 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

Figure 6B, HCT116 cells were treated for 2 hrs with 100 nM Thapsigargin in the presence or absence of shRNA targeting USP22. Cells were harvested, fractionated, and fractions were subjected to immunoblotting with the indicated antibodies.C, In vitro deubiquitylation UbiTest of endogenously ubiquitylated MED24. HCT116 cells were treated with MG132 in the presence or absence of shRNA targeting USP22. Lysates were generated using buffer containing protease cocktail inhibitor, pan-DUB inhibitor PR619, and the JAMM protease inhibitor o-phenanthroline. Ubiquitylated proteins were purified on ubiquitin binding resin and eluates were either undigested (lanes 1 and 2) or digested with USP2 to strip polyubiquitin (lanes 3 and 4) and reduce target proteins to unit length. Digestion reactions were subjected to immunoblotting with the indicated antibodies.


Figure 7A , Genome browser image of ChIP - seq for middle and tail Mediator subunits and H3K4me3 HiChIP tracks before and after induction of ER stress at ER stress response gene BHLHE40 . H3K4me1 and H3K27ac tracks are from the ENCODE Consortium ( GEO accessions GSM945858 and GSM945853 ) . ATF4 / Xbp1s binding sites are from publicly available datasets ( GEO accessions GSE69304 and GSE49952 ) .

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

Figure 7A, Genome browser image of ChIP-seq for middle and tail Mediator subunits and H3K4me3 HiChIP tracks before and after induction of ER stress at ER stress response gene BHLHE40. H3K4me1 and H3K27ac tracks are from the ENCODE Consortium (GEO accessions GSM945858 and GSM945853). ATF4/Xbp1s binding sites are from publicly available datasets (GEO accessions GSE69304 and GSE49952).


Figure 2A Immunoblot analysis with the indicated antibodies of immunoprecipitates ( IP ) prepared from the SSU fraction of eIF3D - V5 / eIF4E - FLAG dKI HEK293T cells ( eIF3D / 4E dKI ) or wild - type ( WT ) cells with antibodies to the epitope tags . The SSU fraction ( input ) was also analyzed directly . Cells were fixed with formalin , cell lysates were treated with RNase , and the SSU and RS fractions were separated by sucrose density gradient centrifugation before immunopurification . Molecular sizes are indicated in kilodaltons . B , C Immunoblot analysis with the indicated antibodies of immunoprecipitates prepared as in A from the SSU fraction of eIF4A1 - FLAG KI HEK293T cells ( eIF4A1 KI ) ( B ) or ASCC3 - FLAG KI HEK293T cells ( ASCC3 KI ) ( C ) . Immunoblot analysis with the indicated antibodies of immunoprecipitates prepared as in A from the RS fraction of RPL7A - HA KI HEK293T cells ( RPL7A KI ) ( F ) .

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 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 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

Figure 2A Immunoblot analysis with the indicated antibodies of immunoprecipitates (IP) prepared from the SSU fraction of eIF3D-V5/eIF4E-FLAG dKI HEK293T cells (eIF3D/4E dKI) or wild-type (WT) cells with antibodies to the epitope tags. The SSU fraction (input) was also analyzed directly. Cells were fixed with formalin, cell lysates were treated with RNase, and the SSU and RS fractions were separated by sucrose density gradient centrifugation before immunopurification. Molecular sizes are indicated in kilodaltons.B, C Immunoblot analysis with the indicated antibodies of immunoprecipitates prepared as in A from the SSU fraction of eIF4A1-FLAG KI HEK293T cells (eIF4A1 KI) (B) or ASCC3-FLAG KI HEK293T cells (ASCC3 KI) (C).Immunoblot analysis with the indicated antibodies of immunoprecipitates prepared as in A from the RS fraction of RPL7A-HA KI HEK293T cells (RPL7A KI) (F).


Figure 4A RNA - seq results for eIF4A1 , eIF4A2 , ASCC2 , and ASCC3 in HEK293T cells transfected with siRNAs specific for eIF4A1 and eIF4A2 ( eIF4A1 / A2 dKD # 1 or # 2 ) or for ASCC3 ( ASCC3 KD # 1 or # 2 ) relative to cells transfected with a control siRNA . The dashed line indicates a fold change of 1 . B Immunoblot analysis of the siRNA - transfected cells with the indicated antibodies . Volcano plots for differential TE ( D ) The changes in TE and SRO for eIF4A1 / A2 dKD # 1 and # 2 and for ASCC3 KD # 1 and # 2 HEK293T cells compared with control cells were analyzed with RiboDiff . The gray dashed lines indicate an adjusted P value of 0 . 05 . P values were calculated by chi - square test and adjusted by Benjamini - Hochberg method .

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 B-PANEL_START 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

Figure 4A RNA-seq results for eIF4A1, eIF4A2, ASCC2, and ASCC3 in HEK293T cells transfected with siRNAs specific for eIF4A1 and eIF4A2 (eIF4A1/A2 dKD #1 or #2) or for ASCC3 (ASCC3 KD #1 or #2) relative to cells transfected with a control siRNA. The dashed line indicates a fold change of 1.B Immunoblot analysis of the siRNA-transfected cells with the indicated antibodies.Volcano plots for differential TE (D) The changes in TE and SRO for eIF4A1/A2 dKD #1 and #2 and for ASCC3 KD #1 and #2 HEK293T cells compared with control cells were analyzed with RiboDiff. The gray dashed lines indicate an adjusted P value of 0.05. P values were calculated by chi-square test and adjusted by Benjamini-Hochberg method.


Figure 5D , E Cumulative fraction for fold change in TE in eIF4A1 / A2 dKD or ASCC3 KD cells relative to control cells . Results are shown for all coding transcripts and for transcripts with a decreased ( D ) or increased ( E ) SRO . The P values were calculated with the Mann - Whitney U 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

Figure 5D, E Cumulative fraction for fold change in TE in eIF4A1/A2 dKD or ASCC3 KD cells relative to control cells. Results are shown for all coding transcripts and for transcripts with a decreased (D) or increased (E) SRO. The P values were calculated with the Mann-Whitney U test.


Figure 6D Cumulative fraction for fold change in TE in eIF4A1 / A2 dKD or ASCC3 KD cells relative to control cells . Results are shown for all coding transcripts and skewed transcripts . The P values were calculated with the Mann - Whitney U test . E Number of transcripts showing significant down - regulation of TE among skewed transcripts in eIF4A1 / A2 dKD and ASCC3 KD cells .

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 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

Figure 6D Cumulative fraction for fold change in TE in eIF4A1/A2 dKD or ASCC3 KD cells relative to control cells. Results are shown for all coding transcripts and skewed transcripts. The P values were calculated with the Mann-Whitney U test.E Number of transcripts showing significant down-regulation of TE among skewed transcripts in eIF4A1/A2 dKD and ASCC3 KD cells.


Figure 7Luciferase assays for RADX , DNAJB6 , and LDHA constructs ( B ) in control and ASCC3 - depleted HEK293T cells . The results of the luciferase assays ( right ) and schematic representations of the constructs ( left ) are shown . Rluc , Renilla luciferase ; Fluc , firefly luciferase . Assay data are means ± s . d . for three independent experiments . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , NS ( two - sided Student ' s t test ) . Luciferase assays for corresponding deletion mutants of the RADX and DNAJB6 constructs ( C ) in control and ASCC3 - depleted HEK293T cells . The results of the luciferase assays ( right ) and schematic representations of the constructs ( left ) are shown . The deleted regions of the RADX and DNAJB6 constructs are indicated by dashed white boxes . Rluc , Renilla luciferase ; Fluc , firefly luciferase . Assay data are means ± s . d . for three independent experiments . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 7Luciferase assays for RADX, DNAJB6, and LDHA constructs (B) in control and ASCC3-depleted HEK293T cells. The results of the luciferase assays (right) and schematic representations of the constructs (left) are shown. Rluc, Renilla luciferase; Fluc, firefly luciferase. Assay data are means ± s.d. for three independent experiments. *P < 0.05, **P < 0.01, *** P < 0.001, NS (two-sided Student's t test).Luciferase assays for corresponding deletion mutants of the RADX and DNAJB6 constructs (C) in control and ASCC3-depleted HEK293T cells. The results of the luciferase assays (right) and schematic representations of the constructs (left) are shown. The deleted regions of the RADX and DNAJB6 constructs are indicated by dashed white boxes. Rluc, Renilla luciferase; Fluc, firefly luciferase. Assay data are means ± s.d. for three independent experiments. *P < 0.05, **P < 0.01, *** P < 0.001


f1BrdU incorporation reflecting DNA synthesis and SA - b - gal activity as markers of CTX - induced senescence in Em - myc transgeniccontrol ; Bcl2 versus Suv39h12 ; Bcl2lymphomas in vivo . Numbers represent mean percentages of positive cells6s . d . ( n55 each ) . b , Representative whole - body FLT - and FDG - PET scans of an individual control ; Bcl2 lymphoma - bearing mouse at tumour manifestation ( untreated ) and at day 6 after a single dose of CTX . Red circles , areas of specific tracer retention ; green circles , matching areas with lost retentions . Specific tracer uptake was assessed by calculation of tumour - to - background ratios . Data represent mean ratios ± s . d . ( n = 5 mice for FLT - PET scans and n = 6 mice for FDG - PET scans ) . Insets show representative Ki67 staining and colour - calibrated ( red , highest intensities ; blue , lowest intensities ) bioluminescence images of glucose levels in tissue sections of the respective groups . c , Intracellular glucose concentrations in untreated and ADR - treated control ; Bcl2 ( that is , senescent ) versus Suv39h1 - ; Bcl2 ( that is , non - senescent ) lymphomas in vitro ( as in d - g ) . Results represent means ± s . d . ( n = 5 each ) . d , Extracellular levels of the specified metabolite - to - glucose ratios in the indicated lymphoma cells groups relative to untreated control ; Bcl2 lymphoma cells after 18 h of cultivation . Data represent mean ratios ± s . d . ( n = 4 each , measurements were carried out in duplicate ) . Note that glucose - 6 - phosphate is not detectable ( n . d . ) in the medium . Insets show ( ADR - treated relative to untreated ) intracellular levels of the respective metabolites after labelling with 13C6 - glucose for one representative lymphoma from each group . Data represent mean ratios ( see Supplementary Fig . 5 for an assessment of the heterogeneity of the TIS - associated hypermetabolism and a comparison of the metabolic parameters of individual lymphomas ) . e , Immunoblot analyses of phosphorylated AMP - activated protein kinase ( pAMPK ) , total AMPK , pyruvate kinase isoforms M1 and M2 in ADR - senescent control ; Bcl2 versus ADR - treated Suv39h1 - ; Bcl2 lymphoma cells in vitro ( n = 3 each ; α - tubulin as a loading control . Gels were cropped to ease presentation ; full - length blots are provided in Supplementary Fig . 16 ) . f , Mean OCRs in ADR - senescent control ; Bcl2 versus ADR - treated Suv39h1 − ; Bcl2 lymphoma cells in vitro . Results represent means ± s . d . ( n = 5 each ) . g , Intracellular ATP concentrations in cells as in f . Results represent means ± s . d . ( n = 5 each ) . * P < 0 . 05 .

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 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 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 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 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 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 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

f1BrdU incorporation reflecting DNA synthesis and SA-b-gal activity as markers of CTX-induced senescence in Em-myc transgeniccontrol;Bcl2 versus Suv39h12;Bcl2lymphomas in vivo. Numbers represent mean percentages of positive cells6s.d. (n55 each).b, Representative whole-body FLT- and FDG-PET scans of an individual control;Bcl2 lymphoma-bearing mouse at tumour manifestation (untreated) and at day 6 after a single dose of CTX. Red circles, areas of specific tracer retention; green circles, matching areas with lost retentions. Specific tracer uptake was assessed by calculation of tumour-to-background ratios. Data represent mean ratios ± s.d. (n = 5 mice for FLT-PET scans and n = 6 mice for FDG-PET scans). Insets show representative Ki67 staining and colour-calibrated (red, highest intensities; blue, lowest intensities) bioluminescence images of glucose levels in tissue sections of the respective groups.c, Intracellular glucose concentrations in untreated and ADR-treated control;Bcl2 (that is, senescent) versus Suv39h1-;Bcl2 (that is, non-senescent) lymphomas in vitro (as in d-g). Results represent means ± s.d. (n = 5 each).d, Extracellular levels of the specified metabolite-to-glucose ratios in the indicated lymphoma cells groups relative to untreated control;Bcl2 lymphoma cells after 18 h of cultivation. Data represent mean ratios ± s.d. (n = 4 each, measurements were carried out in duplicate). Note that glucose-6-phosphate is not detectable (n.d.) in the medium. Insets show (ADR-treated relative to untreated) intracellular levels of the respective metabolites after labelling with 13C6-glucose for one representative lymphoma from each group. Data represent mean ratios (see Supplementary Fig. 5 for an assessment of the heterogeneity of the TIS-associated hypermetabolism and a comparison of the metabolic parameters of individual lymphomas).e, Immunoblot analyses of phosphorylated AMP-activated protein kinase (pAMPK), total AMPK, pyruvate kinase isoforms M1 and M2 in ADR-senescent control;Bcl2 versus ADR-treated Suv39h1-;Bcl2 lymphoma cells in vitro (n = 3 each; α-tubulin as a loading control. Gels were cropped to ease presentation; full-length blots are provided in Supplementary Fig. 16).f, Mean OCRs in ADR-senescent control;Bcl2 versus ADR-treated Suv39h1−;Bcl2 lymphoma cells in vitro. Results represent means ± s.d. (n = 5 each).g, Intracellular ATP concentrations in cells as in f. Results represent means ± s.d. (n = 5 each). *P<0.05.


f2a - e , Cell viability by trypan blue dye exclusion in ADR - senescent control ; Bcl2 versus ADR - treated Suv39h1 - ; Bcl2 lymphoma cells at day 5 compared to the untreated condition in various biochemical , pharmacological or genetic interventional settings in vitro . All results in this figure represent means ± s . d . ( n = 5 each ) . a , Inhibition of glucose metabolism by phloretin , cytochalasin B , and sodium oxamate . b , Exposure to 2DG or switch to glucose - free medium with or without glucose replenishment . c , Lentiviral knockdown of hexokinase 2 ( Hk2 ) by short hairpin RNA ( shHk2 ) . d , Inhibition of AMPK by compound C . e , Block of fatty acid oxidation by the carnitine palmitoyltransferase I inhibitor etomoxir . f , Inhibition of the electron transport chain by antimycin A . * P 0 . 01 , except b ( n = 8 each ) and e , in which * P 0 . 05 .

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 B-PANEL_START 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

f2a-e, Cell viability by trypan blue dye exclusion in ADR-senescent control;Bcl2 versus ADR-treated Suv39h1-;Bcl2 lymphoma cells at day 5 compared to the untreated condition in various biochemical, pharmacological or genetic interventional settings in vitro. All results in this figure represent means ± s.d. (n = 5 each). a, Inhibition of glucose metabolism by phloretin, cytochalasin B, and sodium oxamate.b, Exposure to 2DG or switch to glucose-free medium with or without glucose replenishment.c, Lentiviral knockdown of hexokinase 2 (Hk2) by short hairpin RNA (shHk2). d, Inhibition of AMPK by compound C. e, Block of fatty acid oxidation by the carnitine palmitoyltransferase I inhibitor etomoxir. f, Inhibition of the electron transport chain by antimycin A. *P  0.01, except b (n = 8 each) and e, in which *P  0.05.


f3Proteostasis analysis in TIS control versus senescence - incapable lymphoma cells in vitro , as in Fig . 2 , or versus SASP - attenuated TISlymphoma cells . a , Global protein synthesis rate , measured by incorporation of green - fluorescent methionine . Mean fluorescent intensities reflect newly synthesized protein within 2 h ( DAPI used as a counterstain ) . Data represent means ± s . d . ( n = 5 each ) . b , Rate of global protein synthesis ( measured as in a ) in ADR - senescent relative to untreated ( ut ) control ; Bcl2 lymphoma cells ; either empty vector - infected cells or cells stably expressing the NF - kB super - repressor IkBaDN ( SR , ref . 18 ) . Results represent means ± s . d . ( n = 5 each ) . c , Relative intracellular glucose concentrations in cells as in b . d , Relative OCR in cells as in b . e , Relative intracellular ATP concentrations in cells as in bf , Expression of the transcription factors Xbp1 ( by RT - PCR ( PCR with reverse transcription ; top ) ; note that the lower band reflects the UPR - related splice form ) , ATF4 , CHOP , phospho - Jun N - terminal kinase ( JNK - P - Thr183 / Thr185 ) and total JNK ( by immunoblot analysis , with a - tubulin as a loading control ; bottom ) in lymphoma cells as indicated ( n = 3 each , gels were cropped to ease presentation ; full - length blots are provided in Supplementary Fig . 16 ) . g , Global protein ubiquitination , expression of p62 / SQSTM1 ( whose transcript levels were also selectively induced in TIS cells by approximately 4 . 8 - fold ( data not shown ) ) and V - ATPase subunit A1 , and conversion of MAP1 - LC3 - I to MAP1 - LC3 - II by immunoblot analysis , demonstrating autophagosome formation in lymphoma cells as in f ( α - tubulin was used as a loading control , gels were cropped to ease presentation ; full - length blots are provided in Supplementary Fig . 16 ) . * P < 0 . 05 .

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

f3Proteostasis analysis in TIS control versus senescence-incapable lymphoma cells in vitro, as in Fig. 2, or versus SASP-attenuated TISlymphoma cells. a, Global protein synthesis rate, measured by incorporation of green-fluorescent methionine. Mean fluorescent intensities reflect newly synthesized protein within 2 h (DAPI used as a counterstain). Data represent means ± s.d. (n = 5 each). b, Rate of global protein synthesis (measured as in a) in ADR-senescent relative to untreated (ut) control;Bcl2 lymphoma cells; either empty vector-infected cells or cells stably expressing the NF-kB super-repressor IkBaDN(SR, ref. 18). Results represent means ± s.d. (n = 5 each). c, Relative intracellular glucose concentrations in cells as in b.d, Relative OCR in cells as in b. e, Relative intracellular ATP concentrations in cells as in bf, Expression of the transcription factors Xbp1 (by RT-PCR (PCR with reverse transcription; top); note that the lower band reflects the UPR-related splice form), ATF4, CHOP, phospho-Jun N-terminal kinase (JNK-P-Thr183/Thr185) and total JNK(by immunoblot analysis, with a-tubulin as a loading control; bottom) in lymphoma cells as indicated (n = 3 each, gels were cropped to ease presentation; full-length blots are provided in Supplementary Fig. 16).g, Global protein ubiquitination, expression of p62/SQSTM1 (whose transcript levels were also selectively induced in TIS cells by approximately 4.8-fold (data not shown)) and V-ATPase subunit A1, and conversion of MAP1-LC3-I to MAP1-LC3-II by immunoblot analysis, demonstrating autophagosome formation in lymphoma cells as in f (α-tubulin was used as a loading control, gels were cropped to ease presentation; full-length blots are provided in Supplementary Fig. 16). *P<0.05.


f4a , Cellular viability of super - repressor - or empty - vector - infected control ; Bcl2 lymphomas treated with ADR or untreated for 5 days , then exposed for 2 days to the inhibitors 2DG or bafilomycin A1 , compared to no inhibitor ( ut ) in vitro . Results represent means ± s . d . ( n = 5 each ) . b , Immunoblot analyses of ATF4 , global protein ubiquitination , p62 / SQSTM1 , and MAP1 - LC3 - I to MAP1 - LC3 - II conversion in control ; Bcl2 lymphomas with no ADR treatment , in TIS cells , and in TIS cells after exposure to 2DG or bafilomycin A1in vitro ; representative blot with α - tubulin as a loading control ( gels were cropped to ease presentation ; full - length blots are provided in Supplementary Fig . 16 ) . c , Cleavage ( activation ) of caspase 12 and detection of cleaved caspase 3 by immunoblot analysis of control ; Bcl2 lymphomas as in b ; representative blot with α - tubulin as a loading control ( gels were cropped to ease presentation ; full - length blots are provided in Supplementary Fig . 16 ) . d , Cellular viability of ADR - treated control ; Bcl2 lymphoma cells infected with short - hairpin RNAs against either caspase 12 ( shCasp12 ) or caspase 3 ( shCasp3 ) compared to empty vector - infected lymphoma cells treated as in a . Results represent means ± s . d . ( n = 4 each ) . e , TIS cells in primary human acute myeloid leukaemia ( AML ) samples by SA - b - gal staining of treated ( 0 . 1 µg ml − 1 ADR for 5 days ) versus untreated cells in vitro . Data represent means ± s . d . ( n = 5 each ) . f , Intracellular glucose ( left ) and ATP ( right ) concentrations in senescent AML cells as in e . g , Cellular viability of primary AML samples treated as in e and exposed to inhibitors as in a . Results represent means ± s . d . ( n = 5 each ) . h , Colour - coded whole - body fluorescence imaging to visualize green fluorescent protein ( GFP ) - tagged tumour burden in time - courses of three representative mice harbouring CTX - senescent control ; Bcl2 lymphomas at day 5 ( see Figure 1a ) , and two days later , after the administration of 2DG ( n = 3 mice ) , bafilomycin A1 ( n = 4 mice ) or mock treatment ( n = 4 mice ) starting from day 5 post CTX ( red , highest intensities ; blue , lowest intensities ) . TdT - mediated dUTP nick end labelling ( assay ) ( TUNEL ) staining of lymph node sections in situ to visualize apoptosis at day 7 . Numbers represent means ± s . d . ( n = 3 ) . i , Overall survival of 5 day - CTX - treated control ; Bcl2 or Suv - 39h1 - ; Bcl2 lymphoma - bearing mice randomly assigned to sequential exposure to bafilomycin A1 or mock as in h ( control ; Bcl2 : n = 12 and Suv39h1 - ; Bcl2 : n = 10 ) , presented as matched pair analyses ( P < 0 . 001 for the control ; Bcl2 comparison ; P = 0 . 221 for the Suv39h1 - ; Bcl2 comparison ) . * P < 0 . 05 .

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 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 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 B-PANEL_START 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 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

f4a, Cellular viability of super-repressor- or empty-vector-infected control;Bcl2 lymphomas treated with ADR or untreated for 5 days, then exposed for 2 days to the inhibitors 2DG or bafilomycin A1, compared to no inhibitor (ut) in vitro. Results represent means ± s.d. (n = 5 each).b, Immunoblot analyses of ATF4, global protein ubiquitination, p62/SQSTM1, and MAP1-LC3-I to MAP1-LC3-II conversion in control;Bcl2 lymphomas with no ADR treatment, in TIS cells, and in TIS cells after exposure to 2DG or bafilomycin A1in vitro; representative blot with α-tubulin as a loading control (gels were cropped to ease presentation; full-length blots are provided in Supplementary Fig. 16). c, Cleavage (activation) of caspase 12 and detection of cleaved caspase 3 by immunoblot analysis of control;Bcl2 lymphomas as in b; representative blot with α-tubulin as a loading control (gels were cropped to ease presentation; full-length blots are provided in Supplementary Fig. 16).d, Cellular viability of ADR-treated control;Bcl2 lymphoma cells infected with short-hairpin RNAs against either caspase 12 (shCasp12) or caspase 3 (shCasp3) compared to empty vector-infected lymphoma cells treated as in a. Results represent means ± s.d. (n = 4 each).e, TIS cells in primary human acute myeloid leukaemia (AML) samples by SA-b-gal staining of treated (0.1 µg ml−1 ADR for 5 days) versus untreated cells in vitro. Data represent means ± s.d. (n = 5 each).f, Intracellular glucose (left) and ATP (right) concentrations in senescent AML cells as in e.g, Cellular viability of primary AML samples treated as in e and exposed to inhibitors as in a. Results represent means ± s.d. (n = 5 each).h, Colour-coded whole-body fluorescence imaging to visualize green fluorescent protein (GFP)-tagged tumour burden in time-courses of three representative mice harbouring CTX-senescent control;Bcl2 lymphomas at day 5 (see Figure 1a), and two days later, after the administration of 2DG (n = 3 mice), bafilomycin A1 (n = 4 mice) or mock treatment (n = 4 mice) starting from day 5 post CTX (red, highest intensities; blue, lowest intensities). TdT-mediated dUTP nick end labelling (assay) (TUNEL) staining of lymph node sections in situ to visualize apoptosis at day 7. Numbers represent means ± s.d. (n = 3).i, Overall survival of 5 day-CTX-treated control;Bcl2 or Suv-39h1-;Bcl2 lymphoma-bearing mice randomly assigned to sequential exposure to bafilomycin A1 or mock as in h (control;Bcl2: n = 12 and Suv39h1-;Bcl2: n = 10), presented as matched pair analyses (P<0.001 for the control; Bcl2 comparison; P = 0.221 for the Suv39h1-;Bcl2 comparison). *P<0.05.


Figure 1F - G ) HeLa cells were treated as in B ) and then fixed and stained with ULK1 ( green ) ( F ) or ATG13 ( red ) antibodies ( G ) respectively . Hoechst was used to stain nuclei . Graphs ( right ) show the average of both ULK1 ( F ) ( n = 3 independent experiments , n = 11 fields analysed ) or ATG13 ( n = 3 independent experiments , n ≥ 16 fields analysed ) ( G ) puncta number per cell and both ULK1 ( n = 3 independent experiments ) ( F ) or ATG13 ( n = 3 independent experiments ) ( G ) fold of increase of the average size respectively . White arrows indicate most representative puncta of analysed conditions . All data are reported as the mean value ± SEM . Statistical analyses were performed by two - way ANOVA followed by Tukey ' s multiple comparison test . * * , P < 0 , 01 ; * * * * , P < 0 . 0001 or by unpaired Student ' s t ‐ test when two groups were compared . * , P < 0 . 05 ; * * * * , P < 0 . 0001 . Scale bar 5 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 1F-G) HeLa cells were treated as in B) and then fixed and stained with ULK1 (green) (F) or ATG13 (red) antibodies (G) respectively. Hoechst was used to stain nuclei. Graphs (right) show the average of both ULK1 (F) (n=3 independent experiments, n=11 fields analysed) or ATG13 (n=3 independent experiments, n≥16 fields analysed) (G) puncta number per cell and both ULK1 (n=3 independent experiments) (F) or ATG13 (n=3 independent experiments) (G) fold of increase of the average size respectively. White arrows indicate most representative puncta of analysed conditions. All data are reported as the mean value ± SEM. Statistical analyses were performed by two-way ANOVA followed by Tukey's multiple comparison test. **, P<0,01; ****, P<0.0001 or by unpaired Student's t‐test when two groups were compared. *, P<0.05; ****, P<0.0001. Scale bar 5 μm.


Figure 2HeLa cells were transfected with specific RNAi oligonucleotides ( siTFG ) or unrelated oligonucleotides as a negative control ( siCTR ) and grown in fed or starved conditions for 1h . Cells were fixed and co - labelled with ULK1 ( red ) antibody and A ) anti - CALNEXIN ( CANX ) ( green ) antibody to highlight ER structures ; ( n = 3 independent experiments , n ≥ 16 fields analysed ) B ) GOLGIN97 ( green ) antibody to label GOLGI complex ; ( n = 3 independent experiments , n = 12 fields analysed ) C ) SEC31A ( green ) to point out COPII vesicles . ( n = 3 independent experiments , n ≥ 16 fields analysed ) D ) ERGIC53 ( green ) to mark ERGIC compartment . ( n = 3 independent experiments , n ≥ 14 fields analysed ) . Analysis of ULK1 localization with each compartment is reported in each graph . Co - localization analyses were performed by ImageJ plugin Jacop . Values of Mander ' s coefficient for ULK1 are expressed in percentage as mean ± SEM . Statistical analyses were performed by two - way ANOVA followed by Tukey ' s multiple comparison test . * * , P < 0 , 01 ; * * * , P < 0 , 001 ; * * * * , P < 0 , 0001 ; ns , not significant . Scale bars 5 μm . White arrowheads point at ULK1 puncta associated to the analysed markers ; N , nucleus . F ) HeLa cells were co - transfected with control siRNA and an empty vector ( EV ) , or 3 ' UTR TFG siRNA together with an EV or HA - TFG plasmids . Cells grown for 1h in starvation medium then fixed , permeabilized , and labelled with anti - ULK1 ( green ) and anti - ERGIC53 ( red ) antibodies respectively . Hoechst was used to stain nuclei . Co - localization analysis were performed as in D ) . Values of Mander ' s coefficient for ULK1 , expressed as percentage , are mean ± SEM . Statistical analyses were performed by one - way ANOVA followed by Dunnetts ' s multiple comparison test . * , P < 0 , 05 . ( n = 3 ) ( n = 3 independent experiments , n ≥ 9 fields analysed ) . Scale bar 5 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 O O O O O O O O O O O O O O O O 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 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

Figure 2HeLa cells were transfected with specific RNAi oligonucleotides (siTFG) or unrelated oligonucleotides as a negative control (siCTR) and grown in fed or starved conditions for 1h. Cells were fixed and co-labelled with ULK1 (red) antibody and A) anti-CALNEXIN (CANX) (green) antibody to highlight ER structures; (n=3 independent experiments, n≥16 fields analysed) B) GOLGIN97 (green) antibody to label GOLGI complex; (n=3 independent experiments, n=12 fields analysed) C) SEC31A (green) to point out COPII vesicles. (n=3 independent experiments, n≥16 fields analysed)D) ERGIC53 (green) to mark ERGIC compartment. (n=3 independent experiments, n≥14 fields analysed). Analysis of ULK1 localization with each compartment is reported in each graph. Co-localization analyses were performed by ImageJ plugin Jacop. Values of Mander's coefficient for ULK1 are expressed in percentage as mean ± SEM. Statistical analyses were performed by two-way ANOVA followed by Tukey's multiple comparison test. **, P<0,01; ***, P<0,001; ****, P<0,0001; ns, not significant. Scale bars 5 μm. White arrowheads point at ULK1 puncta associated to the analysed markers; N, nucleus.F) HeLa cells were co-transfected with control siRNA and an empty vector (EV), or 3'UTR TFG siRNA together with an EV or HA-TFG plasmids. Cells grown for 1h in starvation medium then fixed, permeabilized, and labelled with anti-ULK1 (green) and anti-ERGIC53 (red) antibodies respectively. Hoechst was used to stain nuclei. Co-localization analysis were performed as in D). Values of Mander's coefficient for ULK1, expressed as percentage, are mean ± SEM. Statistical analyses were performed by one-way ANOVA followed by Dunnetts's multiple comparison test. *, P<0,05. (n=3) (n=3 independent experiments, n≥9 fields analysed). Scale bar 5 μm.


Figure 3A ) HeLa cells were transfected with specific RNAi oligonucleotides ( siTFG ) or unrelated oligonucleotides as a negative control ( siCTR ) and grown in fed ( FED ) or starvation conditions ( STV ) in the presence or absence of CLQ for 1h . Cells were then fixed and stained using LC3B - II ( red ) antibody . Analysis of LC3B puncta number is reported ( bottom ) . All data are expressed as the mean value ± SEM . Statistical analysis were performed by two - way ANOVA followed by Tukey ' s multiple comparison test . * * * * , P < 0 . 0001 . ( n = 4 independent experiments , n ≥ 25 fields analysed ) . Scale bar 5 μm . B ) HeLa cells were transfected as in A ) and cultured in starvation conditions ( STV ) in the presence or absence of Bafilomycin A1 ( Baf A1 ) for 1h . Cells were then fixed and stained using LC3B - II ( green ) and LAMP1 ( red ) antibodies . Hoechst was used to mark nuclei . Co - localization analyses were performed by ImageJ plugin Jacop . Values of Mander ' s coefficient for LC3B - II are expressed as mean ± SEM . Statistical analysis were performed by two - way ANOVA followed by Tukey ' s multiple comparison test . * , P < 0 , 05 ; ( n = 3 independent experiments , n ≥ 13 fields analysed ) . Scale bar 5 μm . C ) HeLa cells were transfected as in A ) and grown in fed ( FED ) or starvation conditions ( STV ) for 1h . Cells were then fixed and stained using ATG16L1 ( yellow ) antibody . ATG16L1 puncta number ( upper right ) and the average size of ATG16L1 as fold increase ( bottom right ) were reported . All values are expressed as mean ± SEM . Statistical analyses were performed by two - way ANOVA followed by Tukey ' s multiple comparison test . * * , P < 0 , 01 ; * * * * , P < 0 . 0001 or by unpaired Student ' s t ‐ test when two groups were compared . ns , not significant . ( n = 3 independent experiments , n ≥ 9 fields analysed ) . Scale bar 5 μm . D ) HEK293 cells stably expressing GFP - DFCP1 were transfected as in A ) and cultured in starved conditions for 40 ' . Cells were visualised with cellSens microscope for live - cell imaging . Pictures show the images taken at the same number of frames for each video . Analyses of the number of DFCP1 structures per cell ( upper right ) and the mean of omegasomes structures diameter ( μm ) ( bottom right ) were reported . N , nucleus . All values are expressed as mean ± SEM . Statistical analyses were performed by unpaired Student ' s t ‐ test . * , P < 0 , 05 ; * * * , P < 0 . 001 . ( n = 3 independent experiments ) . Scale bar 5 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 O O O O O O O O O O O O O O O O O O O O O O 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 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 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

Figure 3A) HeLa cells were transfected with specific RNAi oligonucleotides (siTFG) or unrelated oligonucleotides as a negative control (siCTR) and grown in fed (FED) or starvation conditions (STV) in the presence or absence of CLQ for 1h. Cells were then fixed and stained using LC3B-II (red) antibody. Analysis of LC3B puncta number is reported (bottom). All data are expressed as the mean value ± SEM. Statistical analysis were performed by two-way ANOVA followed by Tukey's multiple comparison test. ****, P<0.0001. (n=4 independent experiments, n≥25 fields analysed). Scale bar 5 μm.B) HeLa cells were transfected as in A) and cultured in starvation conditions (STV) in the presence or absence of Bafilomycin A1 (Baf A1) for 1h. Cells were then fixed and stained using LC3B-II (green) and LAMP1 (red) antibodies. Hoechst was used to mark nuclei. Co-localization analyses were performed by ImageJ plugin Jacop. Values of Mander's coefficient for LC3B-II are expressed as mean ± SEM. Statistical analysis were performed by two-way ANOVA followed by Tukey's multiple comparison test. *, P<0,05; (n=3 independent experiments, n≥13 fields analysed). Scale bar 5 μm.C) HeLa cells were transfected as in A) and grown in fed (FED) or starvation conditions (STV) for 1h. Cells were then fixed and stained using ATG16L1 (yellow) antibody. ATG16L1 puncta number (upper right) and the average size of ATG16L1 as fold increase (bottom right) were reported. All values are expressed as mean ± SEM. Statistical analyses were performed by two-way ANOVA followed by Tukey's multiple comparison test. **, P<0,01; ****, P<0.0001 or by unpaired Student's t‐test when two groups were compared. ns, not significant. (n=3 independent experiments, n≥9 fields analysed). Scale bar 5 μm.D) HEK293 cells stably expressing GFP-DFCP1 were transfected as in A) and cultured in starved conditions for 40'. Cells were visualised with cellSens microscope for live-cell imaging. Pictures show the images taken at the same number of frames for each video. Analyses of the number of DFCP1 structures per cell (upper right) and the mean of omegasomes structures diameter (μm) (bottom right) were reported. N, nucleus. All values are expressed as mean ± SEM. Statistical analyses were performed by unpaired Student's t‐test. *, P<0,05; ***, P<0.001. (n=3 independent experiments). Scale bar 5 μm.


Figure 4A ) HeLa cells were transfected with specific RNAi oligonucleotides ( siTFG ) or unrelated oligonucleotides as a negative control ( siCTR ) ; after culturing cells in fed ( FED ) or starvation conditions ( STV ) for 1h , cells were fixed and labelled with the indicated antibodies . Co - localization analysis was performed by using ImageJ Jacop plugin and mean ± SEM of Mander ' s coefficients of ULK1 , expressed as percentage , is reported ( bottom ) . White arrowheads point at co - localization events between ULK1 and LC3B ; white arrows track puncta not co - localizing ; N , nucleus . Statistical analyses were performed by two - way ANOVA followed by Tukey ' s multiple comparison test . * , P < 0 , 05 ; * * , P < 0 . 01 ; * * * * , P < 0 . 0001 . ( n = 3 independent experiments , n ≥ 16 fields analysed ) . Scale bar 5 μm . HEK293 cells stably expressing either GFP - ATG13 ( B ) were transiently transfected with specific RNAi oligonucleotides ( siTFG ) or unrelated oligonucleotides as a negative control ( siCTR ) , together with CFP - LC3B plasmid ( visualized in red ) and cultured in starved conditions for 40 ' . Wide - field live - cell imaging of starved cells were taken by cellSens microscope . Representative images of lifespan of ATG13 ( B ) are reported . Images of ATG13 ( B ) forming association with LC3B - II are also shown . The appearance of LC3B on ATG13 ( B ) ( bottom right ) ( C ) puncta were quantified and reported . All values are expressed in seconds ( sec ) as mean ± SEM . Yellow arrowheads point at the DFCP1 and LC3B particles in the first frame from their onset , white arrowheads and white arrows show omegasomes and autophagosomes as indicated . Statistical analyses were performed by unpaired Student ' s t ‐ test . * , P < 0 , 05 ; * * , P < 0 . 01 . ( n = 3 independent experiments , n = 50 events / condition analysed ) . Scale bar 1 μm . HEK293 cells stably expressing either GFP - DFCP1 ( C ) were transiently transfected with specific RNAi oligonucleotides ( siTFG ) or unrelated oligonucleotides as a negative control ( siCTR ) , together with CFP - LC3B plasmid ( visualized in red ) and cultured in starved conditions for 40 ' . Wide - field live - cell imaging of starved cells were taken by cellSens microscope . Representative images of lifespan of both ATG13 ( C ) are reported . Images of DFCP1 ( C ) forming association with LC3B - II are also shown . The appearance of LC3B on on DFCP1 ( bottom right ) ( C ) puncta were quantified and reported . All values are expressed in seconds ( sec ) as mean ± SEM . Yellow arrowheads point at the DFCP1 and LC3B particles in the first frame from their onset , white arrowheads and white arrows show omegasomes and autophagosomes as indicated . Statistical analyses were performed by unpaired Student ' s t ‐ test . * , P < 0 , 05 ; * * , P < 0 . 01 . ( n = 3 independent experiments , n = 50 events / condition analysed ) . Scale bar 1 μm . D ) HeLa cells were transfected as in A ) . Cells were imaged by TEM . Representative electron micrographs ( x26500 ) are reported . The average number of abnormal structures per cell is shown ( bottom graph ) . N , nucleus ; M , mitochondria ; ER , endoplasmic reticulum . The average number of abnormal vacuoles per cell was evaluated ( upper right ) . Statistical analysis was performed by unpaired Student ' s t ‐ test . * * * , P < 0 , 001 . A minimum of 50 cells were observed . Cell count was performed ( n = 3 independent experiments , n ≥ 51 cells analysed ) . Data are presented as mean ± SEM . Scale bar 500 nm as indicated . E ) HeLa cells stably expressing GFP - ATG13 and CFP - ATG16 were transiently co - transfected with DsRed - Rab7 and TFG siRNA ( siTFG ) . Cells were cultured in starved conditions for 1h . Representative electron micrographs ( x3900 ) and merge of the three fluorophores CLEM are shown . Micrographs at higher magnification ( x13500 ) of a region showing the three fluorophores split and merged is reported . Scale bars : 5 μm upper panels , 1 μm bottom panels . G ) Control ( TFG - WT ) and patient ' s ( TFG - R106C ) fibroblasts were grown in fed or starvation conditions for 1h . Cells were fixed , permeabilized , and labelled with anti - ULK ( red ) antibody . Hoechst was used to stain nuclei . The average of ULK1 puncta number per cell is shown ( bottom ) . All data are expressed as the mean value ± SEM . Statistical analysis was performed by two - way ANOVA followed by Tukey ' s multiple comparison test . * , P < 0 . 05 ; * * * , P < 0 , 001 ; * * * * ; P < 0 , 0001 . ( n = 3 independent experiments , n ≥ 14 fields analysed ) . Scale bar 5 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 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 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

Figure 4A) HeLa cells were transfected with specific RNAi oligonucleotides (siTFG) or unrelated oligonucleotides as a negative control (siCTR); after culturing cells in fed (FED) or starvation conditions (STV) for 1h, cells were fixed and labelled with the indicated antibodies. Co-localization analysis was performed by using ImageJ Jacop plugin and mean ± SEM of Mander's coefficients of ULK1, expressed as percentage, is reported (bottom). White arrowheads point at co-localization events between ULK1 and LC3B; white arrows track puncta not co-localizing; N, nucleus. Statistical analyses were performed by two-way ANOVA followed by Tukey's multiple comparison test. *, P<0,05; **, P<0.01; ****, P<0.0001. (n=3 independent experiments, n≥16 fields analysed). Scale bar 5 μm.HEK293 cells stably expressing either GFP-ATG13 (B) were transiently transfected with specific RNAi oligonucleotides (siTFG) or unrelated oligonucleotides as a negative control (siCTR), together with CFP-LC3B plasmid (visualized in red) and cultured in starved conditions for 40'. Wide-field live-cell imaging of starved cells were taken by cellSens microscope. Representative images of lifespan of ATG13 (B) are reported. Images of ATG13 (B) forming association with LC3B-II are also shown. The appearance of LC3B on ATG13 (B) (bottom right) (C) puncta were quantified and reported. All values are expressed in seconds (sec) as mean ± SEM. Yellow arrowheads point at the DFCP1 and LC3B particles in the first frame from their onset, white arrowheads and white arrows show omegasomes and autophagosomes as indicated. Statistical analyses were performed by unpaired Student's t‐test. *, P<0,05; **, P<0.01.(n=3 independent experiments, n=50 events/condition analysed). Scale bar 1 μm.HEK293 cells stably expressing either GFP-DFCP1 (C) were transiently transfected with specific RNAi oligonucleotides (siTFG) or unrelated oligonucleotides as a negative control (siCTR), together with CFP-LC3B plasmid (visualized in red) and cultured in starved conditions for 40'. Wide-field live-cell imaging of starved cells were taken by cellSens microscope. Representative images of lifespan of both ATG13 (C) are reported. Images of DFCP1 (C) forming association with LC3B-II are also shown. The appearance of LC3B on on DFCP1 (bottom right) (C) puncta were quantified and reported. All values are expressed in seconds (sec) as mean ± SEM. Yellow arrowheads point at the DFCP1 and LC3B particles in the first frame from their onset, white arrowheads and white arrows show omegasomes and autophagosomes as indicated. Statistical analyses were performed by unpaired Student's t‐test. *, P<0,05; **, P<0.01.(n=3 independent experiments, n=50 events/condition analysed). Scale bar 1 μm.D) HeLa cells were transfected as in A). Cells were imaged by TEM. Representative electron micrographs (x26500) are reported. The average number of abnormal structures per cell is shown (bottom graph). N, nucleus; M, mitochondria; ER, endoplasmic reticulum. The average number of abnormal vacuoles per cell was evaluated (upper right). Statistical analysis was performed by unpaired Student's t‐test. ***, P<0,001. A minimum of 50 cells were observed. Cell count was performed (n=3 independent experiments, n≥51 cells analysed). Data are presented as mean ± SEM. Scale bar 500 nm as indicated.E) HeLa cells stably expressing GFP-ATG13 and CFP-ATG16 were transiently co-transfected with DsRed-Rab7 and TFG siRNA (siTFG). Cells were cultured in starved conditions for 1h. Representative electron micrographs (x3900) and merge of the three fluorophores CLEM are shown. Micrographs at higher magnification (x13500) of a region showing the three fluorophores split and merged is reported. Scale bars: 5 μm upper panels, 1 μm bottom panels.G) Control (TFG-WT) and patient's (TFG-R106C) fibroblasts were grown in fed or starvation conditions for 1h. Cells were fixed, permeabilized, and labelled with anti-ULK (red) antibody. Hoechst was used to stain nuclei. The average of ULK1 puncta number per cell is shown (bottom). All data are expressed as the mean value ± SEM. Statistical analysis was performed by two-way ANOVA followed by Tukey's multiple comparison test. *, P<0.05; ***, P<0,001; ****; P<0,0001. (n=3 independent experiments, n≥14 fields analysed). Scale bar 5 μm.


Figure 5HeLa cells stably expressing untagged TFGwtLIR or TFGmutLIR3 were transfected with 3 ' UTR TFG siRNA , starved for 1h , fixed , permeabilized and immunolabelled with ULK1 ( red ) Hoechst was used to stain nuclei . ULK1 puncta number ( G ) was analysed and reported as mean ± SEM in the graph ( right ) ( n = 3 independent experiments , n = 24 fields analysed ) . Values of Mander ' s coefficient for ULK1 , expressed as percentage , are reported as mean ± SEM ( bottom graph ) . ( n = 3 independent experiments , n = 19 fields analysed ) White arrowheads point at co - localization events between ULK1 and ERGIC . Statistical analyses were performed by unpaired Student ' s t ‐ test . * * , P < 0 , 01 ; * * * , 0 , 001 . Scale bar 5 μm . HeLa cells stably expressing untagged TFGwtLIR or TFGmutLIR3 were transfected with 3 ' UTR TFG siRNA , starved for 1h , fixed , permeabilized and immunolabelled with ULK1 ( red ) alone or ULK1 ( red ) and ERGIC53 ( green ) ( H ) . Hoechst was used to stain nuclei . analysed ) . Co - localization analyses ( H ) were performed by Jacop plugin . Values of Mander ' s coefficient for ULK1 , expressed as percentage , are reported as mean ± SEM ( bottom graph ) . ( n = 3 independent experiments , n = 19 fields analysed ) White arrowheads point at co - localization events between ULK1 and ERGIC . Statistical analyses were performed by unpaired Student ' s t ‐ test . * * , P < 0 , 01 ; * * * , 0 , 001 . Scale bar 5 μm . M ) HeLa cells stably expressing untagged TFGwtLIR or TFGmutLIR3 were co - transfected with HA - LC3C and Myc - ULK1 plasmids as indicated . Cells were starved for 30 ' and protein extracts were immunoprecipitated using an anti - HA antibody or IgG as negative control . Protein extracts were analysed by WB to detect ULK1 , LC3C , TFG and ACTIN as indicated .

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 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 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

Figure 5HeLa cells stably expressing untagged TFGwtLIR or TFGmutLIR3 were transfected with 3'UTR TFG siRNA, starved for 1h, fixed, permeabilized and immunolabelled with ULK1 (red) Hoechst was used to stain nuclei. ULK1 puncta number (G) was analysed and reported as mean ± SEM in the graph (right) (n=3 independent experiments, n=24 fields analysed). Values of Mander's coefficient for ULK1, expressed as percentage, are reported as mean ± SEM (bottom graph). (n=3 independent experiments, n=19 fields analysed) White arrowheads point at co-localization events between ULK1 and ERGIC. Statistical analyses were performed by unpaired Student's t‐test. **, P<0,01; ***, 0,001. Scale bar 5 μm.HeLa cells stably expressing untagged TFGwtLIR or TFGmutLIR3 were transfected with 3'UTR TFG siRNA, starved for 1h, fixed, permeabilized and immunolabelled with ULK1 (red) alone or ULK1 (red) and ERGIC53 (green) (H). Hoechst was used to stain nuclei. analysed). Co-localization analyses (H) were performed by Jacop plugin. Values of Mander's coefficient for ULK1, expressed as percentage, are reported as mean ± SEM (bottom graph). (n=3 independent experiments, n=19 fields analysed) White arrowheads point at co-localization events between ULK1 and ERGIC. Statistical analyses were performed by unpaired Student's t‐test. **, P<0,01; ***, 0,001. Scale bar 5 μm.M) HeLa cells stably expressing untagged TFGwtLIR or TFGmutLIR3 were co-transfected with HA-LC3C and Myc-ULK1 plasmids as indicated. Cells were starved for 30' and protein extracts were immunoprecipitated using an anti-HA antibody or IgG as negative control. Protein extracts were analysed by WB to detect ULK1, LC3C, TFG and ACTIN as indicated.


Figure 1 The SnRK2 OST1 activates ABFs for expression of a luciferase reporter ( LUC ) in yeast . Luciferase activity is given on a Log10 scale as relative counts of light emission per second ( rcps ) normalized to the cell density . The OST1 - mediated induction factor is indicated above the columns . In the absence of ABFs , the LUC activity was 3 x 103 rcps . Combining the ABA signaling components ABF2 , OST1 , the PP2C ABI1 , and RCAR11 provides ABA - controlled LUC expression . The reporter is under the control of a minimal promoter that contains ABA - responsive cis elements ( ABREs ) . Core ABA signaling involves the RCAR receptor that inhibits the ABA coreceptor ( PP2C ) in the presence of ABA and allows the activation of the protein kinase SnRK2 . SnRK2 phosphorylates and activates the key transcription factor ABF , which targets promoters containing ABRE . Reporter activity was determined 16 h after exogenous ABA ( 0 . 1 mM ) administration and referred to samples expressing ABF2 alone .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 1 The SnRK2 OST1 activates ABFs for expression of a luciferase reporter (LUC) in yeast. Luciferase activity is given on a Log10 scale as relative counts of light emission per second (rcps) normalized to the cell density. The OST1-mediated induction factor is indicated above the columns. In the absence of ABFs, the LUC activity was 3 x 103 rcps.  Combining the ABA signaling components ABF2, OST1, the PP2C ABI1, and RCAR11 provides ABA-controlled LUC expression. The reporter is under the control of a minimal promoter that contains ABA-responsive cis elements (ABREs). Core ABA signaling involves the RCAR receptor that inhibits the ABA coreceptor (PP2C) in the presence of ABA and allows the activation of the protein kinase SnRK2. SnRK2 phosphorylates and activates the key transcription factor ABF, which targets promoters containing ABRE. Reporter activity was determined 16 h after exogenous ABA (0.1 mM) administration and referred to samples expressing ABF2 alone.


Figure 2 In yeast , the OST1 - and ABF2 - mediated reporter activity was inhibited by ABI1 ( ‑ RCAR ) . The subsequent recovery of the response was determined by co - expressed ABA receptors RCAR1 - RCAR14 belonging to three subfamilies , in the presence or absence of 30 µM ABA . The ABA response is expressed relative to the response obtained by replacing ABI1 with the catalytically inactive ABI1D177A . Regulation of an ABA - responsive LUC reporter in Arabidopsis protoplasts by ectopic expression of 1 µg ABI1 - effector in combination with 5 µg ABA receptor DNA ( ± 10 µM ABA ) per sample containing 105 protoplasts .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 2 In yeast, the OST1- and ABF2-mediated reporter activity was inhibited by ABI1 (‑RCAR). The subsequent recovery of the response was determined by co-expressed ABA receptors RCAR1-RCAR14 belonging to three subfamilies, in the presence or absence of 30 µM ABA. The ABA response is expressed relative to the response obtained by replacing ABI1 with the catalytically inactive ABI1D177A. Regulation of an ABA-responsive LUC reporter in Arabidopsis protoplasts by ectopic expression of 1 µg ABI1-effector in combination with 5 µg ABA receptor DNA (± 10 µM ABA) per sample containing 105 protoplasts.


Figure 3 The ABA - dependent regulation of the PP2Cs ABI1 ( A , D , G ) , ABI2 ( B , E , H ) , and HAB1 ( C , F , I ) by RCAR12 and RCAR13 were analyzed in yeast ( A - C ) , Arabidopsis protoplasts ( D - F ) , and in vitro ( G - I ) . A - C ) The analysis in yeast was conducted as mentioned in Fig . 2A except that other PP2C - RCAR combinations were used and the ABA concentrations varied as indicated . D - F ) Analysis of protoplasts was performed in the ABA - deficient background aba2 - 1 in the presence of different exogenous ABA concentrations . The ABA response was approximately tenfold inhibited by ectopic expression of the respective PP2Cs ( Tischer et al , 2017 ) . The induction of the reporter gene is related to protoplasts without ectopic RCAR expression , and in the absence of exogenous ABA . G - I ) The in vitro analysis was performed by using 50 nM PP2C and 100 nM RCAR at different ABA concentrations . Maximum inhibition of PP2C activity by ABA was set to 100 % . Data information : In ( A - C ) each data point represents the mean ± s . d . ; n = 9 biological replicates derived from three independent yeast transformants . In ( D - F ) data represent the mean ± s . d . of three independent transfections . In ( G - I ) data represent the mean ± s . d . of three replicates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 3 The ABA-dependent regulation of the PP2Cs ABI1 (A,D,G), ABI2 (B,E,H), and HAB1 (C,F,I) by RCAR12 and RCAR13 were analyzed in yeast (A-C), Arabidopsis protoplasts (D-F), and in vitro (G-I). A-C) The analysis in yeast was conducted as mentioned in Fig. 2A except that other PP2C - RCAR combinations were used and the ABA concentrations varied as indicated. D-F) Analysis of protoplasts was performed in the ABA-deficient background aba2-1 in the presence of different exogenous ABA concentrations. The ABA response was approximately tenfold inhibited by ectopic expression of the respective PP2Cs (Tischer et al, 2017). The induction of the reporter gene is related to protoplasts without ectopic RCAR expression, and in the absence of exogenous ABA. G -I) The in vitro analysis was performed by using 50 nM PP2C and 100 nM RCAR at different ABA concentrations. Maximum inhibition of PP2C activity by ABA was set to 100%. Data information: In (A-C) each data point represents the mean ± s.d.; n = 9 biological replicates derived from three independent yeast transformants. In (D-F) data represent the mean ± s.d. of three independent transfections. In (G-I) data represent the mean ± s.d. of three replicates.