Figure 2 ( A - B ) Representative MA currents recorded in whole cell patch clamp experiments at - 60 mV in HEK293 cells transiently transfected with Piezo2 + GABAB1 + GABAB2 + GFP ( for visualization ) in response to repetitive mechanical stimulation with a blunt glass probe displaced 2 - 5 . 6 µm in 0 . 4 μm increments every 15 s before ( black ) and after ( red ) exposure to 25 µM baclofen . Not every trace is shown for clarity . ( C ) Quantification of normalized MA currents of HEK293 cells transfected with Piezo2 + GABAB1 + GABAB2 + GFP [ C , n = 13 , * P < 0 . 05 , * * P < 0 . 01 , before ( black ) and after ( red ) baclofen treatment , Repeated measures ANOVA with student ' s t - test no corrections ] . Data are shown as mean ± SEM . ( D ) Quantification of MA current inactivation rate ( Tau ) in HEK293 cells transfected with Piezo2 + GABAB1 + GABAB2 + GFP before ( gray ) and after ( red ) baclofen application ( n = 13 , NS , not significant ; Paired t - test ) . Data are shown as mean ± SEM and scatter plots . Quantification of normalized MA currents of HEK293 cells transfected Piezo2 + GFP ( E , n = 7 ) before ( black ) and after ( red ) application of 25 µM baclofen . Data are shown as mean ± SEM . Quantification of normalized MA currents of HEK293 cells transfected Piezo2 + GABAB1 + GABAB2 + βARKct - mCherry ( F , n = 8 ) before ( black ) and after ( red ) application of 25 µM baclofen . Data are shown as mean ± SEM .

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 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 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 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

Figure 2(A-B) Representative MA currents recorded in whole cell patch clamp experiments at -60 mV in HEK293 cells transiently transfected with Piezo2 +GABAB1+ GABAB2 + GFP (for visualization) in response to repetitive mechanical stimulation with a blunt glass probe displaced 2-5.6 µm in 0.4 μm increments every 15 s before (black) and after (red) exposure to 25 µM baclofen. Not every trace is shown for clarity.(C) Quantification of normalized MA currents of HEK293 cells transfected with Piezo2+GABAB1+ GABAB2 + GFP [C, n=13, *P<0.05, **P<0.01, before (black) and after (red) baclofen treatment, Repeated measures ANOVA with student's t-test no corrections]. Data are shown as mean ± SEM.(D) Quantification of MA current inactivation rate (Tau) in HEK293 cells transfected with Piezo2+GABAB1+ GABAB2 + GFP before (gray) and after (red) baclofen application (n=13, NS, not significant; Paired t-test). Data are shown as mean ± SEM and scatter plots.Quantification of normalized MA currents of HEK293 cells transfected Piezo2 + GFP (E, n=7) before (black) and after (red) application of 25 µM baclofen. Data are shown as mean ± SEM.Quantification of normalized MA currents of HEK293 cells transfected Piezo2+GABAB1+ GABAB2 + βARKct-mCherry (F, n=8) before (black) and after (red) application of 25 µM baclofen. Data are shown as mean ± SEM.


Figure 3Quantification of normalized MA Piezo2 currents in HEK293 cells transiently transfected with Piezo2 + D2RS - YFP ( A , n = 13 ) before ( black ) and after ( red ) application of quinpirole 200 nM . * P < 0 . 05 , Repeated measures ANOVA with student ' s t - test no corrections . Data are shown as mean ± SEM . Quantification of normalized MA Piezo2 currents in HEK293 cells transfected with Piezo2 and GFP ( B , n = 5 ) before ( black ) and after ( red ) application of quinpirole 200 nM . * P < 0 . 05 , Repeated measures ANOVA with student ' s t - test no corrections . Data are shown as mean ± SEM . ( C ) Quantification of Piezo2 current inactivation rate ( Tau ) in HEK cells transfected with Piezo2 + D2RS - YFP . Data are shown as mean ± SEM and scatter plots ( n = 13 ) ( D , E ) Quantification of normalized MA currents in responding large DRG neurons ( D , n = 5 ) versus non - responding DRG neurons ( E , n = 3 ) before ( black ) and after ( red ) application of sumatriptan 10 µM to activate the Gi - coupled Htr1d receptors . * P < 0 . 05 , Repeated measures ANOVA with student ' s t - test no corrections . Data are shown as mean ± SEM . ( F ) Quantification of MA current inactivation rate ( Tau ) measured for responding ( R ) large DRG neurons ( n = 5 , Paired t - test ) . Data are shown as mean ± SEM and scatter plots . ( G - H ) Quantification of normalized MA currents recorded from large DRG neurons dialyzed with 50 ng / ml Gβγ ( G , n = 7 ) or with 0 . 0001 % Lubrol ( vehicle for Gβγ ) ( H , n = 6 ) . * P < 0 . 05 , Repeated measures ANOVA with student ' s t - test no corrections . Data are shown as mean ± SEM . ( I , J ) Quantification of MA current inactivation rate ( Tau ) measured for large DRG neurons dialyzed with Gβγ ( n = 7 , * * P < 0 . 01 , Paired t - test ) and vehicle ( Paired t - test ) . Data are shown as mean ± SEM and scatter plots .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O 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 3Quantification of normalized MA Piezo2 currents in HEK293 cells transiently transfected with Piezo2+D2RS-YFP (A, n=13) before (black) and after (red) application of quinpirole 200 nM. *P<0.05, Repeated measures ANOVA with student's t-test no corrections. Data are shown as mean ± SEM.Quantification of normalized MA Piezo2 currents in HEK293 cells transfected with Piezo2 and GFP (B, n=5) before (black) and after (red) application of quinpirole 200 nM. *P<0.05, Repeated measures ANOVA with student's t-test no corrections. Data are shown as mean ± SEM.(C) Quantification of Piezo2 current inactivation rate (Tau) in HEK cells transfected with Piezo2+D2RS-YFP. Data are shown as mean ± SEM and scatter plots (n=13)(D,E) Quantification of normalized MA currents in responding large DRG neurons (D, n=5) versus non-responding DRG neurons (E, n=3) before (black) and after (red) application of sumatriptan 10 µM to activate the Gi-coupled Htr1d receptors. *P<0.05, Repeated measures ANOVA with student's t-test no corrections. Data are shown as mean ± SEM.(F) Quantification of MA current inactivation rate (Tau) measured for responding (R) large DRG neurons (n=5, Paired t-test). Data are shown as mean ± SEM and scatter plots.(G-H) Quantification of normalized MA currents recorded from large DRG neurons dialyzed with 50 ng/ml Gβγ (G, n=7) or with 0.0001% Lubrol (vehicle for Gβγ) (H, n=6). *P<0.05, Repeated measures ANOVA with student's t-test no corrections. Data are shown as mean ± SEM.(I,J) Quantification of MA current inactivation rate (Tau) measured for large DRG neurons dialyzed with Gβγ (n=7, **P<0.01, Paired t-test) and vehicle (Paired t-test). Data are shown as mean ± SEM and scatter plots.


Figure 4Quantification of normalized MA Piezo2 currents in HEK293 cells transiently transfected with Piezo2 + GABAB1 + GABAB2 + GFP ( A , n = 12 ) Quantification of normalized MA Piezo2 currents in HEK293 cells transiently transfected with Piezo2 + GABAB1 + GABAB2 + GFP ( A , n = 12 ) , treated with the PI3K inhibitor Wortmannin 50 nM ( B , n = 10 ) Cells were treated with wortmannin 30 min before the experiments , and were present throughout the measurements . Quantification of normalized MA Piezo2 currents in HEK293 cells transiently transfected with Piezo2 + GABAB1 + GABAB2 + GFP ( A , n = 12 ) , treated with the MAPK inhibitor U0126 10 µM ( C , n = 7 ) . Cells were treated with U0126 for 30 min before the experiments , and were present throughout the measurements .

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

Figure 4Quantification of normalized MA Piezo2 currents in HEK293 cells transiently transfected with Piezo2+ GABAB1+ GABAB2 + GFP (A, n=12)Quantification of normalized MA Piezo2 currents in HEK293 cells transiently transfected with Piezo2+ GABAB1+ GABAB2 + GFP (A, n=12), treated with the PI3K inhibitor Wortmannin 50 nM (B, n=10) Cells were treated with wortmannin 30 min before the experiments, and were present throughout the measurements.Quantification of normalized MA Piezo2 currents in HEK293 cells transiently transfected with Piezo2+ GABAB1+ GABAB2 + GFP (A, n=12), treated with the MAPK inhibitor U0126 10 µM (C, n=7). Cells were treated with U0126 for 30 min before the experiments, and were present throughout the measurements.


Figure 5Mice were injected in the plantar surface of the hind paw with sumatriptan , or vehicle ± the combination of wortmannin and U0126 , as described in the methods section . Mechanical sensitivity was assessed 30 min later using von Frey filaments in an ascending order . ( A - C ) Percentage of withdrawal response for female mice using von Frey filaments at : 0 . 07 g ( A ) , 0 . 16 g ( B ) and 0 . 4 g ( C ) . ( n = 11 - 16 per group , * * * P < 0 . 001 , * * * * P < 0 . 0001 , One way - ANOVA with Tukey ' s multiple comparison test ) . Data are shown as mean ± SEM and scatter plots . Mice were injected in the plantar surface of the hind paw with sumatriptan , or vehicle ± the combination of wortmannin and U0126 , as described in the methods section . Mechanical sensitivity was assessed 30 min later using von Frey filaments in an ascending order . ( D - F ) Percentage of withdrawal response for male mice using von Frey filaments at : 0 . 07 g ( D ) , 0 . 16 g ( E ) and 0 . 4 g ( F ) . ( n = 12 - 16 per group , * * * P < 0 . 001 , * * * * P < 0 . 0001 , One way - ANOVA with Tukey ' s multiple comparison test ) . Data are shown as mean ± SEM and scatter plots .

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 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

Figure 5Mice were injected in the plantar surface of the hind paw with sumatriptan, or vehicle ± the combination of wortmannin and U0126, as described in the methods section. Mechanical sensitivity was assessed 30 min later using von Frey filaments in an ascending order. (A-C) Percentage of withdrawal response for female mice using von Frey filaments at: 0.07 g (A), 0.16 g (B) and 0.4 g (C). (n= 11-16 per group, ***P<0.001, ****P<0.0001, One way- ANOVA with Tukey's multiple comparison test). Data are shown as mean ± SEM and scatter plots. Mice were injected in the plantar surface of the hind paw with sumatriptan, or vehicle ± the combination of wortmannin and U0126, as described in the methods section. Mechanical sensitivity was assessed 30 min later using von Frey filaments in an ascending order. (D-F) Percentage of withdrawal response for male mice using von Frey filaments at: 0.07 g (D), 0.16 g (E) and 0.4 g (F). (n= 12-16 per group, ***P<0.001, ****P<0.0001, One way- ANOVA with Tukey's multiple comparison test). Data are shown as mean ± SEM and scatter plots.


Figure 1 ( A ) Purification of the Halo - tagged cohesin tetramer . Human cohesin tetramer tagged by Halo was purified from insect cells and Alexa488 dye was conjugated to Halo - tag ( cohesinHalo488 ) . ( B ) CohesinHalo488 loaded onto DNA in the presence of Scc2 - Scc4 was incubated in the buffer with or without TEV protease . After high - salt washing , CohesinHalo488 intensities on DNA were measured . Red bars denote the median , lower , and upper quartile values ( n ≥ 98 ; * p < 0 . 0001 , two - tailed Mann - Whitney test ) . ( C ) Kymograph of a single DNA - interacting cohesinHalo488 particle . CohesinHalo488 is loaded onto DNA in the presence of Scc2 - Scc4 and , after high - salt washing , single cohesin particles were observed in the presence of ATP and 100 mM KCl . Scale bar , 2 μm . ( D ) Histogram of the net displacement ( xfinal - xinitial ) of cohesin particles treated as in ( C ) . The magenta curve represents the Gaussian fit . ( E ) CohesinHalo488 was loaded and washed in high salt as in ( C ) , and the cohesin particles were observed in the presence of ATP and 100 mM or 750 mM KCl . Kymograph of DNA - associated cohesin particles under high - salt conditions ( 750 mM KCl , right ) and the mean square displacement ( MSD ) versus time ( left ) are shown . Dotted lines are regression lines used to estimate the diffusion coefficient ( D ) . Scale bar , 2 μm . ( F ) MSD versus time for DNA - bound cohesinWT - Halo488 or cohesinKA - Halo488 particles . CohesinWT - Halo488 or cohesinKA - Halo488 was loaded onto DNA in the presence of Scc2 - 4 and ATP . After high - salt washing , the cohesin particles on DNA were observed in the presence of 1 mM AMP - PCP or ATP and 100 mM KCl . D indicates the diffusion coefficient ( n = 45 , mean ± s . e . m . ) .

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

Figure 1(A) Purification of the Halo-tagged cohesin tetramer. Human cohesin tetramer tagged by Halo was purified from insect cells and Alexa488 dye was conjugated to Halo-tag (cohesinHalo488).(B) CohesinHalo488 loaded onto DNA in the presence of Scc2-Scc4 was incubated in the buffer with or without TEV protease. After high-salt washing, CohesinHalo488 intensities on DNA were measured. Red bars denote the median, lower, and upper quartile values (n ≥ 98; *p < 0.0001, two-tailed Mann-Whitney test).(C) Kymograph of a single DNA-interacting cohesinHalo488 particle. CohesinHalo488 is loaded onto DNA in the presence of Scc2-Scc4 and, after high-salt washing, single cohesin particles were observed in the presence of ATP and 100 mM KCl. Scale bar, 2 μm.(D) Histogram of the net displacement (xfinal - xinitial) of cohesin particles treated as in (C). The magenta curve represents the Gaussian fit.(E) CohesinHalo488 was loaded and washed in high salt as in (C), and the cohesin particles were observed in the presence of ATP and 100 mM or 750 mM KCl. Kymograph of DNA-associated cohesin particles under high-salt conditions (750 mM KCl, right) and the mean square displacement (MSD) versus time (left) are shown. Dotted lines are regression lines used to estimate the diffusion coefficient (D). Scale bar, 2 μm.(F) MSD versus time for DNA-bound cohesinWT-Halo488 or cohesinKA-Halo488 particles. CohesinWT-Halo488 or cohesinKA-Halo488 was loaded onto DNA in the presence of Scc2-4 and ATP. After high-salt washing, the cohesin particles on DNA were observed in the presence of 1 mM AMP-PCP or ATP and 100 mM KCl. D indicates the diffusion coefficient (n = 45, mean ± s.e.m.).


Figure 2 ( A ) Kymograph of CohesinHalo488 in the presence or absence of Wapl - Pds5 . CohesinHalo488 was loaded onto DNA in the presence of Scc2 - Scc4 and washed in high - salt buffer . DNA - bound cohesin was further incubated with or without Wapl - Pds5 , and then cohesinHalo488 particles were observed in the presence of ATP and 100 mM KCl . Scale bar , 1 μm . ( B ) MSD versus time of cohesin particles in ( A ) are shown . D indicates the diffusion coefficient ( n = 45 , mean ± s . e . m . ) . ( C ) Acetylated - or unacetylated - cohesinHalo488 was loaded onto DNA in the presence of Scc2 - Scc4 and washed in high - salt buffer . DNA - bound cohesin was further incubated with Wapl - Pds5 , and then cohesinHalo488 particles were observed in the presence of ATP and 100 mM KCl . MSD versus time is shown . D indicates the diffusion coefficient ( n = 45 , mean ± s . e . m . ) . ( D ) Acetylated cohesinHalo488 was treated as in ( C ) , the DNA - bound cohesin was further incubated with buffer , Wapl - Pds5 , or Wapl - Pds5 plus Sororin , and then cohesinHalo488 particles were observed in the presence of ATP and 100 mM KCl . MSD versus time is shown . D indicates the diffusion coefficient ( n = 45 , mean ± s . e . m . ) .

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

Figure 2(A) Kymograph of CohesinHalo488 in the presence or absence of Wapl-Pds5. CohesinHalo488 was loaded onto DNA in the presence of Scc2-Scc4 and washed in high-salt buffer. DNA-bound cohesin was further incubated with or without Wapl-Pds5, and then cohesinHalo488 particles were observed in the presence of ATP and 100 mM KCl. Scale bar, 1 μm.(B) MSD versus time of cohesin particles in (A) are shown. D indicates the diffusion coefficient (n = 45, mean ± s.e.m.).(C) Acetylated- or unacetylated-cohesinHalo488 was loaded onto DNA in the presence of Scc2-Scc4 and washed in high-salt buffer. DNA-bound cohesin was further incubated with Wapl-Pds5, and then cohesinHalo488 particles were observed in the presence of ATP and 100 mM KCl. MSD versus time is shown. D indicates the diffusion coefficient (n = 45, mean ± s.e.m.).(D) Acetylated cohesinHalo488 was treated as in (C), the DNA-bound cohesin was further incubated with buffer, Wapl-Pds5, or Wapl-Pds5 plus Sororin, and then cohesinHalo488 particles were observed in the presence of ATP and 100 mM KCl. MSD versus time is shown. D indicates the diffusion coefficient (n = 45, mean ± s.e.m.).


Figure 3 ( A ) Acetylated cohesinHalo488 was loaded onto DNA in the presence of Scc2 - Scc4 and washed in high - salt buffer . DNA - bound cohesin was further incubated with Wapl - Pds5 plus Sororin , and then treated with buffer , Plk1 , or Plk1 plus BI4834 . CohesinHalo488 particles were observed in the presence of ATP and 100 mM KCl . MSD versus time is shown . D indicates the diffusion coefficient ( n = 45 , mean ± s . e . m . ) . ( B ) Acetylated cohesinHalo488 was treated as in ( A ) , or treated with CDK1 or Aurora B rather than Plk1 . Sororin intensity on DNA was measured after immunofluorescence microscopy with anti - Sororin antibody ( n > 100 , * p < 0 . 0001 , two - tailed Mann - Whitney test ) . ( C ) Acetylated cohesinHalo488 was treated as in ( A ) but treated with Aurora B rather than Plk1 . CohesinHalo488 particles were observed in the presence of ATP and 100 mM KCl . MSD versus time is shown . D indicates the diffusion coefficient ( n = 45 , mean ± s . e . m . ) .

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 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

Figure 3(A) Acetylated cohesinHalo488 was loaded onto DNA in the presence of Scc2-Scc4 and washed in high-salt buffer. DNA-bound cohesin was further incubated with Wapl-Pds5 plus Sororin, and then treated with buffer, Plk1, or Plk1 plus BI4834. CohesinHalo488 particles were observed in the presence of ATP and 100 mM KCl. MSD versus time is shown. D indicates the diffusion coefficient (n = 45, mean ± s.e.m.).(B) Acetylated cohesinHalo488 was treated as in (A), or treated with CDK1 or Aurora B rather than Plk1. Sororin intensity on DNA was measured after immunofluorescence microscopy with anti-Sororin antibody (n > 100, *p < 0.0001, two-tailed Mann-Whitney test).(C) Acetylated cohesinHalo488 was treated as in (A) but treated with Aurora B rather than Plk1. CohesinHalo488 particles were observed in the presence of ATP and 100 mM KCl. MSD versus time is shown. D indicates the diffusion coefficient (n = 45, mean ± s.e.m.).


Figure 4 ( A ) Stretched DNA was incubated in the HSS in the presence or absence of geminin . After washing the HSS , endogenous Smc3 ( green ) and Mcm2 ( magenta ) were detected by immunofluorescence staining . DNA was counterstained with SYTOX ( gray ) . Scale bar , 2 µm . ( B ) Numbers of Smc3 and Mcm2 signals were counted on 10 μm DNA shown in ( A ) . ( 22 DNAs per condition , n = 3 , mean ± s . e . m . * p < 0 . 0001 , two - tailed Mann - Whitney test ) . ( C ) Chromatin formed in the HSS in the presence of geminin , and cohesinHalo488 ( green ) was loaded onto the chromatin with Scc2 - Scc4 . Chromatin was detected by immunostaining of histone H3 ( magenta ) . DNA was counterstained with SYTOX ( gray ) . The distributions of cohesin particles and histone H3 are shown as line - scan plots ( bottom ) . Scale bar , 5 μm . ( D ) Cohesin particles observed in ( C ) were categorized as indicated ( > 37 particles per condition . n = 6 , mean ± s . e . m . ) . ( E ) Cohesin particle numbers for each condition . Red bars denote the median , lower , and upper quartile values ( n ≥ 12 , * p < 0 . 0004 , unpaired t - test ) . ( F ) Colocalization of Mcm2 with Smc3 was quantified ( 50 Mcm2 particles per condition , n = 3 , mean ± s . e . m . )

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Figure 4(A) Stretched DNA was incubated in the HSS in the presence or absence of geminin. After washing the HSS, endogenous Smc3 (green) and Mcm2 (magenta) were detected by immunofluorescence staining. DNA was counterstained with SYTOX (gray). Scale bar, 2 µm.(B) Numbers of Smc3 and Mcm2 signals were counted on 10 μm DNA shown in (A). (22 DNAs per condition, n = 3, mean ± s.e.m. *p < 0.0001, two-tailed Mann-Whitney test).(C) Chromatin formed in the HSS in the presence of geminin, and cohesinHalo488 (green) was loaded onto the chromatin with Scc2-Scc4. Chromatin was detected by immunostaining of histone H3 (magenta). DNA was counterstained with SYTOX (gray). The distributions of cohesin particles and histone H3 are shown as line-scan plots (bottom). Scale bar, 5 μm.(D) Cohesin particles observed in (C) were categorized as indicated (> 37 particles per condition. n = 6, mean ± s.e.m.).(E) Cohesin particle numbers for each condition. Red bars denote the median, lower, and upper quartile values (n ≥ 12, *p < 0.0004, unpaired t-test).(F) Colocalization of Mcm2 with Smc3 was quantified (50 Mcm2 particles per condition, n = 3, mean ± s.e.m.)


Figure 5 ( A ) Stretched DNA was incubated in the HSS . Endogenous Smc3 ( green ) and Mcm2 ( magenta ) were detected by immunofluorescence staining . DNA was counterstained with SYTOX ( gray ) . Colocalization of Smc3 and Mcm2 was quantified ( bottom , n = 3 , mean ± s . e . m . ) . Scale bar , 2 μm . ( B ) Fluorescently labeled Xenopus cohesins ( xCohesinHaloTMR ) were loaded onto DNA in the HSS , and DNA - bound cohesin particles were observed in the HSS with or without 5 mM AMP - PNP supplementation . On the kymograph , the magenta lines indicate the duration of DNA binding of each cohesin particle . DNA was counterstained with SYTOX . Scale bar , 5 μm . ( C ) Kymographs of DNA - bound xCohesinHaloTMR ( green ) in HSS . After time - lapse imaging of xCohesinHaloTMR , nucleosomes were immunostained by anti - H3 antibody ( magenta ) . Result of H3 immunostaining was merged with the kymograph . DNA was counterstained with SYTOX . Scale bar , 5 μm . ( D ) HSS were subjected to mock or XEco2 immunodepletion and human Esco1 - His ( hEsco1 ) was added back to XEco2 - depleted HSS . Sperm chromatin was incubated in these extracts and chromatin - bound proteins were analyzed by immunoblotting . ( E ) Kymographs of DNA - bound xCohesinHaloTMR in mock - , XEco2 - depleted , or hEsco1 - added XEco2 - depleted HSS . Scale bar , 2 μm . ( F ) MSD versus time of cohesin particle in the experiment in ( E ) . D indicates the diffusion coefficient ( n = 15 , mean ± s . e . m . ) .

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Figure 5(A) Stretched DNA was incubated in the HSS. Endogenous Smc3 (green) and Mcm2 (magenta) were detected by immunofluorescence staining. DNA was counterstained with SYTOX (gray). Colocalization of Smc3 and Mcm2 was quantified (bottom, n = 3, mean ± s.e.m.). Scale bar, 2 μm.(B) Fluorescently labeled Xenopus cohesins (xCohesinHaloTMR) were loaded onto DNA in the HSS, and DNA-bound cohesin particles were observed in the HSS with or without 5 mM AMP-PNP supplementation. On the kymograph, the magenta lines indicate the duration of DNA binding of each cohesin particle. DNA was counterstained with SYTOX. Scale bar, 5 μm.(C) Kymographs of DNA-bound xCohesinHaloTMR (green) in HSS. After time-lapse imaging of xCohesinHaloTMR, nucleosomes were immunostained by anti-H3 antibody (magenta). Result of H3 immunostaining was merged with the kymograph. DNA was counterstained with SYTOX. Scale bar, 5 μm.(D) HSS were subjected to mock or XEco2 immunodepletion and human Esco1-His (hEsco1) was added back to XEco2-depleted HSS. Sperm chromatin was incubated in these extracts and chromatin-bound proteins were analyzed by immunoblotting.(E) Kymographs of DNA-bound xCohesinHaloTMR in mock-, XEco2-depleted, or hEsco1-added XEco2-depleted HSS. Scale bar, 2 μm.(F) MSD versus time of cohesin particle in the experiment in (E). D indicates the diffusion coefficient (n = 15, mean ± s.e.m.).


Figure 6 ( B ) Kymographs of examples for cohesin motion during DNA replication . xCohesinHaloTMR ( magenta ) was loaded onto DNA in the HSS , and NPE was introduced to allow DNA replication . DNA replication was monitored by xFen1D179A - PAGFP ( green ) . Thirthy - four particles were categorized into five groups . Arrowheads indicate the time points when cohesin was dissociated from chromatin . Scale bar , 2μm .

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Figure 6(B) Kymographs of examples for cohesin motion during DNA replication. xCohesinHaloTMR (magenta) was loaded onto DNA in the HSS, and NPE was introduced to allow DNA replication. DNA replication was monitored by xFen1D179A-PAGFP (green). Thirthy-four particles were categorized into five groups. Arrowheads indicate the time points when cohesin was dissociated from chromatin. Scale bar, 2μm.


Figure 1B Analysis of cell differentiation ( left ) and proliferation ( right ) in the intestine after CDK8 deletion as in ( A ) . Olfm4 , Lysozyme , PAS and Dclk1 staining was used to reveal , respectively , stem , Paneth , goblet and tuft cells . β - Catenin staining allows detection of cancer cells ( cytoplasmic vs nuclear localisation ) . Cell proliferation was assessed by PCNA , Ki - 67 and BrdU ( 2h pulse ) staining . Scatter plots represent the percentage of the area stained by each antibody ( relative to the area occupied by hematoxylin ) . For Paneth cells , BrdU , PCNA and Ki67 , only crypts were analysed . For goblet cells , crypts and villi were analysed . For Tuft cell quantification , Dclk1 - positive cells were counted in 50 villi . Colour code depicts small intestine ( green ) , proximal colon ( blue ) , and distant colon ( red ) . Mean ± SEM is shown . P - value of unpaired two - tailed t - test is indicated ( ns , not significant ; p > 0 . 05 ) . Scale bars , 25μm ( Olfm4 , Lysozyme , Dclk1 and β - Catenin ) and 50μm ( PAS , BrdU , Ki - 67 and PCNA ) . ( n = 9 biological replicates ) C Analysis of mouse colon after AOM / DSS treatment . Quantification of the number of neoplastic lesions ( n = 10 for Cdk8 + / + , and n = 7 for Cdk8 - / - mice ) . P - value of unpaired t - test is indicated : ns , not significant ( p > 0 . 05 ) . Mean ± SD is shown . D Quantification of the percentage of the colon surface occupied by tumours . Intestine samples were stained for β - Catenin , and tumour regions with nuclear β - Catenin localisation were quantified using NDP . view software . Two - tailed p - value of unpaired t - test is indicated ; ns , not significant ( p > 0 . 05 ) . Mean ± SD is shown . ( n = 6 biological replicates ) E Example of IHC with β - Catenin staining of tumour - free regions with membrane β - catenin localisation ( left ) and tumour regions with nuclear β - catenin localisation ( right ) . Scale bars , 50μm .

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Figure 1B Analysis of cell differentiation (left) and proliferation (right) in the intestine after CDK8 deletion as in (A). Olfm4, Lysozyme, PAS and Dclk1 staining was used to reveal, respectively, stem, Paneth, goblet and tuft cells. β-Catenin staining allows detection of cancer cells (cytoplasmic vs nuclear localisation). Cell proliferation was assessed by PCNA, Ki-67 and BrdU (2h pulse) staining. Scatter plots represent the percentage of the area stained by each antibody (relative to the area occupied by hematoxylin). For Paneth cells, BrdU, PCNA and Ki67, only crypts were analysed. For goblet cells, crypts and villi were analysed. For Tuft cell quantification, Dclk1-positive cells were counted in 50 villi. Colour code depicts small intestine (green), proximal colon (blue), and distant colon (red). Mean ± SEM is shown. P-value of unpaired two-tailed t-test is indicated (ns, not significant; p > 0.05). Scale bars, 25μm (Olfm4, Lysozyme, Dclk1 and β-Catenin) and 50μm (PAS, BrdU, Ki-67 and PCNA). (n=9 biological replicates)C Analysis of mouse colon after AOM/DSS treatment. Quantification of the number of neoplastic lesions (n = 10 for Cdk8+/+, and n = 7 for Cdk8 -/- mice). P-value of unpaired t-test is indicated: ns, not significant (p > 0.05). Mean ± SD is shown.D Quantification of the percentage of the colon surface occupied by tumours. Intestine samples were stained for β-Catenin, and tumour regions with nuclear β-Catenin localisation were quantified using NDP.view software. Two-tailed p-value of unpaired t-test is indicated; ns, not significant (p > 0.05). Mean ± SD is shown. (n=6 biological replicates)E Example of IHC with β-Catenin staining of tumour-free regions with membrane β-catenin localisation (left) and tumour regions with nuclear β-catenin localisation (right). Scale bars, 50μm.


Figure 2A Genotyping confirms the loss of Cdk8 exon 2 in Cdk8 - / - and Cdk8 - / - / Cdk19 - / - organoids after 7 days of OH - tamoxifen treatment . B WB of organoid samples after 7 days of OH - tamoxifen treatment ; β - actin was used as loading control . C Phase contrast images of organoids before and after 6 days of OH - tamoxifen treatment . Scale bars , 150μm . D Quantification of organoid size at day 6 , shown in ( C ) ( mean ± SEM are shown ) . P - value , one - way ANOVA ( Bonferroni test ) : ( * ) p ≤ 0 . 05 , ( * * * ) p ≤ 0 . 001 ; ns , not significant ( p > 0 . 05 ) . ( n = 47 biological replicates ) E IHC staining of organoids ( day 7 of OH - tamoxifen treatment ) with Ki - 67 antibody . Scale bars , 100μm . F Quantification of Ki - 67 positive area ( % of the total area of the organoids ; mean ± SEM ) in the four different genotypes presented in ( E ) . Areas with positive Ki - 67 signal were detected and quantified using QuPath and ImageJ programs . Four technical replicates from 2 biological replicates of each genotype were analysed . Adjusted p - values of ordinary one - way ANOVA ( Bonferroni test ) are indicated : ( * * ) p - values ≤ 0 . 01 ; ( * ) p - values ≤ 0 . 05 ; ns , not significant ( p > 0 . 05 ) .

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Figure 2A Genotyping confirms the loss of Cdk8 exon 2 in Cdk8 -/- and Cdk8 -/-/Cdk19 -/- organoids after 7 days of OH-tamoxifen treatment.B WB of organoid samples after 7 days of OH-tamoxifen treatment; β-actin was used as loading control.C Phase contrast images of organoids before and after 6 days of OH-tamoxifen treatment. Scale bars, 150μm. D Quantification of organoid size at day 6, shown in (C) (mean ± SEM are shown). P-value, one-way ANOVA (Bonferroni test): (*) p ≤ 0.05, (***) p ≤ 0.001; ns, not significant (p > 0.05). (n=47 biological replicates)E IHC staining of organoids (day 7 of OH-tamoxifen treatment) with Ki-67 antibody. Scale bars, 100μm. F Quantification of Ki-67 positive area (% of the total area of the organoids; mean ± SEM) in the four different genotypes presented in (E). Areas with positive Ki-67 signal were detected and quantified using QuPath and ImageJ programs. Four technical replicates from 2 biological replicates of each genotype were analysed. Adjusted p-values of ordinary one-way ANOVA (Bonferroni test) are indicated: (**) p-values ≤ 0.01; (*) p-values ≤ 0.05; ns, not significant (p > 0.05).


Figure 3C Left , IHC staining of WT and Cdk8 - / - / Cdk19 - / - organoids with Olfm4 ( stem cells ) , Lysozyme ( Paneth cells ) , Muc2 ( goblet cells ) , and Dclk1 ( tuft cells ) antibodies . Scale bars , 50μm ( Olfm4 , Muc2 , Dclk1 ) and 25μm ( Lysozyme ) . Right , quantification of Olfm4 , Lysozyme and Muc2 positive area ( % of the total organoid area as indicated by hematoxylin staining ; mean ± SEM ) , or the number of Dclk1 positive cells per organoid . P - value , unpaired two - tailed t - test : ( * * * ) p - value ≤ 0 . 001 ; ns , not significant ( p > 0 . 05 ) . 20 different organoids from 2 biological replicates of each genotype were used for the analysis .

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Figure 3C Left, IHC staining of WT and Cdk8 -/-/Cdk19 -/- organoids with Olfm4 (stem cells), Lysozyme (Paneth cells), Muc2 (goblet cells), and Dclk1 (tuft cells) antibodies. Scale bars, 50μm (Olfm4, Muc2, Dclk1) and 25μm (Lysozyme). Right, quantification of Olfm4, Lysozyme and Muc2 positive area (% of the total organoid area as indicated by hematoxylin staining; mean ± SEM), or the number of Dclk1 positive cells per organoid. P-value, unpaired two-tailed t-test: (***) p-value ≤ 0.001; ns, not significant (p > 0.05). 20 different organoids from 2 biological replicates of each genotype were used for the analysis.


Figure 4D WB of indicated proteins extracted from Wt and Cdk8 - / - / Cdk19 - / - organoids . Included are CFTR positive and negative control cell line samples obtained from the Cystic Fibrosis Foundation ( CFF ) . Amidoblack was used as loading control .

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Figure 4D WB of indicated proteins extracted from Wt and Cdk8 -/-/Cdk19 -/- organoids. Included are CFTR positive and negative control cell line samples obtained from the Cystic Fibrosis Foundation (CFF). Amidoblack was used as loading control.


Figure 5A Histological PAS staining of organoids treated for 7 days with OH - tamoxifen . Scale bar , 50μm . B Quantification of PAS signal ( % of total organoid area ; mean ± SD are shown ) in the four different genotypes presented in ( A ) . Areas containing positive PAS staining were detected and quantified using QuPath and ImageJ programs . Adjusted p - values of ordinary one - way Anova followed by Tukey ' s multiple comparison test are indicated : ( * * * ) p - value ≤ 0 . 001 ; ns : not significant ( p > 0 . 05 ) . Four technical replicates from 2 biological replicates of each genotype were analysed . C Representative phase contrast images of organoids at the indicated time points after 7 days of OH - tamoxifen treatment are shown . Scale bar , 100 μm . D Quantification of the time needed for mucus release ( observed as a dark staining in the center of the organoid ; mean ± SD are shown ) . Adjusted p - values of ordinary one - way Anova followed by Tukey ' s multiple comparison test are indicated : ( * * * ) p - value ≤ 0 . 001 ; ns : not significant ( p > 0 . 05 ) ; ( n = 17 for Wt , 11 for Cdk8 - / - , 18 for Cdk19 - / - , and 12 for Cdk8 - / - / Cdk19 - / - , all biological replicates ) . E Fluorescence confocal microscopy images of Calcein green - labeled WT and Cdk8 - / - / Cdk19 - / - organoids treated with forskolin . Scale bars , 100 μm . F Quantification of forskolin - induced swelling in WT organoids treated for 1hour or 24 hours with 0 . 1μM , 1μM or 10μM Senexin B ( SenB ) , as indicated , or double KO organoids ; DMSO vehicle was used as control . The surface area of individual organoids at different time points relative to the area at t = 0 ( 100 % ) was measured ( mean ± SD , n = 8 biological replicates ) . Linear regression lines are shown . H WB of indicated proteins extracted from Senexin B - treated ( 10μM , 2h and 24h ) organoids ( left ) , and WT and Cdk8 - / - / Cdk19 - / - organoids ( right ) . Amidoblack was used as loading control .

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Figure 5A Histological PAS staining of organoids treated for 7 days with OH-tamoxifen. Scale bar, 50μm. B Quantification of PAS signal (% of total organoid area; mean ± SD are shown) in the four different genotypes presented in (A). Areas containing positive PAS staining were detected and quantified using QuPath and ImageJ programs. Adjusted p-values of ordinary one-way Anova followed by Tukey's multiple comparison test are indicated: (***) p-value ≤ 0.001; ns: not significant (p > 0.05). Four technical replicates from 2 biological replicates of each genotype were analysed.C Representative phase contrast images of organoids at the indicated time points after 7 days of OH-tamoxifen treatment are shown. Scale bar, 100 μm. D Quantification of the time needed for mucus release (observed as a dark staining in the center of the organoid; mean ± SD are shown). Adjusted p-values of ordinary one-way Anova followed by Tukey's multiple comparison test are indicated: (***) p-value ≤ 0.001; ns: not significant (p > 0.05); (n= 17 for Wt, 11 for Cdk8 -/-, 18 for Cdk19 -/-, and 12 for Cdk8 -/-/ Cdk19 -/- , all biological replicates).E Fluorescence confocal microscopy images of Calcein green-labeled WT and Cdk8-/-/Cdk19-/- organoids treated with forskolin. Scale bars, 100 μm. F Quantification of forskolin-induced swelling in WT organoids treated for 1hour or 24 hours with 0.1μM, 1μM or 10μM Senexin B (SenB), as indicated, or double KO organoids; DMSO vehicle was used as control. The surface area of individual organoids at different time points relative to the area at t = 0 (100%) was measured (mean ± SD, n=8 biological replicates). Linear regression lines are shown.H WB of indicated proteins extracted from Senexin B-treated (10μM, 2h and 24h) organoids (left), and WT and Cdk8 -/-/ Cdk19 -/- organoids (right). Amidoblack was used as loading control.


Figure 6A WB of indicated proteins extracted from intestinal epithelium of control mice or mice treated with Senexin 631 ( Snx 631 ) for 4 or 11 days . B Left , immunohistochemical PAS and hematoxylin staining of mouse small intestines collected at 4 and 11 days of Senexin 631 ( Snx 631 ) treatment ; scale bars 50μm . Right , quantification of PAS - and hematoxilin ( Hema ) - positive area ( mean ± SEM ) , using QuPath and ImageJ software . Adjusted p - values of unpaired t - test are shown : ( * ) p < 0 . 05 ; ns , not significant ( p > 0 . 05 ) . 8 mice ( 4 treated with Senexin 631 and and 4 control ) were used in this quantification .

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Figure 6A WB of indicated proteins extracted from intestinal epithelium of control mice or mice treated with Senexin 631 (Snx 631) for 4 or 11 days.B Left, immunohistochemical PAS and hematoxylin staining of mouse small intestines collected at 4 and 11 days of Senexin 631 (Snx 631) treatment; scale bars 50μm. Right, quantification of PAS- and hematoxilin (Hema)-positive area (mean ± SEM), using QuPath and ImageJ software. Adjusted p-values of unpaired t-test are shown: (*) p < 0.05; ns, not significant (p > 0.05). 8 mice (4 treated with Senexin 631 and and 4 control) were used in this quantification.


Figure 1A , Representative immunofluorescence staining of normal , tumor , and residual 3D organoids grown from primary mammary cells of a preclinical mouse model of breast cancer ( Methods ) . Similar morphology of normal and residual organoids shown on the left with polarity markers ITGA6 ( red ) , ZO - 1 ( green ) , GM - 130 ( magenta ) ; DAPI ( blue ) . Right : human MYC oncoprotein ( green ) is expressed only in tumor cells , CDH1 ( red ) . Scale bar : 25 μm . Data information : Number of replicates corresponds to individual mice used to derive organoids .

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Figure 1A, Representative immunofluorescence staining of normal, tumor, and residual 3D organoids grown from primary mammary cells of a preclinical mouse model of breast cancer (Methods). Similar morphology of normal and residual organoids shown on the left with polarity markers ITGA6 (red), ZO-1 (green), GM-130 (magenta); DAPI (blue). Right: human MYC oncoprotein (green) is expressed only in tumor cells, CDH1 (red). Scale bar: 25 μm. Data information: Number of replicates corresponds to individual mice used to derive organoids.


Figure 3 , Selective secreted metabolites with significant change linked to urea cycle components from healthy ( n = 3 ; blue ) and regressed ( n = 3 ; green ) mammary glands . Values represent metabolite abundance levels as quantified by the area under the curve ( AUC ) of the marker fragment ions / transitions for each metabolite . Values are plotted on the log2 scale . Statistics were calculated using the limma package n R with the significance threshold corresponding to a Benjamini - Hochberg adjusted p - value ≤ 0 . 05 ( residual compared to normal ) . ata information : umbers of replicates correspond to individual mice or humans . ox plots : midline , median ; box , 25 - 75th percentile ; whisker , minimum to maximum . umbers marking comparisons ( gray lines ) show p values ( corresponding statistical tests are described in individual panel legends ) . C , Left , Quantification of cells expressing ARG1 ( top ) , an enzyme converting arginine to urea and ornithine , and intensity of ARG1 ( bottom ) in normal cells from healthy ( n = 5 , 2921 cells analyzed ; blue ) and residual cells from regressed ( n = 5 , 2241 cells analyzed ; green ) mammary gland tissue sections . Statistical differences were calculated with the Mann - Whitney U test ( Wilcoxon rank - sum test ) . Right , Representative images of immunofluorescence staining in normal ( top ) and residual ( bottom ) duct stained for ARG1 ( green ) , CDH1 ( red ) , and DAPI ( blue ) . Scale bar : 20 μm . Data information : Numbers of replicates correspond to individual mice or humans . Box plots : midline , median ; box , 25 - 75th percentile ; whisker , minimum to maximum . Numbers marking comparisons ( gray lines ) show p values ( corresponding statistical tests are described in individual panel legends ) . D , Nitric oxide synthase ( NOS ) activity , an enzyme involved in arginine metabolism , in healthy ( n = 3 ; blue ) and in residual ( n = 3 ; green ) mouse mammary glands . The difference is statistically significant by unpaired two - samples t - test . E , Fractional labeling of lactate after cultivation of isolated regressed ( n = 3 ; green ) and healthy ( n = 3 ; blue ) mouse mammary glands in cell growth medium supplemented with [ U - 13C ] glucose for 8 h ( Methods ) . The three - carbon labeled ( 13C ) isotopologue ( M + 3 ) of lactate is depicted and shows enrichment in the residual cells of the regressed mammary glands . The difference is statistically significant by unpaired two - samples t - test . Data information : Numbers of replicates correspond to individual mice or humans . Numbers marking comparisons ( gray lines ) show p values ( corresponding statistical tests are described in individual panel legends ) .

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 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 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

Figure 3, Selective secreted metabolites with significant change linked to urea cycle components from healthy (n=3; blue) and regressed (n=3; green) mammary glands. Values represent metabolite abundance levels as quantified by the area under the curve (AUC) of the marker fragment ions/transitions for each metabolite. Values are plotted on the log2 scale. Statistics were calculated using the limma package n R with the significance threshold corresponding to a Benjamini-Hochberg adjusted p-value ≤ 0.05 (residual compared to normal). ata information: umbers of replicates correspond to individual mice or humans. ox plots: midline, median; box, 25-75th percentile; whisker, minimum to maximum. umbers marking comparisons (gray lines) show p values (corresponding statistical tests are described in individual panel legends).C, Left, Quantification of cells expressing ARG1 (top), an enzyme converting arginine to urea and ornithine, and intensity of ARG1 (bottom) in normal cells from healthy (n=5, 2921 cells analyzed; blue) and residual cells from regressed (n=5, 2241 cells analyzed; green) mammary gland tissue sections. Statistical differences were calculated with the Mann-Whitney U test (Wilcoxon rank-sum test). Right, Representative images of immunofluorescence staining in normal (top) and residual (bottom) duct stained for ARG1 (green), CDH1 (red), and DAPI (blue). Scale bar: 20 μm. Data information: Numbers of replicates correspond to individual mice or humans. Box plots: midline, median; box, 25-75th percentile; whisker, minimum to maximum. Numbers marking comparisons (gray lines) show p values (corresponding statistical tests are described in individual panel legends).D, Nitric oxide synthase (NOS) activity, an enzyme involved in arginine metabolism, in healthy (n=3; blue) and in residual (n=3; green) mouse mammary glands. The difference is statistically significant by unpaired two-samples t-test. E, Fractional labeling of lactate after cultivation of isolated regressed (n=3; green) and healthy (n=3; blue) mouse mammary glands in cell growth medium supplemented with [U-13C] glucose for 8 h (Methods). The three-carbon labeled (13C) isotopologue (M+3) of lactate is depicted and shows enrichment in the residual cells of the regressed mammary glands. The difference is statistically significant by unpaired two-samples t-test. Data information: Numbers of replicates correspond to individual mice or humans. Numbers marking comparisons (gray lines) show p values (corresponding statistical tests are described in individual panel legends).


Figure 4A , ( Top ) Experimental design and ( bottom ) quantification of cell death ( Methods ) of normal , tumor , and residual cells after 72 - h treatment with 3 - BP at the indicated doses ( n = 5 mice per condition ) . Two - way ANOVA with Tukey ' s HSD multiple comparison testing was utilized to calculate statistical significance . Dox , doxycycline . Data information : Box plots : midline , median ; box , 25 - 75th percentile ; whiskers , minimum to maximum . Number of replicates corresponds to individual mice used to derive organoids . Numbers marking comparisons ( gray lines ) show p values ( corresponding statistical tests are described in individual panel legends ) . B , Representative bright - field images of normal ( left ) , tumor ( middle ) , and residual ( right ) organoids , treated with vehicle ( top ) or with 50 μM 3 - BP ( bottom ) . Scale bar , 100 μm . C , ( Top ) Experimental design and ( bottom ) cell - death quantification of passaged residual and normal cells after 48 h of treatment with 3 - BP ( n = 5 mice per condition ) . Statistics were calculated with multiple t - tests . Data information : Box plots : midline , median ; box , 25 - 75th percentile ; whiskers minimum to maximum . Number of replicates corresponds to individual mice used to derive organoids . Numbers marking comparisons ( gray lines ) show p values ( corresponding statistical tests are described in individual panel legends ) . D , ( Main ) Extracellular glucose abundance changes upon treatment with 3 - BP in all three populations ( n = 4 mice per condition ) . Values represent the ratio of glucose abundance in 3 - BP - treated versus untreated cells . Statistics were calculated using the limma package in R with the significance threshold corresponding to a Benjamini - Hochberg adjusted p - value ≤ 0 . 05 ( residual compared to normal ) . ( Insert ) Fractional labeling of lactate in untreated and 3 - BP treated normal ( n = 2 ) and residual ( n = 2 ) cells following cultivation in growth medium supplemented with [ U - 13C ] glucose . The three - carbon labeled ( 13C ) isotopologue ( M + 3 ) of lactate is depicted . Statistics were calculated with unpaired two - samples t - tests . Data information : , Box plots : midline , median ; box , 25 - 75th percentile minimum to maximum . Number of replicates corresponds to individual mice used to derive organoids . Numbers marking comparisons ( gray lines ) show p values ( corresponding statistical tests are described in individual panel legends ) .

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 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 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, (Top) Experimental design and (bottom) quantification of cell death (Methods) of normal, tumor, and residual cells after 72-h treatment with 3-BP at the indicated doses (n=5 mice per condition). Two-way ANOVA with Tukey's HSD multiple comparison testing was utilized to calculate statistical significance. Dox, doxycycline. Data information: Box plots: midline, median; box, 25-75th percentile; whiskers , minimum to maximum. Number of replicates corresponds to individual mice used to derive organoids. Numbers marking comparisons (gray lines) show p values (corresponding statistical tests are described in individual panel legends).B, Representative bright-field images of normal (left), tumor (middle), and residual (right) organoids, treated with vehicle (top) or with 50 μM 3-BP (bottom). Scale bar, 100 μm.C, (Top) Experimental design and (bottom) cell-death quantification of passaged residual and normal cells after 48 h of treatment with 3-BP (n=5 mice per condition). Statistics were calculated with multiple t-tests. Data information: Box plots: midline, median; box, 25-75th percentile; whiskers minimum to maximum. Number of replicates corresponds to individual mice used to derive organoids. Numbers marking comparisons (gray lines) show p values (corresponding statistical tests are described in individual panel legends).D, (Main) Extracellular glucose abundance changes upon treatment with 3-BP in all three populations (n=4 mice per condition). Values represent the ratio of glucose abundance in 3-BP-treated versus untreated cells. Statistics were calculated using the limma package in R with the significance threshold corresponding to a Benjamini-Hochberg adjusted p-value ≤ 0.05 (residual compared to normal). (Insert) Fractional labeling of lactate in untreated and 3-BP treated normal (n=2) and residual (n=2) cells following cultivation in growth medium supplemented with [U-13C] glucose. The three-carbon labeled (13C) isotopologue (M+3) of lactate is depicted. Statistics were calculated with unpaired two-samples t-tests. Data information: , Box plots: midline, median; box, 25-75th percentile minimum to maximum. Number of replicates corresponds to individual mice used to derive organoids. Numbers marking comparisons (gray lines) show p values (corresponding statistical tests are described in individual panel legends).


Figure 1 ( B ) GST , GST - MCM2N ( human ) and GST - Spt5N ( yeast ) were mixed as indicated with yeast histones H3 - H4 , in the presence of glutathione agarose beads ( see STAR method for further details ) . The input and pulldown samples were analysed by SDS - PAGE before staining with colloidal Coomassie blue . The asterisks denote truncations of GST - Spt5N . ( C ) Similar experiment to ( B ) but with histones H2A - H2B . ( D ) Similar experiment to ( B ) to compare histone binding by GST - MCM2N and GST - MCM2N - 2A ( with Y81A Y90A mutations of conserved residues ) . ( H ) . Budding yeast cells expressing fusions of Spt5N and Spt5N - 3A to Protein A were grown in parallel with cells expressing the equivalent wild type and mutant versions of fission yeast Spt5N ( Sp Spt5N and Sp Spt5N - 3A - the latter contained the F146A , E150A and V153A mutations ) .

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Figure 1(B) GST, GST-MCM2N (human) and GST-Spt5N (yeast) were mixed as indicated with yeast histones H3-H4, in the presence of glutathione agarose beads (see STAR method for further details). The input and pulldown samples were analysed by SDS-PAGE before staining with colloidal Coomassie blue. The asterisks denote truncations of GST-Spt5N. (C) Similar experiment to (B) but with histones H2A-H2B. (D) Similar experiment to (B) to compare histone binding by GST-MCM2N and GST-MCM2N-2A (with Y81A Y90A mutations of conserved residues).(H). Budding yeast cells expressing fusions of Spt5N and Spt5N-3A to Protein A were grown in parallel with cells expressing the equivalent wild type and mutant versions of fission yeast Spt5N (Sp Spt5N and Sp Spt5N-3A - the latter contained the F146A, E150A and V153A mutations).


Figure 2 ( C ) Yeast cells were generated that expressed Protein A - tagged versions of wild type Spt5 ( full length or the indicated truncations ) , or full length Spt5 - 3A , under the control of the GAL1 , 10 promoter . Cells were grown in medium containing galactose and the ProteinA - tagged Spt5 proteins were isolated from cell extracts on magnetic beads coated with IgG . Association of the tagged Spt5 proteins with the elongating form of Pol II was monitored by immunoblotting , using antibodies specific to the indicated phosphorylation sites in the C - Terminal Domain repeats of the Rpb1 subunit of Pol II . ( E ) Serial dilutions of control cells or spt5 - AID cells , expressing the indicated versions of Spt5 from the GAL1 , 10 promoter , were grown on the indicated medium .

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Figure 2(C) Yeast cells were generated that expressed Protein A-tagged versions of wild type Spt5 (full length or the indicated truncations), or full length Spt5-3A, under the control of the GAL1,10 promoter. Cells were grown in medium containing galactose and the ProteinA-tagged Spt5 proteins were isolated from cell extracts on magnetic beads coated with IgG. Association of the tagged Spt5 proteins with the elongating form of Pol II was monitored by immunoblotting, using antibodies specific to the indicated phosphorylation sites in the C-Terminal Domain repeats of the Rpb1 subunit of Pol II.(E) Serial dilutions of control cells or spt5-AID cells, expressing the indicated versions of Spt5 from the GAL1,10 promoter, were grown on the indicated medium.


Figure 3 ( B ) Immunoblot analysis at the indicated times for the samples described in ( A ) . ( C ) Cells from the end of the experiment described in ( A ) were fixed and processed for ChIP - Seq , using antibodies specific for phosphorylated Serine 5 in the C - Terminal Domain of the Pol II catalytic subunit Rpb1 ( CTD - Ser5P ) . For each of the three indicated strains , the histograms represent the normalized read density ( DNA sequence read count per million reads , or RPM ) , across a sample gene body .

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Figure 3(B) Immunoblot analysis at the indicated times for the samples described in (A).(C) Cells from the end of the experiment described in (A) were fixed and processed for ChIP-Seq, using antibodies specific for phosphorylated Serine 5 in the C-Terminal Domain of the Pol II catalytic subunit Rpb1 (CTD-Ser5P). For each of the three indicated strains, the histograms represent the normalized read density (DNA sequence read count per million reads, or RPM), across a sample gene body.


Figure 4 ( A ) Samples from the experiment described in Figure 3A were also processed for MNase - Seq . For each of the three indicated strains , the histograms represent the normalized DNA sequence read count per million reads ( RPM ) , across a sample gene body . ( C ) Samples from the same experiment were further processed for ChIP - Seq using antibodies to histone H3K4me3 . For each of the three indicated strains , the normalised read count per million reads is presented across a sample gene body .

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Figure 4(A) Samples from the experiment described in Figure 3A were also processed for MNase-Seq. For each of the three indicated strains, the histograms represent the normalized DNA sequence read count per million reads (RPM), across a sample gene body.(C) Samples from the same experiment were further processed for ChIP-Seq using antibodies to histone H3K4me3. For each of the three indicated strains, the normalised read count per million reads is presented across a sample gene body.


Figure 2 ( F ) Volcano plot showing the expression levels of BRAFi - resistant genes ( identified in 501Mel by CRISPR - SAM screen ) in 12 human melanoma cell lines . The fold change corresponds to the ratio of expression levels found in resistant and sensitive cell lines . In red : selected genes . EGFR is considered as positive control and SMAD3 , BIRC3 and SLC9A5 ; selected as favorite genes for the next steps . SMAD3 , BIRC3 , SLC9A5 and AFAP1 are BRAFi - resistance genes and potent tumor - promoting genes ( BRAFi for

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Figure 2(F) Volcano plot showing the expression levels of BRAFi-resistant genes (identified in 501Mel by CRISPR-SAM screen) in 12 human melanoma cell lines. The fold change corresponds to the ratio of expression levels found in resistant and sensitive cell lines. In red: selected genes. EGFR is considered as positive control and SMAD3, BIRC3 and SLC9A5; selected as favorite genes for the next steps. SMAD3, BIRC3, SLC9A5 and AFAP1 are BRAFi-resistance genes and potent tumor-promoting genes (BRAFi for PBV


Figure 4 ( B ) Tumor growth curves from BRAFi - persister cells ( monitored during 5 months ) V for Vem - resistant cells and PB for PLX8394 - resistant cells .

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Figure 4(B) Tumor growth curves from BRAFi-persister cells (monitored during 5 months) V for Vem-resistant cells and PB for PLX8394-resistant cells.


Figure 5 ( G ) SMAD3 depletion ( siRNA # 1 & # 2 ) decreased cell density and increased BRAFi effect ( vemurafenib ) on BRAFi - resistant cells ( SKMel28R ) . CTR for non - targeting siRNA . DMSO for dimethylsulfoxide ( solvent of vemurafenib ; BRAFi ) . n = 3 biologically independent experiments . Each histogram represents the mean + s . d . ( H ) SMAD3 depletion ( siRNA # 1 & # 2 ) decreased cell density and increased BRAFi effect ( vemurafenib , BRAFi ) on BRAFi - resistant cells ( Me1402 ) . CTR for non - targeting siRNA . DMSO for dimethylsulfoxide ( solvent of vemurafenib ) . n = 3 biologically independent experiments . Each histogram represents the mean + s . d . ( L ) SMAD3 , Phospho - SMAD3 Ser423 / 425 and BIRC3 expression levels in melanoma cell lines . Four subtypes of melanoma cells ( U , undifferentiated ; NC , neural crest - like ; T , transitory ; M , melanocytic ) have been compared . 501Mel and M249 cells are melanocytic cells in contrast to dedifferentiated BRAFi - resistant cells ( R ) . Three couples of melanoma cell lines ( Sensitive ( S ) and ( R ) ) have been used to illustrate the SMAD3 and BIRC3 increases in BRAFi - resistant cell lines . HEK293 cells are used as control ( kidney ) . HSC70 serves as loading control for western - blot experiments . Each antibody has been evaluated on individual membrane ( explaining the three HSC70 , loading controls ) . ( O ) The chemical inhibition of SMAD3 by SIS3 ( SMAD3i ) improved current therapy effect ( BRAFi + MEKi ; Vem 5µM and Cobi 1µM ) on BRAFi - resistant cells ( SKMel28R , M229R & M238R ) . Cells have been

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

Figure 5(G) SMAD3 depletion (siRNA#1 & #2) decreased cell density and increased BRAFi effect (vemurafenib) on BRAFi-resistant cells (SKMel28R). CTR for non-targeting siRNA. DMSO for dimethylsulfoxide (solvent of vemurafenib; BRAFi). n=3 biologically independent experiments. Each histogram represents the mean + s.d. (H) SMAD3 depletion (siRNA#1 & #2) decreased cell density and increased BRAFi effect (vemurafenib, BRAFi) on BRAFi-resistant cells (Me1402). CTR for non-targeting siRNA. DMSO for dimethylsulfoxide (solvent of vemurafenib). n=3 biologically independent experiments. Each histogram represents the mean + s.d.(L) SMAD3, Phospho-SMAD3 Ser423/425 and BIRC3 expression levels in melanoma cell lines. Four subtypes of melanoma cells (U, undifferentiated; NC, neural crest-like; T, transitory; M, melanocytic) have been compared. 501Mel and M249 cells are melanocytic cells in contrast to dedifferentiated BRAFi-resistant cells (R). Three couples of melanoma cell lines (Sensitive (S) and (R)) have been used to illustrate the SMAD3 and BIRC3 increases in BRAFi-resistant cell lines. HEK293 cells are used as control (kidney). HSC70 serves as loading control for western-blot experiments. Each antibody has been evaluated on individual membrane (explaining the three HSC70, loading controls).(O) The chemical inhibition of SMAD3 by SIS3 (SMAD3i) improved current therapy effect (BRAFi+MEKi; Vem 5µM and Cobi 1µM) on BRAFi-resistant cells (SKMel28R, M229R & M238R). Cells have been treated


Figure 6 ( B ) AhR activation by exogenous and endogenous ligands promotes SMAD3 induction . 501Mel cells AhR wild - type or knock - out have been exposed to exogenous and endogenous AhR ligands ; TCDD ( 5nM ) or ITE ( 10µM ) or the solvent ( DMSO ) during 10 days . n = 5 biologically independent experiments for AhR WT cells and n = 3 for AhR KO cells . Each histogram represents the mean + s . d . ( C ) AhR activation by exogenous and endogenous AhR ligands promotes TIPARP induction . 501Mel cells have been treated as described in B . n = 5 biologically independent experiments for AhR WT cells and n = 3 for AhR KO cells . Each histogram represents the mean + s . d . ; ( E ) Loss of AhR reduces SMAD3 expression levels . SMAD3 expression has been investigated in SKMel28 cells AhR wild - type ( WT ) or knock - out ( KO ) . SKMel28R , have been obtained from SKMel28S by chronic exposure to non - lethal doses of BRAFi R for BRAFi - resistant SKMel28 cells and S for sensitive .

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Figure 6(B) AhR activation by exogenous and endogenous ligands promotes SMAD3 induction. 501Mel cells AhR wild-type or knock-out have been exposed to exogenous and endogenous AhR ligands; TCDD (5nM) or ITE (10µM) or the solvent (DMSO) during 10 days. n=5 biologically independent experiments for AhR WT cells and n=3 for AhR KO cells. Each histogram represents the mean + s.d. (C) AhR activation by exogenous and endogenous AhR ligands promotes TIPARP induction. 501Mel cells have been treated as described in B. n=5 biologically independent experiments for AhR WT cells and n=3 for AhR KO cells. Each histogram represents the mean + s.d.;(E) Loss of AhR reduces SMAD3 expression levels. SMAD3 expression has been investigated in SKMel28 cells AhR wild-type (WT) or knock-out (KO). SKMel28R, have been obtained from SKMel28S by chronic exposure to non-lethal doses of BRAFi R for BRAFi-resistant SKMel28 cells and S for sensitive.


Figure 7 ( E ) Heatmap depicting mRNA levels of SMAD3 - signature in BRAF ( V600E ) non - treated melanoma patients ( dataset from TCGA ; SKCM , BRAF ( V600E ) mutated : n = 118 ) . Three pigmentation genes ( MITF , MLANA and TYR ) have been added to highlight the differentiation states of tumors . ~ 20 % of tumors are considered as dedifferentiated tumors ( red box ) with a high SMAD3 - signature . ( H ) SMAD3 depletion decreases expression of SMAD3 - regulated genes . SMAD3 knock - down by siRNA decreased mRNA expression of BRAFi - resistance genes in SKMel28R and Me1402 . NRP1 mRNA was not detected in our experimental conditions .

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Figure 7(E) Heatmap depicting mRNA levels of SMAD3-signature in BRAF(V600E) non-treated melanoma patients (dataset from TCGA; SKCM, BRAF(V600E) mutated: n=118). Three pigmentation genes (MITF, MLANA and TYR) have been added to highlight the differentiation states of tumors. ~20% of tumors are considered as dedifferentiated tumors (red box) with a high SMAD3-signature.(H) SMAD3 depletion decreases expression of SMAD3-regulated genes. SMAD3 knock-down by siRNA decreased mRNA expression of BRAFi-resistance genes in SKMel28R and Me1402. NRP1 mRNA was not detected in our experimental conditions.


Figure 1 . siRNA depletion of COPI increases autophagy . ( A ) siRNA depletion of β ′ - , β - , and α - COPI ( top ) and Sec23A and B ( bottom ) in 293 / GFP - LC3 cells for 48 h . Decreased protein levels of the corresponding subunits is shown by immunoblots . ( B ) Loss of β ′ - , α - , and β - COP but not subunits of COPII ( Sec 23A and B ) increase GFP - LC3 - II . In A and B , tubulin was the loading control . ( C ) GFP - LC3 - positive vesicles were detectable in 293 / GFP - LC3 cells after siRNA depletion of β ′ - , α - , and β - COP but only basal levels of GFP - LC3 - positive vesicles are observed in control siRNA - depleted cells .

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 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

Figure 1.siRNA depletion of COPI increases autophagy. (A) siRNA depletion of β′-, β-, and α-COPI (top) and Sec23A and B (bottom) in 293/GFP-LC3 cells for 48 h. Decreased protein levels of the corresponding subunits is shown by immunoblots.(B) Loss of β′-, α-, and β-COP but not subunits of COPII (Sec 23A and B) increase GFP-LC3-II. In A and B, tubulin was the loading control.(C) GFP-LC3-positive vesicles were detectable in 293/GFP-LC3 cells after siRNA depletion of β′-, α-, and β-COP but only basal levels of GFP-LC3-positive vesicles are observed in control siRNA-depleted cells.


Figure 2 . Time course of COPI depletion shows that Golgi disperses before AVs are formed , but Golgi dispersal does not cause AV formation . ( A ) COPI subunits were depleted in 293 / GFP - LC3 cells and analyzed 24 , 36 , and 48 h after addition of siRNA . Immunoblotting for β ′ - and α - COP confirms loss of COP subunits . ( B ) GFP - LC3 - I and - II were monitored by immunoblots in parallel lysates using anti - GFP antibodies at the indicated times . ( C ) After 24 h siRNA treatment , depletion of β ′ - COP caused morphological changes and a reduction in perinuclear population of GM130 . Box indicates enlarged area . ( D ) Rab1a / b was depleted using siRNAs for 48 h . Lysates were probed with anti - Rab1 to confirm depletion . ( E ) β ′ - COP or Rab1a / b were depleted as in D , and analyzed by indirect immunofluorescence using anti - GM130 and GFP fluorescence . In β ′ - COP panel , the asterisk indicates cells that did not show an accumulation of GFP - LC3 - positive AVs .

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 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 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 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

Figure 2.Time course of COPI depletion shows that Golgi disperses before AVs are formed, but Golgi dispersal does not cause AV formation. (A) COPI subunits were depleted in 293/GFP-LC3 cells and analyzed 24, 36, and 48 h after addition of siRNA. Immunoblotting for β′- and α-COP confirms loss of COP subunits.(B) GFP-LC3-I and -II were monitored by immunoblots in parallel lysates using anti-GFP antibodies at the indicated times.(C) After 24 h siRNA treatment, depletion of β′-COP caused morphological changes and a reduction in perinuclear population of GM130. Box indicates enlarged area.(D) Rab1a/b was depleted using siRNAs for 48 h. Lysates were probed with anti-Rab1 to confirm depletion.(E) β′-COP or Rab1a/b were depleted as in D, and analyzed by indirect immunofluorescence using anti-GM130 and GFP fluorescence. In β′-COP panel, the asterisk indicates cells that did not show an accumulation of GFP-LC3-positive AVs.


Figure 3 . AV formation after siRNA depletion of COPI requires Atg5 and Atg7 . 293 / GFP - LC3 cells were incubated with siRNA for β ′ - , α - COP , or control siRNA alone or combined with siRNA against Atg5 or Atg7 for 48 h , then ( A ) lysed and analyzed by immunoblot for Atg5 , Atg7 , GFP - LC3 , and tubulin ( loading control ) , ( B ) fixed for immunofluorescence analysis using anti - ERGIC - 53 antibodies , or GFP fluorescence . In A , the asterisk indicates nonspecific band ; in B and C , asterisk indicates cells that did not show a fragmented ERGIC - 53 .

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Figure 3.AV formation after siRNA depletion of COPI requires Atg5 and Atg7. 293/GFP-LC3 cells were incubated with siRNA for β′-, α-COP, or control siRNA alone or combined with siRNA against Atg5 or Atg7 for 48 h, then (A) lysed and analyzed by immunoblot for Atg5, Atg7, GFP-LC3, and tubulin (loading control),(B) fixed for immunofluorescence analysis using anti-ERGIC-53 antibodies, or GFP fluorescence. In A, the asterisk indicates nonspecific band; in B and C, asterisk indicates cells that did not show a fragmented ERGIC-53.


Figure 4 . Correlative light - electron microscopy analysis of GFP - LC3 puncta demonstrates they are AVs . ( A ) Phase , ( B ) fluorescent , and ( C ) TEM of the same field of 293 / GFP - LC3 cells 48 h after siRNA depletion of α - COP . In B the image was inverted to allow better visualization of the GFP - LC3 - positive structures . ( D ) Higher magnification of cell shown with asterisk in A and C . Top region of cell of interest is enlarged in E to show the two AVs structures indicated by arrows in B . ( F and G ) Higher magnification of AVs shown by arrows in E .

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Figure 4.Correlative light-electron microscopy analysis of GFP-LC3 puncta demonstrates they are AVs. (A) Phase, (B) fluorescent, and (C) TEM of the same field of 293/GFP-LC3 cells 48 h after siRNA depletion of α-COP. In B the image was inverted to allow better visualization of the GFP-LC3-positive structures. (D) Higher magnification of cell shown with asterisk in A and C. Top region of cell of interest is enlarged in E to show the two AVs structures indicated by arrows in B. (F and G) Higher magnification of AVs shown by arrows in E.


Figure 5 . Ub and p62 accumulate in α - and β ′ - COP - depleted 293 / GFP - LC3 cells . ( A ) 293 / GFP - LC3 cells were treated with control or β ′ - COP siRNA for 48 h , then fixed and labeled with anti - p62 and anti - ubiquitin antibodies for colocalization with GFP - LC3 . ( B ) HEK293 cells treated as in A and labeled with anti - ubiquitin and anti - p62 antibodies . ( C ) Cryosections from β ′ - COP - depleted cells were labeled with anti - Ub antibodies followed by 10 - nm protein A - gold . AV , autophagosomes .

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

Figure 5.Ub and p62 accumulate in α- and β′-COP-depleted 293/GFP-LC3 cells. (A) 293/GFP-LC3 cells were treated with control or β′-COP siRNA for 48 h, then fixed and labeled with anti-p62 and anti-ubiquitin antibodies for colocalization with GFP-LC3.(B) HEK293 cells treated as in A and labeled with anti-ubiquitin and anti-p62 antibodies.(C) Cryosections from β′-COP-depleted cells were labeled with anti-Ub antibodies followed by 10-nm protein A-gold. AV, autophagosomes.


Figure 6 . AVs in COPI - depleted cells are not degradative . ( A ) Long - lived protein degradation was assessed 72 h after transfection with control or β ′ - COP siRNA in 293 / GFP - LC3 cells . Cells were either starved of amino acids for 2 h ( St ) or incubated in fresh growth medium ( Fed ) for 2 h . The amount of protein degradation is presented as mean ± SEM . ( B ) siControl - treated cells and β ′ - and α - COP were depleted for 48 h , then incubated with Lysotracker red ( 50 nM ) for 30 min before fixation . Merged image shows very little colocalization of GFP - LC3 AV - positive structures . Asterisk shows cell enlarged in inset ( C ) After β ′ - and α - COP siRNA depletion , Magic Red - RR2 ( MR ) was used to detect active cathepsin B in cells which were fed or starved ( St ) for 2 h . The intensity of MR labeling was measured in live cells by flow cytometry .

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 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 O O O O O O O O O O O

Figure 6.AVs in COPI-depleted cells are not degradative. (A) Long-lived protein degradation was assessed 72 h after transfection with control or β′-COP siRNA in 293/GFP-LC3 cells. Cells were either starved of amino acids for 2 h (St) or incubated in fresh growth medium (Fed) for 2 h. The amount of protein degradation is presented as mean ± SEM.(B) siControl-treated cells and β′- and α-COP were depleted for 48 h, then incubated with Lysotracker red (50 nM) for 30 min before fixation. Merged image shows very little colocalization of GFP-LC3 AV-positive structures. Asterisk shows cell enlarged in inset(C) After β′- and α-COP siRNA depletion, Magic Red-RR2 (MR) was used to detect active cathepsin B in cells which were fed or starved (St) for 2 h. The intensity of MR labeling was measured in live cells by flow cytometry.


Figure 7 . GFP - LC3 AVs are LAMP2 positive . ( A ) 293 / GFP - LC3 cells were incubated with control or β ′ - or α - COP siRNA for 48 h , fixed and labeled with anti - LAMP2 antibodies , followed by Alexa 555 anti - mouse antibodies . After COPI depletion many GFP - LC3 - positive AVs are positive for LAMP2 . Asterisk indicates cell enlarged in inset . ( B ) Cryosections of β ′ - COP - depleted cells labeled with anti - GFP ( left ) or anti - GFP and anti - LAMP2 ( right ) followed by protein A - gold as indicated . AV , autophagosomes , M , mitochondria .

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Figure 7.GFP-LC3 AVs are LAMP2 positive. (A) 293/GFP-LC3 cells were incubated with control or β′- or α-COP siRNA for 48 h, fixed and labeled with anti-LAMP2 antibodies, followed by Alexa 555 anti-mouse antibodies. After COPI depletion many GFP-LC3-positive AVs are positive for LAMP2. Asterisk indicates cell enlarged in inset.(B) Cryosections of β′-COP-depleted cells labeled with anti-GFP (left) or anti-GFP and anti-LAMP2 (right) followed by protein A-gold as indicated. AV, autophagosomes, M, mitochondria.


Figure 8 . GFP - LC3 - positive AVs colocalize TGN46 and early endocytotic markers after COPI depletion . ( A ) 293 / GFP - LC3 cells were incubated with siRNA for β ′ - COP or control siRNA ( Cont ) for 48 h , processed for immunofluorescence , and labeled with antibodies for TGN46 . ( B ) siRNA - treated cells were fixed and labeled , or starved for 2 h ( St ) , and labeled with antibodies to EEA1 . ( ( C ) Control - starved HEK293 cells were labeled for endogenous LC3 and EEA1 by double labeling after methanol fixation . ( D ) After siRNA treatment , rhodamine - labeled dextran was internalized for 20 min , followed by a 40 - min chase after which the 293 / GFP - LC3 cells were fixed and analyzed by confocal microscopy .

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 B-PANEL_START 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

Figure 8.GFP-LC3-positive AVs colocalize TGN46 and early endocytotic markers after COPI depletion. (A) 293/GFP-LC3 cells were incubated with siRNA for β′-COP or control siRNA (Cont) for 48 h, processed for immunofluorescence, and labeled with antibodies for TGN46.(B) siRNA-treated cells were fixed and labeled, or starved for 2 h (St), and labeled with antibodies to EEA1. ((C) Control-starved HEK293 cells were labeled for endogenous LC3 and EEA1 by double labeling after methanol fixation.(D) After siRNA treatment, rhodamine-labeled dextran was internalized for 20 min, followed by a 40-min chase after which the 293/GFP-LC3 cells were fixed and analyzed by confocal microscopy.


Figure 9 . Transferrin and EGF trafficking is inhibited by COPI depletion . 293 / GFP - LC3 cells were treated with β ′ - COP siRNA or control siRNA . After 48 h cells were incubated with Alexa 555 transferrin ( A ) for 2 min , ( B ) 30 min , or ( C ) 30 min followed by a 30 - min chase , then washed and fixed and analyzed by confocal microscopy . ( D and E ) 125I - EGF was added to HeLa cells after 48 h of siRNA depletion of β ′ - COP , or control siRNA for 10 min . The cells were then washed as described in the Materials and methods , and chased for 30 , 60 , 120 , and 180 min ( including 10 - min incubation ) . ( D ) The amount of 125I - EGF internalized was shown as a percentage of the total 125I - EGF taken up in 10 min . ( E ) The amount of degraded 125I - EGF was determined as a percentage of the total radioactivity present after TCA precipitation over the total 125I - EGF internalized . The experiment was performed three times in duplicate and the data shown is the mean ± SEM . The significance was determined by Student ' s t test . * , P ≤ 0 . 05 .

O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 9.Transferrin and EGF trafficking is inhibited by COPI depletion. 293/GFP-LC3 cells were treated with β′-COP siRNA or control siRNA. After 48 h cells were incubated with Alexa 555 transferrin (A) for 2 min, (B) 30 min, or (C) 30 min followed by a 30-min chase, then washed and fixed and analyzed by confocal microscopy.(D and E) 125I-EGF was added to HeLa cells after 48 h of siRNA depletion of β′-COP, or control siRNA for 10 min. The cells were then washed as described in the Materials and methods, and chased for 30, 60, 120, and 180 min (including 10-min incubation). (D) The amount of 125I-EGF internalized was shown as a percentage of the total 125I-EGF taken up in 10 min.(E) The amount of degraded 125I-EGF was determined as a percentage of the total radioactivity present after TCA precipitation over the total 125I-EGF internalized. The experiment was performed three times in duplicate and the data shown is the mean ± SEM. The significance was determined by Student's t test. *, P ≤ 0.05.


f1a , b , THP1 cells were stimulated with rotenone ( 10 µM ) , TTFA ( 10 µM ) or antimycin ( 40 µg ml − 1 ) for 6 h and then stained with Mitotracker green and Mitotracker deep red ( a ) or MitoSOX ( b ) for 30 min and analysed by flow cytometry . c , THP1 cells were stimulated for 6 h with the indicated amounts of rotenone , TTFA or antimycin . Supernatants ( SN ) and cell extracts ( input ) were analysed by western blotting as indicated . d , LPS - primed bone - marrow - derived macrophages ( BMDMs ) from wild - type ( WT ) , Nlrp3 or Ipaf deficient mice were stimulated for 6 h with MSU ( 150 µg ml − 1 ) , rotenone ( 10 µM for THP1 cells and 40 µM for BMDMs ) or antimycin ( 40 µg ml − 1 for THP1 cells and 10 µg ml − 1 for BMDMs ) . The release of caspase - 1 ( western blot ) was then determined . Data shown are representative of three independent experiments .

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

f1a, b, THP1 cells were stimulated with rotenone (10 µM), TTFA (10 µM) or antimycin (40 µg ml−1) for 6 h and then stained with Mitotracker green and Mitotracker deep red (a) or MitoSOX (b) for 30 min and analysed by flow cytometry.c, THP1 cells were stimulated for 6 h with the indicated amounts of rotenone, TTFA or antimycin. Supernatants (SN) and cell extracts (input) were analysed by western blotting as indicated.d, LPS-primed bone-marrow-derived macrophages (BMDMs) from wild-type (WT), Nlrp3 or Ipaf deficient mice were stimulated for 6 h with MSU (150 µg ml−1), rotenone (10 µM for THP1 cells and 40 µM for BMDMs) or antimycin (40 µg ml−1 for THP1 cells and 10 µg ml−1 for BMDMs). The release of caspase-1 (western blot) was then determined. Data shown are representative of three independent experiments.


f2a , BMDMs expressing GFP - LC3 were stimulated with rotenone ( 40 µM ) for 3 h and the co - localization of mitochondria and GFP - LC3 dots were analysed using confocal microscopy . b , THP1 cells stimulated with 3 - methyladenine ( 3 - MA , 10 mM ) for 24 h or THP1 cells stably expressing shRNA against beclin 1 or ATG5 were stained with Mitotracker green and Mitotracker deep red for 30 min , and analysed by flow cytometry . c , LPS - primed BMDMs from wild - type , Nlrp3 − / − , Asc − / − or Ipaf − / − knockout mice were stimulated with MSU ( 150 µg ml − 1 ) or 3 - MA ( 10 mM ) for 6 h and the release of active caspase - 1 and IL - 1b was determined . d , THP1 cells stably expressing shRNA against beclin 1 or ATG5 were incubated for 24 h and media supernatants ( SN ) and inputs were analysed by western blotting . e , THP1 cells stably expressing shRNA against beclin 1 or ATG5 were stimulated with MSU or nigericin for 6 h . Data shown are representative of three independent experiments .

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 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 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

f2a, BMDMs expressing GFP-LC3 were stimulated with rotenone (40 µM) for 3 h and the co-localization of mitochondria and GFP-LC3 dots were analysed using confocal microscopy.b, THP1 cells stimulated with 3-methyladenine (3-MA, 10 mM) for 24 h or THP1 cells stably expressing shRNA against beclin 1 or ATG5 were stained with Mitotracker green and Mitotracker deep red for 30 min, and analysed by flow cytometry.c, LPS-primed BMDMs from wild-type, Nlrp3−/−, Asc−/− or Ipaf −/− knockout mice were stimulated with MSU (150 µg ml−1) or 3-MA (10 mM) for 6 h and the release of active caspase-1 and IL-1b was determined.d, THP1 cells stably expressing shRNA against beclin 1 or ATG5 were incubated for 24 h and media supernatants (SN) and inputs were analysed by western blotting.e, THP1 cells stably expressing shRNA against beclin 1 or ATG5 were stimulated with MSU or nigericin for 6 h. Data shown are representative of three independent experiments.


f3a - d , THP1 cells expressing Flag - NLRP3 ( a , b ) or Flag - ASC ( c , d ) were analysed for the co - localization of Flag - NLRP3 with the endoplasmic reticulum ( ER ) marker calreticulin or mitochondria ( Mitotracker ) using confocal microscopy . b , THP1 cells expressing Flag - NLRP3 were analysed for the co - localization of Flag - NLRP3 with the endoplasmic reticulum ( ER ) marker calreticulin using confocal microscopy . e , THP1 cells were stimulated with MSU or nigericin and then fractionated . The cytosolic , ER , MAM and mitochondrial fractions were analysed by western blotting as indicated in the text . f , THP1 cells stably expressing shRNA against NLRP3 were stimulated with nigericin and subcellular fractions analysed as in e . g , TXNIP associates with mitochondria after NLRP3 inflammasome activation in a ROS - dependent manner . THP1 cells were stimulated with MSU and nigericin , and TXNIP localization was investigated in the presence or absence of the antioxidant APDC . Data shown are representative of two or three independent experiments . Scale bars : a - g , 20 µm .

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

f3a-d, THP1 cells expressing Flag-NLRP3 (a, b) or Flag-ASC (c, d) were analysed for the co-localization of Flag-NLRP3 with the endoplasmic reticulum (ER) marker calreticulin or mitochondria (Mitotracker) using confocal microscopy.b, THP1 cells expressing Flag-NLRP3 were analysed for the co-localization of Flag-NLRP3 with the endoplasmic reticulum (ER) marker calreticulin using confocal microscopy.e, THP1 cells were stimulated with MSU or nigericin and then fractionated. The cytosolic, ER, MAM and mitochondrial fractions were analysed by western blotting as indicated in the text.f, THP1 cells stably expressing shRNA against NLRP3 were stimulated with nigericin and subcellular fractions analysed as in e.g, TXNIP associates with mitochondria after NLRP3 inflammasome activation in a ROS-dependent manner. THP1 cells were stimulated with MSU and nigericin, and TXNIP localization was investigated in the presence or absence of the antioxidant APDC. Data shown are representative of two or three independent experiments. Scale bars: a-g, 20 µm.


f4a , THP1 cells stably expressing shVDAC1 were stimulated with various NLRP inflammasome activators and then analysed for caspase - 1 activation and IL - 1b secretion . b , THP1 cells expressing shRNA against VDAC2 were stimulated with MSU , R837 , silica , alum or nigericin and analysed for IL - 1b secretion . c , THP1 cells expressing shRNA against VDAC1 were stimulated with MSU and nigericin , and NLRP3 localization was analysed by confocal microscopy . Scale bars : 20 µm .

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f4a, THP1 cells stably expressing shVDAC1 were stimulated with various NLRP inflammasome activators and then analysed for caspase-1 activation and IL-1b secretion.b, THP1 cells expressing shRNA against VDAC2 were stimulated with MSU, R837, silica, alum or nigericin and analysed for IL-1b secretion.c, THP1 cells expressing shRNA against VDAC1 were stimulated with MSU and nigericin, and NLRP3 localization was analysed by confocal microscopy. Scale bars: 20 µm.