( E ) LC3 immunoblots of epithelial spheroids treated with 100 μM Dynasore and 100 nM BafA1 as indicated . GAPDH is a loading control . Representative image is shown from n = 3 experiments .

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

(E) LC3 immunoblots of epithelial spheroids treated with 100 μM Dynasore and 100 nM BafA1 as indicated. GAPDH is a loading control. Representative image is shown from n=3 experiments.


( F ) Images of wild ‐ type colonic epithelial spheroids labelled with TRITC ‐ UEA ( red ) after treatment with either vehicle or 100 μM Dynasore . Bars = 20 μm .

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

(F) Images of wild‐type colonic epithelial spheroids labelled with TRITC‐UEA (red) after treatment with either vehicle or 100 μM Dynasore. Bars=20 μm.


( G ) Quantification of average mucin area / goblet cell ( n = 6 samples / group ; 150 cells were quantified / sample ) . Error bars indicate s . e . m . * * * P0 . 001 as determined by the Student ' s t ‐ test .

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

(G) Quantification of average mucin area/goblet cell (n=6 samples/group; 150 cells were quantified/sample). Error bars indicate s.e.m. ***P0.001 as determined by the Student's t‐test.


( A ) Colonic epithelial monolayers from a wild ‐ type and p22 mut mouse stained with LC3β ( green ) and EEA1 ( red ) . Bar = 5 μm .

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

(A) Colonic epithelial monolayers from a wild‐type and p22 mut mouse stained with LC3β (green) and EEA1 (red). Bar=5 μm.


( B , C ) Quantification of average number of LC3β dots per cell ( B ) and the percentage of LC3β dots that co ‐ localized with EEA1 per cell ( C ) ( n = 3 experiments / group ; 25 - 30 cells / experiment ) .

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

(B, C) Quantification of average number of LC3β dots per cell (B) and the percentage of LC3β dots that co‐localized with EEA1 per cell (C) (n=3 experiments/group; 25-30 cells/experiment).


( D ) LC3 immunoblots of either wild ‐ type or p22 mut epithelial spheroids treated with 100 nM BafA1 as indicated . Representative image is shown from n = 3 experiments .

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

(D) LC3 immunoblots of either wild‐type or p22 mut epithelial spheroids treated with 100 nM BafA1 as indicated. Representative image is shown from n=3 experiments.


( E ) Double label immunogold transmission electron microscopic images for LC3β ( 12μnm particles ) and p22phox ( 18 nm particles ) showing a p22phox ‐ positive single membrane cytoplasmic vesicle ( D ) and a p22phox ‐ , LC3β ‐ positive vacuole ( E ) . Bars = 200μnm .

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

(E) Double label immunogold transmission electron microscopic images for LC3β (12μnm particles) and p22phox (18 nm particles) showing a p22phox‐positive single membrane cytoplasmic vesicle (D) and a p22phox‐, LC3β‐positive vacuole (E). Bars=200μnm.


( F ) Colonic epithelial monolayers from a wild ‐ type mouse stained for LC3β ( green ) and p22phox ( red ) and quantification of percent LC3β dots co ‐ localized with p22phox per cell , Bar = 5 μm .

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

(F) Colonic epithelial monolayers from a wild‐type mouse stained for LC3β (green) and p22phox (red) and quantification of percent LC3β dots co‐localized with p22phox per cell, Bar=5 μm.


( G ) Colonic epithelial cellspheroids from control and p22 mut mice stained with TRITC ‐ UEA to label goblet cellmucin . Bars = 50 μm .

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

(G) Colonic epithelial cellspheroids from control and p22 mut mice stained with TRITC‐UEA to label goblet cellmucin. Bars=50 μm.


( H ) Quantification of average mucin area / goblet cell ( n = 6 samples / group ; 150 cells were quantified / sample ) . Error bars indicate s . e . m . * * P0 . 01 ; * * * * P0 . 0001 as determined by the Student ' s t ‐ test .

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

(H) Quantification of average mucin area/goblet cell (n=6 samples/group; 150 cells were quantified/sample). Error bars indicate s.e.m. **P0.01; ****P0.0001 as determined by the Student's t‐test.


( A ) Colonic epithelial cell spheroids from p22phox − / − , Atg5VC and wild ‐ type mice . Wild ‐ type spheroids were treated with vehicle ( control ) , 10 μM DPI ( NADPH oxidase inhibitor ) or 100 μM Dynasore . Intracellular ROS in live cells were labelled by 5 μM DCF . Bars = 100 μm .

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

(A) Colonic epithelial cell spheroids from p22phox−/−, Atg5VC and wild‐type mice. Wild‐type spheroids were treated with vehicle (control), 10 μM DPI (NADPH oxidase inhibitor) or 100 μM Dynasore. Intracellular ROS in live cells were labelled by 5 μM DCF. Bars=100 μm.


( B ) Quantification of fluorescent DCF normalized to control ( n = 3 independent experiments / group ; 30 spheroids quantified / experiment ) .

O O 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) Quantification of fluorescent DCF normalized to control (n=3 independent experiments/group; 30 spheroids quantified/experiment).


( C ) Quantification of average area of cytoplasmic mucin from wild ‐ type mice treated with 3 % NAC as compared to controls ( n = 6 - 7 mice / group ) . Error bars indicate s . e . m . * P0 . 05 ; * * P0 . 01 ; * * * * P0 . 0001 as determined by the Student ' s t ‐ test .

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

(C) Quantification of average area of cytoplasmic mucin from wild‐type mice treated with 3% NAC as compared to controls (n=6-7 mice/group). Error bars indicate s.e.m. *P0.05; **P0.01; ****P0.0001 as determined by the Student's t‐test.


( D ) TEM images of colonic epithelial monolayers treated with cerium chloride . The presence of electron dense precipitate is indicative of ROS production . Bar = 500 nm .

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

(D) TEM images of colonic epithelial monolayers treated with cerium chloride. The presence of electron dense precipitate is indicative of ROS production. Bar=500 nm.


Exogeneous ROS can restore mucingranule accumulation defects . ( A ) Quantification of fluorescent DCF normalized to controls ( n = 3 independent experiments / group ; 10 - 30 spheroids quantified / culture ) . * P0 . 05 as determined by ANOVA with Tukey ' s multiple post ‐ test

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

Exogeneous ROS can restore mucingranule accumulation defects. (A) Quantification of fluorescent DCF normalized to controls (n=3 independent experiments/group; 10-30 spheroids quantified/culture). *P0.05 as determined by ANOVA with Tukey's multiple post‐test comparisons


( B ) Images of control and Atg5VC colonic epithelial spheroids labelled with TRITC ‐ UEA ( red ) after treatment with either vehicle or 300 μM H2O2 . Bars = 20 μm . ( C ) Quantification of average mucin area / goblet cell ( n = 6 samples / group ; 150 cells were quantified / sample ) . * * * P0 . 001 , * * * * P . 0001 as determined by ANOVA with Tukey ' s multiple post ‐ test comparisons .

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

(B) Images of control and Atg5VC colonic epithelial spheroids labelled with TRITC‐UEA (red) after treatment with either vehicle or 300 μM H2O2. Bars=20 μm. (C) Quantification of average mucin area/goblet cell (n=6 samples/group; 150 cells were quantified/sample). ***P0.001, ****P.0001 as determined by ANOVA with Tukey's multiple post‐test comparisons.


( D ) Relative expression of Muc2 mRNA in differentiated spheroids from control and Atg5VC mice treated with either vehicle or 300 μM H2O2 as measured by qRT - PCR . No statistically significant differences were found in either group as determined by ANOVA .

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

(D) Relative expression of Muc2 mRNA in differentiated spheroids from control and Atg5VC mice treated with either vehicle or 300 μM H2O2 as measured by qRT-PCR. No statistically significant differences were found in either group as determined by ANOVA.


( E , G ) Images of p22 mut or Dynasore ‐ treated colonic epithelial spheroids ( mucin is labelled with TRITC ‐ UEA ; red ) after treatment with either vehicle or 300 μM H2O2 . Bars = 20 μm . ( F , H ) Quantification of average mucin area / goblet cell ( n = 6 sample / group ; 30 - 60 cells were quantified / sample ) corresponding to experiments performed using p22 mut ( E ) and Dynasore ‐ treated wild ‐ type spheroids ( G )

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

(E, G) Images of p22 mut or Dynasore‐treated colonic epithelial spheroids (mucin is labelled with TRITC‐UEA; red) after treatment with either vehicle or 300 μM H2O2. Bars=20 μm. (F, H) Quantification of average mucin area/goblet cell (n=6 sample/group; 30-60 cells were quantified/sample) corresponding to experiments performed using p22 mut (E) and Dynasore‐treated wild‐type spheroids (G)


Atg5VC and p22 mut spheroids were treated with 300 μM H2O2 plus or minus 100 μM BAPTA ‐ AM ( I ) or PMA / ionomycin ( J ) . ( I , J ) Quantification of average mucin area / goblet cell ( n = 5 sample / group ; 150 cells were quantified / sample ) . Error bars indicate s . e . m . * * * * P0 . 0001 as determined by the Student ' s t ‐ test ( I ) or ANOVA with Tukey ' s multiple post ‐ test comparisons ( J ) .

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

Atg5VC and p22 mut spheroids were treated with 300 μM H2O2 plus or minus 100 μM BAPTA‐AM (I) or PMA/ionomycin (J). (I, J) Quantification of average mucin area/goblet cell (n=5 sample/group; 150 cells were quantified/sample). Error bars indicate s.e.m. ****P0.0001 as determined by the Student's t‐test (I) or ANOVA with Tukey's multiple post‐test comparisons (J).


B . In vitro modification reactions containing the six fragments and HeLa NE were performed . After nuclease P1 digestion , the products were analyzed by 1D - TLC with pU and pψ as controls .

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

B. In vitro modification reactions containing the six fragments and HeLa NE were performed. After nuclease P1 digestion, the products were analyzed by 1D-TLC with pU and pψ as controls.


C . WT and mutant 7SK fragment 5 containing U250G were analyzed as in B .

C. WT and mutant 7SK fragment 5 containing U250G were analyzed as in B.


E . 7SK RNA , generated by either in vitro transcription ( IVT ) or purified from HeLa total RNA and then site - specifically labeled at the indicated positions by the procedure in D , was analyzed by 1D - TLC .

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

E. 7SK RNA, generated by either in vitro transcription (IVT) or purified from HeLa total RNA and then site-specifically labeled at the indicated positions by the procedure in D, was analyzed by 1D-TLC.


F . Cyber Gold staining of 7SK obtained by either IVT or immunoprecipitation ( IP ) with the indicated antibodies .

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

F. Cyber Gold staining of 7SK obtained by either IVT or immunoprecipitation (IP) with the indicated antibodies.


G . 2D - TLC analysis of 32p - labeled mononucleotides derived from IVT or CDK9 - bound 7SK RNA , with the first panel depicting a schematic migration pattern of the indicated 5 ' - phosphorylated mononucleotides .

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

G. 2D-TLC analysis of 32p-labeled mononucleotides derived from IVT or CDK9-bound 7SK RNA, with the first panel depicting a schematic migration pattern of the indicated 5'-phosphorylated mononucleotides.


H . 7SK RNA , obtained by either IVT or anti - CDK9 IP and then site - specifically labeled at the indicated positions by the procedure in D , was analyzed by 1D - TLC .

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

H. 7SK RNA, obtained by either IVT or anti-CDK9 IP and then site-specifically labeled at the indicated positions by the procedure in D, was analyzed by 1D-TLC.


C - F ; H - K ; and N - Q . The tagged WT or mutant 7SK RNA was co - transfected with the indicated Flag - tagged proteins into HeLa cells . Anti - Flag or immunoprecipitates ( IP ) were analyzed by immunoblotting for the indicated proteins and primer extension for the bound 7SK RNA .

C-F; H-K; and N-Q. The tagged WT or mutant 7SK RNA was co-transfected with the indicated Flag-tagged proteins into HeLa cells. Anti-Flag or immunoprecipitates (IP) were analyzed by immunoblotting for the indicated proteins and primer extension for the bound 7SK RNA.


C - F ; H - K ; and N - Q . The tagged WT or mutant 7SK RNA was co - transfected with the indicated Flag - - tagged proteins into HeLa cells . Anti - Flag immunoprecipitates ( IP ) were analyzed by immunoblotting for the indicated proteins and primer extension for the bound 7SK RNA .

C-F; H-K; and N-Q. The tagged WT or mutant 7SK RNA was co-transfected with the indicated Flag--tagged proteins into HeLa cells. Anti-Flag immunoprecipitates (IP) were analyzed by immunoblotting for the indicated proteins and primer extension for the bound 7SK RNA.


C - F ; H - K ; and N - Q . The tagged WT or mutant 7SK RNA was co - transfected with the indicated Flag - tagged proteins into HeLa cells . Anti - Flag immunoprecipitates ( IP ) were analyzed by immunoblotting for the indicated proteins and primer extension for the bound 7SK RNA .

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

C-F; H-K; and N-Q. The tagged WT or mutant 7SK RNA was co-transfected with the indicated Flag-tagged proteins into HeLa cells. Anti-Flag immunoprecipitates (IP) were analyzed by immunoblotting for the indicated proteins and primer extension for the bound 7SK RNA.


C - F ; H - K ; and N - Q . The tagged WT or mutant 7SK RNA was co - transfected with the indicated HA - tagged proteins into HeLa cells . Anti - HA immunoprecipitates ( IP ) were analyzed by immunoblotting for the indicated proteins and primer extension for the bound 7SK RNA .

C-F; H-K; and N-Q. The tagged WT or mutant 7SK RNA was co-transfected with the indicated HA-tagged proteins into HeLa cells. Anti-HA immunoprecipitates (IP) were analyzed by immunoblotting for the indicated proteins and primer extension for the bound 7SK RNA.


R . The indicated 7SK were in vitro transcribed in the presence of 32p - UTP and subjected to in vitro modification reactions . The nuclease P1 - digested products were analyzed by 1D - TLC with pU and pΨ as controls .

R. The indicated 7SK were in vitro transcribed in the presence of 32p-UTP and subjected to in vitro modification reactions. The nuclease P1-digested products were analyzed by 1D-TLC with pU and pΨ as controls.


A . Anti - V5immunoprecipitates ( IP ) from HeLa cells expressing nothing ( - ) or V5 - DKC1 were analyzed by immunoblotting for V5 - DKC1 and qRT - PCR for the bound 7SK RNA . The error bars represent mean ± SD from three independent experiments .

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

A. Anti-V5immunoprecipitates (IP) from HeLa cells expressing nothing (-) or V5-DKC1 were analyzed by immunoblotting for V5-DKC1 and qRT-PCR for the bound 7SK RNA. The error bars represent mean ± SD from three independent experiments.


B . Anti - FlagIP from NE of HeLa or the HeLa - derived F1C2 cells expressing CDK9 - F were analyzed by Western blotting ( WB ) for the indicated proteins and primer extension ( PEx ) for 7SK RNA .

O O O O O O O O O O O O O O O O O O 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. Anti-FlagIP from NE of HeLa or the HeLa-derived F1C2 cells expressing CDK9-F were analyzed by Western blotting (WB) for the indicated proteins and primer extension (PEx) for 7SK RNA.


C . WB analysis of DKC1 and CDK9 levels in two HeLa clones inducibly expressing shDKC1 upon doxycycline ( DOX ) treatment for 5 days .

C. WB analysis of DKC1 and CDK9 levels in two HeLa clones inducibly expressing shDKC1 upon doxycycline (DOX) treatment for 5 days.


D . The 7SK RNA levels in these two clones were detected by qRT - PCR before and after the DOX treatment and normalized to the GAPDH mRNA levels . The error bars represent mean ± SD from three independent experiments .

D. The 7SK RNA levels in these two clones were detected by qRT-PCR before and after the DOX treatment and normalized to the GAPDH mRNA levels. The error bars represent mean ± SD from three independent experiments.


E . 1D - TLC analysis of Ψ250 in 7SK RNA from inducible DKC1 KD clone 1 before and after the DOX treatment .

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

E. 1D-TLC analysis of Ψ250 in 7SK RNA from inducible DKC1 KD clone 1 before and after the DOX treatment.


F . NE and anti - CDK9IP from NE of inducible DKC1 KD clone 1 , which was untreated or treated with DOX , were analyzed by WB for the indicated proteins and PEx for 7SK RNA .

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

F. NE and anti-CDK9IP from NE of inducible DKC1 KD clone 1, which was untreated or treated with DOX, were analyzed by WB for the indicated proteins and PEx for 7SK RNA.


G . Clone 1 was treated with DOX for 3 days and then transfected with the Tat - Flag cDNA ( 20 ug / 150 - mm dish ) . Two days later , NE and anti - FlagIP from NE were analyzed as in F .

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

G. Clone 1 was treated with DOX for 3 days and then transfected with the Tat-Flag cDNA (20 ug/150-mm dish). Two days later, NE and anti-FlagIP from NE were analyzed as in F.


A . Luciferase activities were measured in HeLa cells transfected with the indicated reporter constructs , an empty vector ( - ) or the Tat cDNA , and either siDKC1 or a non - target siRNA ( siNT ) . For each reporter , the activity in cells expressing siNT was set to 1 .

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

A. Luciferase activities were measured in HeLa cells transfected with the indicated reporter constructs, an empty vector (-) or the Tat cDNA, and either siDKC1 or a non-target siRNA (siNT). For each reporter, the activity in cells expressing siNT was set to 1.


B , C , and E . The mRNA levels of luciferase ( B ) or Tat ( C E ) in cells expressing the indicated siRNAs were determined by qRT - PCR and normalized to the GAPDH mRNA and plotted .

B, C, and E. The mRNA levels of luciferase (B) or Tat (C E) in cells expressing the indicated siRNAs were determined by qRT-PCR and normalized to the GAPDH mRNA and plotted.


D . NH1 cells containing the integrated HIV - 1 LTR - luciferase reporter and expressing Tat were transfected with siDKC1 or siNT . ChIP - qPCR analysis was performed to determine the levels of the indicated factors bound to the HIV - 1 promoter . The signals were normalized to those of input and plotted .

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

D. NH1 cells containing the integrated HIV-1 LTR-luciferase reporter and expressing Tat were transfected with siDKC1 or siNT. ChIP-qPCR analysis was performed to determine the levels of the indicated factors bound to the HIV-1 promoter. The signals were normalized to those of input and plotted.


B , C , and E . The mRNA levels of Tat ( C E ) in cells expressing the indicated siRNAs were determined by qRT - PCR and normalized to the GAPDH mRNA and plotted .

B, C, and E. The mRNA levels of Tat (C E) in cells expressing the indicated siRNAs were determined by qRT-PCR and normalized to the GAPDH mRNA and plotted.


F . Western analysis of DKC1 and CDK9 levels in a Jurkat 2D10 - derived clone inducibly expressing shDKC1 upon exposure to DOX .

F. Western analysis of DKC1 and CDK9 levels in a Jurkat 2D10-derived clone inducibly expressing shDKC1 upon exposure to DOX.


G and H . The inducible DKC1 KD clone , treated or untreated with DOX for 4 days , was incubated with the indicated concentrations of JQ1 ( G ) or prostratin ( H ) for 24 h . The GFP expression was measured by flow cytometry and expressed as percentages of GFP ( + ) cells of the entire population . The error bars in all panels represent mean ± SD from three independent experiments , and the p values determined by the Student ' s t - test .

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

G and H. The inducible DKC1 KD clone, treated or untreated with DOX for 4 days, was incubated with the indicated concentrations of JQ1 (G) or prostratin (H) for 24 h. The GFP expression was measured by flow cytometry and expressed as percentages of GFP(+) cells of the entire population. The error bars in all panels represent mean ± SD from three independent experiments, and the p values determined by the Student's t-test.


A . Western blot analysis of supernatants and whole cell lysates collected from WT and IQGAP1 KO YAMC cells with and without LPS stimulation for 4 hours or LPS plus ATP ( 4 hours plus 30 minutes ) treatment as indicated .

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

A. Western blot analysis of supernatants and whole cell lysates collected from WT and IQGAP1 KO YAMC cells with and without LPS stimulation for 4 hours or LPS plus ATP (4 hours plus 30 minutes) treatment as indicated.


B . Nanoparticle tracking analysis of supernatant of WT and IQGAP1 KO YAMC cells treated with LPS plus ATP . * * , P < 0 . 01 by unpaired two - tailed t test ( n = 3 biological repeats in one experiments ) .

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

B. Nanoparticle tracking analysis of supernatant of WT and IQGAP1 KO YAMC cells treated with LPS plus ATP. **, P<0.01 by unpaired two-tailed t test (n=3 biological repeats in one experiments).


C . Size distributions for vesicles as analyzed in D .

C. Size distributions for vesicles as analyzed in D.


D . Western blot analysis of enriched extracellular vesicles from supernatants WT and IQGAP1 KO YAMC cells .

D. Western blot analysis of enriched extracellular vesicles from supernatants WT and IQGAP1 KO YAMC cells.


F . Flow cytometry analysis for CD63 + exosomes from supernatants of LPS plus ATP treated WT and IQGAP1 KO supernatants . Quantification was done by mean fluorescence intensity . * * , P < 0 . 01 by unpaired two - tailed t test ( n = 4 ) .

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

F. Flow cytometry analysis for CD63+ exosomes from supernatants of LPS plus ATP treated WT and IQGAP1 KO supernatants. Quantification was done by mean fluorescence intensity. **, P<0.01 by unpaired two-tailed t test (n=4).


B . Co - immunoprecipitation of GSDMD with full - length and deletion mutants of IQGAP1 tested in HEK293 cells .

B. Co-immunoprecipitation of GSDMD with full-length and deletion mutants of IQGAP1 tested in HEK293 cells.


C . Co - immunoprecipitation of GSDMD cleavage mutant D276A with IQGAP1 overexpressed in HEK293 cells .

C. Co-immunoprecipitation of GSDMD cleavage mutant D276A with IQGAP1 overexpressed in HEK293 cells.


D . Co - immunoprecipitation of GSDMD with IQGAP1 in WT and cleavage mutant ( D276A ) restored GSDMD KO YAMC cells . Cells were treated as indicated .

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

D. Co-immunoprecipitation of GSDMD with IQGAP1 in WT and cleavage mutant (D276A) restored GSDMD KO YAMC cells. Cells were treated as indicated.


E . Co - immunoprecipitation of C - terminal GSDMD ( CT ) with IQGAP1 overexpressed in HEK293 cells .

E. Co-immunoprecipitation of C-terminal GSDMD (CT) with IQGAP1 overexpressed in HEK293 cells.


F . Co - immunoprecipitation of pyroptosis mutants full - length ( I105N FL ) and N - terminal GSDMD defective of pyroptosis ( I105N - NT ) with IQGAP1 .

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

F. Co-immunoprecipitation of pyroptosis mutants full-length (I105N FL) and N-terminal GSDMD defective of pyroptosis (I105N-NT) with IQGAP1.


A . Imaging analysis for co - localization of CD63 with IQGAP1 in IQGAP1 ( FL ) and IQ domain truncation ( ΔIQ ) restored YAMC cell . Cells were treated with LPS plus ATP . The IQGAP1 - CD63 co - localization dots ( shown by yellow ) were counted , and Pearson ' s correlation coefficient was calculated for the quantification of co - localization .

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

A. Imaging analysis for co-localization of CD63 with IQGAP1 in IQGAP1 (FL) and IQ domain truncation (ΔIQ) restored YAMC cell. Cells were treated with LPS plus ATP. The IQGAP1-CD63 co-localization dots (shown by yellow) were counted, and Pearson's correlation coefficient was calculated for the quantification of co-localization.


B . Imaging analysis for co - localization of CD63 with GSDMD in IQGAP1 full length ( FL ) and IQ domain truncation restored YAMC cell ( ΔIQ ) after LPS plus ATP treatment . The GSDMD - CD63 co - localized dots were counted and Pearson ' s correlation coefficient was calculated for quantification .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Imaging analysis for co-localization of CD63 with GSDMD in IQGAP1 full length (FL) and IQ domain truncation restored YAMC cell (ΔIQ) after LPS plus ATP treatment. The GSDMD-CD63 co-localized dots were counted and Pearson's correlation coefficient was calculated for quantification.


C . Proximity ligation assay for IQGAP1 and GSDMD in IQGAP1 FL restored cells and ΔIQ restored cells .

C. Proximity ligation assay for IQGAP1 and GSDMD in IQGAP1 FL restored cells and ΔIQ restored cells.


D . Western blots analysis for supernatants collected from IQGAP1 KO YAMC cells , IQGAP1 full length and IQ domain truncation restored IQGAP1 KO YAMC cells . Cells were treated as indicated and the supernatants were subjected to IP with anti - IL - 1β . E . Western blots analysis for whole cell lysate collected from panel D .

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

D. Western blots analysis for supernatants collected from IQGAP1 KO YAMC cells, IQGAP1 full length and IQ domain truncation restored IQGAP1 KO YAMC cells. Cells were treated as indicated and the supernatants were subjected to IP with anti-IL-1β. E. Western blots analysis for whole cell lysate collected from panel D.


F . ZetaView particle analysis for exosomes concentrated from supernatants collected from LPS or LPS and ATP treated cells as indicated .

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

F. ZetaView particle analysis for exosomes concentrated from supernatants collected from LPS or LPS and ATP treated cells as indicated.


G . Vesicle size distributions for exosome samples collected from panel E .

G. Vesicle size distributions for exosome samples collected from panel E.


A . Imaging analysis for early endosome ( shown by marker EEA1 ) change in WT and IQGAP1 KO YAMC cells upon indicated treatments . Quantification was done by counting the EEA1 puncta from each cell . Five field views of cells ' puncta were counted . Three independent experiments were

A. Imaging analysis for early endosome (shown by marker EEA1) change in WT and IQGAP1 KO YAMC cells upon indicated treatments. Quantification was done by counting the EEA1 puncta from each cell. Five field views of cells' puncta were counted. Three independent experiments were repeated


B . Co - localization of CD63 and IQGAP1 in YAMC and GSDMD KO YAMC cells . Yellow dots indicated the co - localization signal .

O O 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. Co-localization of CD63 and IQGAP1 in YAMC and GSDMD KO YAMC cells. Yellow dots indicated the co-localization signal.


C . Co - localization of CD63 and GSDMD in YAMC and IQGAP1 KO YAMC cells .

C. Co-localization of CD63 and GSDMD in YAMC and IQGAP1 KO YAMC cells.


D . Co - localization of CD63 with IQGAP1 - GSDMD complex , as measured by PLA , shown by the yellow dots after LPS and ATP treatment .

D. Co-localization of CD63 with IQGAP1-GSDMD complex, as measured by PLA, shown by the yellow dots after LPS and ATP treatment.


E . Co - localization of CD63 and pro - IL - 1β in YAMC and IQGAP1 KO YAMC cells .

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

E. Co-localization of CD63 and pro-IL-1β in YAMC and IQGAP1 KO YAMC cells.


F . Western blot analysis of extracellular vesicle secretion from WT and TSG101 knockdown cells upon indicated treatments . Secreted proteins were pulled down by IL - 1β in the supernatant and the indicated proteins were probed for .

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

F. Western blot analysis of extracellular vesicle secretion from WT and TSG101 knockdown cells upon indicated treatments. Secreted proteins were pulled down by IL-1β in the supernatant and the indicated proteins were probed for.


G . Total particle count and size distribution for exosomes collected from WT and TSG101 knockdown cell supernatants with indicated treatment .

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

G. Total particle count and size distribution for exosomes collected from WT and TSG101 knockdown cell supernatants with indicated treatment.


H . Western blot analysis for interaction of TSG101 with IQGAP1 in WT and GSDMD KO cells by pulling down IQGAP1 under indicated treatments .

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

H. Western blot analysis for interaction of TSG101 with IQGAP1 in WT and GSDMD KO cells by pulling down IQGAP1 under indicated treatments.


I . Co - immunoprecipitation of GSDMD with TSG101 in WT and IQGAP1 KO cells by pulling down TSG101 under indicated treatments .

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

I. Co-immunoprecipitation of GSDMD with TSG101 in WT and IQGAP1 KO cells by pulling down TSG101 under indicated treatments.


A . Total particle count and size distribution of exosomes secreted in the supernatant from IQGAP1 KO ( IQ1 KO ) , IQGAP1 full length restored ( FL ) and GRD domain truncation restored ( ΔGRD ) YAMC cells upon LPS or LPS plus ATP treatment .

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

A. Total particle count and size distribution of exosomes secreted in the supernatant from IQGAP1 KO (IQ1 KO), IQGAP1 full length restored (FL) and GRD domain truncation restored (ΔGRD) YAMC cells upon LPS or LPS plus ATP treatment.


B . PLA assay for IQGAP1 and CDC42 interaction in IQGAP1 FL restored cell and ΔGRD restored cell . The green dots showed the interaction of IQGAP1 and CDC42 .

O O O O O O 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. PLA assay for IQGAP1 and CDC42 interaction in IQGAP1 FL restored cell and ΔGRD restored cell. The green dots showed the interaction of IQGAP1 and CDC42.


C . Colocalization of CD63 with IQGAP1 - CDC42 complex ( shown by PLA signal ) in WT and GSDMD KO cells under LPS treatment . Quantification was performed by counting PLA green dots .

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

C. Colocalization of CD63 with IQGAP1-CDC42 complex (shown by PLA signal) in WT and GSDMD KO cells under LPS treatment. Quantification was performed by counting PLA green dots.


D . Co - immunoprecipitation analysis of endogenous IQGAP1 with CDC42 in wild - type and GSDMD KO cells upon LPS or LPS plus ATP treatment .

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

D. Co-immunoprecipitation analysis of endogenous IQGAP1 with CDC42 in wild-type and GSDMD KO cells upon LPS or LPS plus ATP treatment.


E . Co - immunoprecipitation analysis of GTP - bound CDC42 in indicated cells following LPS treatment .

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

E. Co-immunoprecipitation analysis of GTP-bound CDC42 in indicated cells following LPS treatment.


F . Co - immunoprecipitation analysis of endogenous GSDMD with CDC42 in wild - type and IQGAP1 KO cells upon LPS or LPS plus ATP treatment .

F. Co-immunoprecipitation analysis of endogenous GSDMD with CDC42 in wild-type and IQGAP1 KO cells upon LPS or LPS plus ATP treatment.


G . Western blots for exosome secretion upon CDC42 GTPase inhibition . Cells were pretreated with and without 10µM ML141 for 24 hours to fully block its GTPase activity , followed by LPS and ATP treatment as indicated . H . Particle analysis and size distribution for exosomes collected from cells with the same treatment as panel G .

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

G. Western blots for exosome secretion upon CDC42 GTPase inhibition. Cells were pretreated with and without 10µM ML141 for 24 hours to fully block its GTPase activity, followed by LPS and ATP treatment as indicated. H. Particle analysis and size distribution for exosomes collected from cells with the same treatment as panel G.


I . Co - immunoprecipitation analysis of endogenous GSDMD with CDC42 in vehicle or ML141 ( 10µM for 24 hours ) treated cells following LPS or LPS plus ATP treatment .

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

I. Co-immunoprecipitation analysis of endogenous GSDMD with CDC42 in vehicle or ML141 (10µM for 24 hours) treated cells following LPS or LPS plus ATP treatment.


A . Histology staining for IQGAP1 and GSDMD in naive and inflamed ( 3 days of DSS - challenge ) colon tissues .

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

A. Histology staining for IQGAP1 and GSDMD in naive and inflamed (3 days of DSS-challenge) colon tissues.


B . Total particle count and size distribution for exosomes collected from colon explant of DSS treated IQGAP1 or GSDMD knockout mice and their respective controls .

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

B. Total particle count and size distribution for exosomes collected from colon explant of DSS treated IQGAP1 or GSDMD knockout mice and their respective controls.


C . Western blot analysis for supernatant collected from colon explants of DSS - treated wild - type ( WT ) , Gsdmd - / - ( GSDMD KO ) and Iqgap1 - / - ( IQGAP1 KO ) mice . Supernatant was first pulled down by IL - 1β antibody and samples were subjected to Western blot analysis . The indicated proteins were probed .

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

C. Western blot analysis for supernatant collected from colon explants of DSS-treated wild-type (WT), Gsdmd-/-(GSDMD KO) and Iqgap1-/- (IQGAP1 KO) mice. Supernatant was first pulled down by IL-1β antibody and samples were subjected to Western blot analysis. The indicated proteins were probed.


D . Immunofluorescent staining with anti - GTP - CDC42 in DSS treated colons from Gsdmd + / + ( WT ) and Gsdmd - / - ( GSDMD KO ) , IQGAP1 + / + and IQGAP1 - / - mice .

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

D. Immunofluorescent staining with anti-GTP-CDC42 in DSS treated colons from Gsdmd+/+ (WT) and Gsdmd-/-(GSDMD KO), IQGAP1+/+ and IQGAP1-/- mice.


( C ) Quantification of AQR position in adult WT , cst - 1 and / or cst - 2 mutant animals . The full length between the URXs and the end of the nematode tail is divided into 10 blocks , and the percent of AQR that stopped within each block is indicated . * * * , P < 0 . 001 by Fisher ' s exact test . N = 50 - 109 .

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

(C) Quantification of AQR position in adult WT, cst-1 and/or cst-2 mutant animals. The full length between the URXs and the end of the nematode tail is divided into 10 blocks, and the percent of AQR that stopped within each block is indicated. ***, P < 0.001 by Fisher's exact test. N = 50 - 109.


( E - F ) Fluorescence time - lapse images of F - actin , plasma membrane and histone during AQR / QR . ap migration in WT ( E ) or cst - 1 ; cst - 2 double mutant ( F ) animals . Merged images , left ; inverted fluorescence images of F - actin , right ; white arrows , migration direction ; asterisks , nucleus ; double headed arrows , migration distance . F - actin is labeled with GFP - tagged with the actin - binding domain of Moesin ( GFP : : Moesin ABD ) , and the plasma membrane and Histone are labeled with mCherry - tagged with a myristoylation signal and a histone . The dotted lines indicate the cell periphery . Time is presented in minutes . Scale bars , 5 μm . ( G - J ) Quantification of the QR . ap migration angle ( G H ) , distance ( I ) and speed ( J ) . Each line in ( G I ) represents the measurement from one time - lapse movie . Error bars indicate SD . N = 10 - 17 . Statistical significance is based on Student ' s t - tests , * * , P 0 . 01 ; * * * , P 0 . 001 .

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

(E - F) Fluorescence time-lapse images of F-actin, plasma membrane and histone during AQR/QR.ap migration in WT (E) or cst-1; cst-2 double mutant (F) animals. Merged images, left; inverted fluorescence images of F-actin, right; white arrows, migration direction; asterisks, nucleus; double headed arrows, migration distance. F-actin is labeled with GFP-tagged with the actin-binding domain of Moesin (GFP::Moesin ABD), and the plasma membrane and Histone are labeled with mCherry-tagged with a myristoylation signal and a histone. The dotted lines indicate the cell periphery. Time is presented in minutes. Scale bars, 5 μm. (G - J) Quantification of the QR.ap migration angle (G H), distance (I) and speed (J). Each line in (G I) represents the measurement from one time-lapse movie. Error bars indicate SD. N = 10 - 17. Statistical significance is based on Student's t-tests, **, P 0.01; ***, P  0.001.


( A ) Fluorescence time - lapse images of F - actin , plasma membrane and histone during QR . ap migration in mig - 2 ( rh17 ) gain - of - function mutant animals . Merged images , left ; inverted fluorescence images of F - actin , right ; white arrows , migration direction ; asterisks , nucleus ; double headed arrow , migration distance . The dotted lines indicate the cell periphery . Time is presented in minutes . Scale bar , 5 μm . ( B - C ) Quantification of the QR . ap migration angle ( B ) and distance ( C ) . N = 10 .

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

(A) Fluorescence time-lapse images of F-actin, plasma membrane and histone during QR.ap migration in mig-2(rh17) gain-of-function mutant animals. Merged images, left; inverted fluorescence images of F-actin, right; white arrows, migration direction; asterisks, nucleus; double headed arrow, migration distance. The dotted lines indicate the cell periphery. Time is presented in minutes. Scale bar, 5 μm. (B - C) Quantification of the QR.ap migration angle (B) and distance (C). N = 10.


( D ) Mass spectrum of a MIG - 2 peptide that was phosphorylated at Serine 139 in C . elegans . The peptide was identified from the GFP : : MIG - 2 immunoprecipitate .

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

(D) Mass spectrum of a MIG-2 peptide that was phosphorylated at Serine 139 in C. elegans. The peptide was identified from the GFP::MIG-2 immunoprecipitate.


( E ) In vitro phosphorylation assay of CST - 1 / 2 to MIG - 2 . Bacterially expressed His - tagged MIG - 2 or His - tagged MIG - 2S139A were incubated with the GST - tagged kinase domain of GST - CST - 1 / 2 ( 1 - 318 aa ) . The kinase may use ATP - γ - S as a phosphodonor to thiophosphorylate its substrate ; thus , the phosphorylation reaction was performed with ATP - γ - S , and anti - thiophosphate ester antibody was used to detect substrate phosphorylation ( Allen et al , 2005 ; Allen et al , 2007 ) . The final reaction was probed via immunoblotting with antibodies as indicated . Thio , thiophosphate ester .

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

(E) In vitro phosphorylation assay of CST-1/2 to MIG-2. Bacterially expressed His-tagged MIG-2 or His-tagged MIG-2S139A were incubated with the GST-tagged kinase domain of GST-CST-1/2 (1-318 aa). The kinase may use ATP-γ-S as a phosphodonor to thiophosphorylate its substrate; thus, the phosphorylation reaction was performed with ATP-γ-S, and anti-thiophosphate ester antibody was used to detect substrate phosphorylation (Allen et al, 2005; Allen et al, 2007). The final reaction was probed via immunoblotting with antibodies as indicated. Thio, thiophosphate ester.


( G H ) Three - dimensional ribbon ( PDB 3TH5 ) representations of the human Rac1 polypeptide which indicate the position of the T135 ( S139 in MIG - 2 ) located between the last two α - helixes ( Krauthammer et al , 2012 ) .

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

(G H) Three-dimensional ribbon (PDB 3TH5) representations of the human Rac1 polypeptide which indicate the position of the T135 (S139 in MIG-2) located between the last two α-helixes (Krauthammer et al, 2012).


( A ) Quantification of AQR position in mig - 2 ( mu28 ) null mutant animals with different rescue transgenes . N = 108 - 114 . * , P < 0 . 05 ; # # , P < 0 . 01 , phenotype enhanced . P values are based on Fisher ' s exact tests .

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

(A) Quantification of AQR position in mig-2(mu28) null mutant animals with different rescue transgenes. N = 108 - 114. *, P < 0.05; ##, P < 0.01, phenotype enhanced. P values are based on Fisher's exact tests.


( B ) Quantification of AQR position in WT and knock - in animals that harbor mig - 2S139 point mutations . N = 100 - 111 . * * * , P < 0 . 001 by Fisher ' s exact test .

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

(B) Quantification of AQR position in WT and knock-in animals that harbor mig-2S139 point mutations. N = 100 - 111. ***, P < 0.001 by Fisher's exact test.


( C - F ) Quantification of the QR . ap migration angle ( C D ) , distance ( E ) and speed ( F ) in mig - 2S139A / E mutant animals . Each line in ( C E ) represents the measurement from one movie of QR . ap migration . Error bars indicate SD . N = 12 - 17 . * * , P 0 . 01 ; * * * , P 0 . 001 by Student ' s t tests .

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

(C - F) Quantification of the QR.ap migration angle (C D), distance (E) and speed (F) in mig-2S139A/E mutant animals. Each line in (C  E) represents the measurement from one movie of QR.ap migration. Error bars indicate SD. N = 12 -17. **, P 0.01; ***, P  0.001 by Student's t tests.


( G ) Inverted fluorescence images ( up ) and schematics ( down ) of QR . ap in WT , cst - 1 ; cst - 2 double or mig - 2 mutant animals . The plasma membrane and histones were labeled with mCherry . Scale bar , 5 μm . ( H ) Area ratio between the cell body and the nucleus in WT and mutant animals . Error bars indicate SD . N = 17 - 22 . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 by Student ' s t tests .

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

(G) Inverted fluorescence images (up) and schematics (down) of QR.ap in WT, cst-1; cst-2 double or mig-2 mutant animals. The plasma membrane and histones were labeled with mCherry. Scale bar, 5 μm. (H) Area ratio between the cell body and the nucleus in WT and mutant animals. Error bars indicate SD. N = 17 - 22. *, P < 0.05; **, P < 0.01; ***, P < 0.001 by Student's t tests.


( I - K ) Representative frames ( upper ) and quantification ( lower ) of F - actin enrichment at the QR . ap periphery in WT ( I ) and mig - 2S139A / E mutant animals ( J K ) . The trace starts from the rear of the QR . ap and moves along the cell periphery to the leading edge ( L ) and back to the rear ( R ) . F - actin was labeled with GFP : : Moesin ABD ; the plasma membrane and histone were marked with mCherry fluorescence . Green lines , F - actin fluorescence intensity ; red lines , plasma membrane fluorescence intensity ; dotted lines , cell periphery . Double - headed arrows indicate the F - actin enrichment region , in which the fluorescence intensity is 2 - fold higher than the background . Scale bars , 5 μm . ( L ) Quantification of F - actin enrichment ratio in WT and mig - 2 mutants . Error bars indicate SD . N = 28 - 68 . * * , P 0 . 01 ; * * * , P 0 . 001 by Student ' s t - tests .

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

(I - K) Representative frames (upper) and quantification (lower) of F-actin enrichment at the QR.ap periphery in WT (I) and mig-2S139A/E mutant animals (J  K). The trace starts from the rear of the QR.ap and moves along the cell periphery to the leading edge (L) and back to the rear (R). F-actin was labeled with GFP::Moesin ABD; the plasma membrane and histone were marked with mCherry fluorescence. Green lines, F-actin fluorescence intensity; red lines, plasma membrane fluorescence intensity; dotted lines, cell periphery. Double-headed arrows indicate the F-actin enrichment region, in which the fluorescence intensity is 2-fold higher than the background. Scale bars, 5 μm. (L) Quantification of F-actin enrichment ratio in WT and mig-2 mutants. Error bars indicate SD. N = 28 - 68. **, P  0.01; ***, P  0.001 by Student's t-tests.


( A - C ) Fluorescence images ( upper ) and quantifications ( lower ) of GFP - tagged CST - 1 ( A B ) or GFP - labeled F - actin ( C ) and mCherry tagged plasma membrane and histone in WT and mig - 2 null mutant animals . The membrane protrusions in the white boxes are enlarged 3 times at the bottom . Yellow dashed lines indicate the cell periphery . White lines indicate the line to measure the fluorescence intensity . Green lines denote the CST - 1 fluorescence intensity ; red lines denote the plasma membrane fluorescence intensity ; double - headed arrows denote the distance between the GFP signal and cell periphery . Scale bars , 5 μm . ( D ) Distance between GFP : : CST - 1 or GFP : : Moesin ABD and the plasma membrane at the leading edge in WT and mig - 2 null mutants . Error bars indicate SD . N = 9 - 14 . * * * , P 0 . 001 by Student ' s t test .

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

(A - C) Fluorescence images (upper) and quantifications (lower) of GFP-tagged CST-1 (A  B) or GFP-labeled F-actin (C) and mCherry tagged plasma membrane and histone in WT and mig-2 null mutant animals. The membrane protrusions in the white boxes are enlarged 3 times at the bottom. Yellow dashed lines indicate the cell periphery. White lines indicate the line to measure the fluorescence intensity. Green lines denote the CST-1 fluorescence intensity; red lines denote the plasma membrane fluorescence intensity; double-headed arrows denote the distance between the GFP signal and cell periphery. Scale bars, 5 μm. (D) Distance between GFP::CST-1 or GFP::Moesin ABD and the plasma membrane at the leading edge in WT and mig-2 null mutants. Error bars indicate SD. N = 9 - 14. ***, P  0.001 by Student's t test.


( E - F ) Fluorescence images ( left ) and quantifications ( right ) of GFP - tagged CST - 1 and mCherry tagged plasma membrane and histone in WT and mig - 2 null mutant animals . Blue lines , sites of measurements ; dotted lines , cell periphery ; Green lines ; CST - 1 fluorescence intensity ; red lines , mCherry fluorescence intensity . Scale bars , 5 μm . ( G ) Quantification of the fluorescence intensity ratio of GFP : : CST - 1 and mCherry - membrane between the leading edge and the lagging edge of AQR in WT and mig - 2 null mutant animals . Error bars indicate SD . N = 20 . * * * , P < 0 . 001 by Student ' s t test .

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

(E - F) Fluorescence images (left) and quantifications (right) of GFP-tagged CST-1 and mCherry tagged plasma membrane and histone in WT and mig-2 null mutant animals. Blue lines, sites of measurements; dotted lines, cell periphery; Green lines; CST-1 fluorescence intensity; red lines, mCherry fluorescence intensity. Scale bars, 5 μm. (G) Quantification of the fluorescence intensity ratio of GFP::CST-1 and mCherry-membrane between the leading edge and the lagging edge of AQR in WT and mig-2 null mutant animals. Error bars indicate SD. N = 20. ***, P < 0.001 by Student's t test.


Statistical analysis of stx mutant stx34 sleep profile compared with that of the Canton - S control . 24 - hour - sleep curve ( y - axis was average sleep time in every 30 minutes ) , n = 16 . Statistical analysis of stx mutant stxd77 sleep profile compared with control w1118 . 24 - hour - sleep curve , n = 16 .

Statistical analysis of stx mutant stx34 sleep profile compared with that of the Canton-S control. 24-hour-sleep curve (y-axis was average sleep time in every 30 minutes), n=16. Statistical analysis of stx mutant stxd77 sleep profile compared with control w1118. 24-hour-sleep curve, n=16.


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

Rescue experiment of stx mutant. The stx mutant phenotype can be rescued by stx over expression driven by stx-Gal4 (in female flies, P) or EB1-Gal4 (Q). For P, from left to right females mean±SEM: 226.0±11.76, n=49; 336.6±14.10, n=86; 382.7±16.43, n=50; 357.8±16.55, n=72; 296.2±16.04, n=68; 484.9±12.97, n=49; 507.6±13.83, n=86; 539.5±12.99, n=50. 577.0±11.34, n=72; 520.7±14.96, n=68; For Q, from left to right mean±SEM; 398.3±9.535, n=63; 378.4±23.78, n=32; 324.4±12.33, n=45; 495.8±16.43, n=28; 478.5±14.65, n=53; 328.7±16.99, n=34; 540.7±7.690, n=63; 489.9±21.39, n=32; 490.9±13.65, n=45; 494.7±11.99, n=28; 537.8±12.07, n=53; 450.3±15.47, n=34.


Statistical analysis of Pc and stx double mutant stxd77 ; ; pcXT109 sleep profile compared with controls . Quantitative RT - PCR of Octβ2R in adult head of stxd77 and w1118 control . ( Data represent mean ± SEM : w1118 , 0 . 8853 ± 0 . 1177 ; N = 3 ; stxd77 / Y , 0 . 3263 ± 0 . 1575 ； N = 3 ) .

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

Statistical analysis of Pc and stx double mutant stxd77;; pcXT109 sleep profile compared with controls. Quantitative RT-PCR of Octβ2R in adult head of stxd77 and w1118 control. (Data represent mean±SEM: w1118, 0.8853±0.1177; N=3; stxd77/Y, 0.3263±0.1575；N=3).


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

Rescue of stxd77 by over expressing Octβ2R in ellipsoid body. From left to right mean±SEM: 399.6±11.49, n=50; 400.3±10.48, n=60; 349.9±14.77, n=48; 495.8±16.43, n=28; 470.1±13.04, n=44; 423.6±14.32, n=47; 548.6±8.707, n=50; 499.0±8.225, n=60; 490.7±16.26, n=48; 494.7±11.99, n=28; 480.2±14.64, n=44; 437.4±14.85, n=47.


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

Rescue of stxd77 by Pc RNAi in ellipsoid body. From left to right mean±SEM: 399.9±11.88, n=48; 409.8±11.50, n=49; 348.7±20.19, n=32; 495.8±16.43, n=28; 459.3±14.78, n=61; 407.5±15.66, n=40; 550.7±8.881, n=48; 516.0±11.76, n=49; 502.3±15.08, n=32; 494.7±11.99, n=28; 546.3±11.35, n=61; 514.2±18.94, n=40. Note that the same data is used for genotype stxd77;; EB1-Gal4 in panel F and G.


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

Sleep phenotype of OctβR double and triple mutants. (From left to right mean±SEM: 392.0±12.91, n=60; 395.1±8.56, n=79; 433.7±13.03, n=58; 422.8±10.34; n=62; 474.4±8.36, n=73; 517.4±6.30, n=119; 513.2±7.10, n=79; 536.6±9.05 n=78; 499.1±11.95, n=60; 546.0±8.51, n=79; 519.6±10.31, n=58; 518.9±13.13; n=62; 584.3±8.59, n=73; 579.2±8.35, n=119; 560.9±6.80, n=79; 606.4±6.38, n=78).


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

Quantitative RT-PCR of stx after OA treatment in w1118 control and Octβ1R, Octβ2R mutants, Octβ3R mutants. (From left to right mean±SEM: 1.000±0.0288, 0.781±0.0176, 1.477±0.043，1.403±0.0578, 1.157±0.0617, 0.881±0.0689, 1.149±0.0398, 0.953±0.0543, 1.017±0.0600, 1.090±0.0378, N=3).


Ellipsoid body knock down of Octβ2R results in sleep rebound increase . In response to OA , ellipsoid knock down of Octβ2R rescues control phenotype . Data represent mean ± SEM : 13 . 80 ± 2 . 439 , n = 31 ; 5 . 465 ± 2 . 036 , n = 25 ; 19 . 55 ± 2 . 275 , n = 27 ; 12 . 47 ± 2 . 684 , n = 24 ; 30 . 03 ± 3 . 428 , n = 25 ; 43 . 67 ± 4 . 616 , n = 25 .

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

Ellipsoid body knock down of Octβ2R results in sleep rebound increase. In response to OA, ellipsoid knock down of Octβ2R rescues control phenotype. Data represent mean±SEM: 13.80±2.439, n=31; 5.465±2.036, n=25; 19.55±2.275, n=27; 12.47±2.684, n=24; 30.03±3.428, n=25; 43.67±4.616, n=25.


Sleep profile of PcXT109 mutant with or without OA treatment . In both control and PcXT109 mutant flies , the treatment of OA results in sleep decrease , n = 16 ( G ) . Quantifications of the decreasing amount showed that PcXT109 mutant fly loss significantly more sleep that control flies ( From left to right mean ± SEM : 13 . 90 ± 1 . 397 ; 21 . 25 ± 1 . 493 , N = 4 ) ( H ) .

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

Sleep profile of PcXT109 mutant with or without OA treatment. In both control and PcXT109 mutant flies, the treatment of OA results in sleep decrease, n=16 (G). Quantifications of the decreasing amount showed that PcXT109 mutant fly loss significantly more sleep that control flies (From left to right mean±SEM: 13.90±1.397; 21.25±1.493, N=4) (H).


Total sleep for Tβh mutant and control flies in video - based method ( From left to right mean ± SEM : 688 . 2 ± 32 . 71 , n = 48 ; 123 . 2 ± 11 . 68 , n = 46 ) .

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

Total sleep for Tβh mutant and control flies in video-based method (From left to right mean±SEM: 688.2±32.71, n=48; 123.2±11.68, n=46).


( A ) Western blot analysis with representative blot including lamin A / C , NLRP3 , caspase 1 , and actin levels in skin fibroblats from patients with HGPS , n = 2 controls and 2 patients . Positive control correspond to THP1 cells stimulated with LPS + ATP .

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

(A) Western blot analysis with representative blot including lamin A/C, NLRP3, caspase 1, and actin levels in skin fibroblats from patients with HGPS, n = 2 controls and 2 patients. Positive control correspond to THP1 cells stimulated with LPS+ATP.


( B ) Immunofluorescence ( IF ) visualization of NLRP3 ( green ) and nuclei ( blue ) in skin fibroblasts from a representative patient and control . Scale bar :

O O O O O O O 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) Immunofluorescence (IF) visualization of NLRP3 (green) and nuclei (blue) in skin fibroblasts from a representative patient and control. Scale bar: 30µm


( C ) Protein expression of NLRP3 and caspase 1 in lymphoblasts from control and one patient after stimulation with uric acid and cholesterol crystal .

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

(C) Protein expression of NLRP3 and caspase 1 in lymphoblasts from control and one patient after stimulation with uric acid and cholesterol crystal.


( D ) IL - 1β and IL - 18 medium release from lymphoblasts which were assessed after a 24 hr incubation with uric acid and cholesterol . * * * P < 0 . 001 , * * P < 0 . 005 , * treatment vs no treatment ; aaaP < 0 . 001 ; aaP < 0 . 01 control cells vs patient cells .

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

(D) IL-1β and IL-18 medium release from lymphoblasts which were assessed after a 24 hr incubation with uric acid and cholesterol. ***P < 0.001, **P < 0.005, * treatment vs no treatment; aaaP < 0.001; aaP < 0.01 control cells vs patient cells.


( E ) Western blot analysis with representative blot including NLRP3 , caspase 1 , IL - 1β and actin levels in heart and liver tissues from wild - type and Zmpste24 - / - mice .

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

(E) Western blot analysis with representative blot including NLRP3, caspase 1, IL-1β and actin levels in heart and liver tissues from wild-type and Zmpste24-/- mice.


( F ) NLRP3 and Caspase 1 transcript expression levels were determined in heart and liver tissues by real - time quantitative RT - PCR . n = 5 for Zmpste24 + / + and n = 5 for Zmpste24 - / - groups respectively . * * * P < 0 . 001 , * * P < 0 . 005 , wild - type vs Zmpste24 - / - mice . Data are showed means ± SD , n = 4 mice per group .

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

(F) NLRP3 and Caspase 1 transcript expression levels were determined in heart and liver tissues by real-time quantitative RT-PCR. n=5 for Zmpste24+/+ and n=5 for Zmpste24-/- groups respectively. ***P < 0.001, **P < 0.005, wild-type vs Zmpste24-/- mice. Data are showed means ± SD, n = 4 mice per group.


( A ) Cell growthn ( lower subpanel left patient 1 and right patient 2 ) and morphological aspect ( upper subpanel ) with MCC950 determined in healthy and representative HGPS fibroblasts . * * P < 0 . 01 , * P < 0 . 05 patient cells no treatment vs treatment . Results are presented as the mean ± SD of three independent experiments .

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

(A) Cell growthn (lower subpanel left patient 1 and right patient 2) and morphological aspect (upper subpanel) with MCC950 determined in healthy and representative HGPS fibroblasts. **P < 0.01, *P < 0.05 patient cells no treatment vs treatment. Results are presented as the mean ± SD of three independent experiments.


( B ) Western blot analysis with representative blots including lamin A / C , NLRP3 , IL - 1β and actin levels in skin fibroblats from control and HGPS patient after 48 hr of vehicle and MCC950 treatment .

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

(B) Western blot analysis with representative blots including lamin A/C, NLRP3, IL-1β and actin levels in skin fibroblats from control and HGPS patient after 48 hr of vehicle and MCC950 treatment.


( C - E ) Representative fluorescence images of HGPS and control fibroblasts to evaluate the effect of the MCC950 in the nuclear morphology ( D ) and NLRP3 expression ( E ) . Scale bar : 30µm . Results are presented as the mean ± SD of three independent experiments . * * * P < 0 . 01 patient vs control cells , aaaP < 0 . 01 patient cells no treatment vs treatment . One - way ANOVA test was used for statistical analysis .

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

(C-E) Representative fluorescence images of HGPS and control fibroblasts to evaluate the effect of the MCC950 in the nuclear morphology (D) and NLRP3 expression (E). Scale bar: 30µm. Results are presented as the mean ± SD of three independent experiments. ***P < 0.01 patient vs control cells, aaaP < 0.01 patient cells no treatment vs treatment. One-way ANOVA test was used for statistical analysis.


( F ) Kaplan - Meier graph showing a significant increase in the maximum lifespan in WT mice compared with Zmpste24 - / - mice . N = 7 per group .

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

(F) Kaplan-Meier graph showing a significant increase in the maximum lifespan in WT mice compared with Zmpste24-/- mice. N=7 per group.


( G ) Body weights evolution of the groups over time . Data are showed means ± SD , n = 6 mice per group .

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

(G) Body weights evolution of the groups over time. Data are showed means ± SD, n = 6 mice per group.


( H ) Analysis of serum concentrations of IL - 1β measured by ELISA . N = 6 per group . Data are shown as means ± SD . * * * P < 0 . 001 , wild - type vs Zmpste24 - / - mice ; aaaP < 0 . 001 ; vehicle vs MCC950 .

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

(H) Analysis of serum concentrations of IL-1β measured by ELISA. N=6 per group. Data are shown as means ± SD. ***P < 0.001, wild-type vs Zmpste24-/- mice; aaaP < 0.001; vehicle vs MCC950.


A . Representative immunoblots and quantifications ( below ) of the indicated proteins in lysates of ARPE - 19 cells treated with 1 mM DFP for the indicated time ( h , hours ) .

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

A. Representative immunoblots and quantifications (below) of the indicated proteins in lysates of ARPE-19 cells treated with 1 mM DFP for the indicated time (h, hours).


B - C . Representative confocal images of ARPE - 19 cells stably expressing the mito - QC reporter ( B ) , or the pexo - QC reporter ( C ) treated with 1 mM DFP for the indicated time ( h , hours ) . Enlarged images of the area outlined in white are shown in the lower corners . The ' red - only ' mask is generated during images analysis and highlights red - only puncta ( autolysosomes ) based on the mCherry / GFP ratio and thresholding criteria . Nuclei were stained in blue ( Hoechst ) . Scale bar : 10μm . At right , quantification of total red - only punctate per cell ( mitolysosomes or pexolysosomes ) .

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

B-C. Representative confocal images of ARPE-19 cells stably expressing the mito-QC reporter (B), or the pexo-QC reporter (C) treated with 1 mM DFP for the indicated time (h, hours). Enlarged images of the area outlined in white are shown in the lower corners. The 'red-only' mask is generated during images analysis and highlights red-only puncta (autolysosomes) based on the mCherry/GFP ratio and thresholding criteria. Nuclei were stained in blue (Hoechst). Scale bar: 10μm. At right, quantification of total red-only punctate per cell (mitolysosomes or pexolysosomes).


D . ARPE - 19 cells were transfected with 50 pmol of non - targeting siRNA ( siNT ) or 50 pmol of siRNA targeting ATG13 or ULK1 . 48 hours post knockdown , cells were treated with 1 mM DFP for an additional 48 hours . Cell lysates were subject to immunoblotting with the indicated antibodies . Quantification is shown on the right .

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

D. ARPE-19 cells were transfected with 50 pmol of non-targeting siRNA (siNT) or 50 pmol of siRNA targeting ATG13 or ULK1. 48 hours post knockdown, cells were treated with 1 mM DFP for an additional 48 hours. Cell lysates were subject to immunoblotting with the indicated antibodies. Quantification is shown on the right.


A . Representative immunoblots of the indicated proteins in lysates of WT or HIF1α KO U2OS cells , treated with 1mM DFP for 48 hours .

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

A. Representative immunoblots of the indicated proteins in lysates of WT or HIF1α KO U2OS cells, treated with 1mM DFP for 48 hours.


B . Representative confocal images of WT or HIF1α KO U2OS cells stably expressing the pexo - QC reporter and treated with 1 mM DFP for 48 hours ( left panel ) and flow cytometry analysis of the mCherry / GFP ratio ( right panel ) .

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

B. Representative confocal images of WT or HIF1α KO U2OS cells stably expressing the pexo-QC reporter and treated with 1 mM DFP for 48 hours (left panel) and flow cytometry analysis of the mCherry/GFP ratio (right panel).


C . Representative immunoblots ( left panel ) and quantification ( right panel ) of the indicated proteins in lysates of WT or two NIX KO ARPE - 19 clones ( Cl ) , treated with 1 mM DFP for the indicated time ( h , hours ) .

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

C. Representative immunoblots (left panel) and quantification (right panel) of the indicated proteins in lysates of WT or two NIX KO ARPE-19 clones (Cl), treated with 1 mM DFP for the indicated time (h, hours).


D - E . Representative confocal images of WT or NIX KO ARPE - 19 cells stably expressing the mito - QC reporter ( D ) or the pexo - QC reporter ( E ) treated with 1 mM DFP for 48 hours ( left panel ) and flow cytometry analysis of the mCherry / GFP ratio ( right panel ) .

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

D-E. Representative confocal images of WT or NIX KO ARPE-19 cells stably expressing the mito-QC reporter (D) or the pexo-QC reporter (E) treated with 1 mM DFP for 48 hours (left panel) and flow cytometry analysis of the mCherry/GFP ratio (right panel).


F . Representative immunoblots ( left panel ) and quantification ( right panel ) of the indicated proteins . NIX KO ARPE - 19 cells ( Cl 31 ) stably expressing a pBabe empty vector ( ∅ ) or a pBabe NIX vector , were treated with 1 mM DFP for 48 hours prior lysis . Two biological replicates are shown in the immunoblot .

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

F. Representative immunoblots (left panel) and quantification (right panel) of the indicated proteins. NIX KO ARPE-19 cells (Cl 31) stably expressing a pBabe empty vector (∅) or a pBabe NIX vector, were treated with 1 mM DFP for 48 hours prior lysis. Two biological replicates are shown in the immunoblot.


A - B . Representative confocal images of untreated ( CTRL ) and DFP - treated ARPE - 19 cells ( 1mM , 48 hours ) , stably expressing GFP - WT NIX ( green ) and stained with anti - catalase antibody ( A , red ) or anti - PMP70 antibody ( B , red ) . At right , quantification of Pearson correlation coefficients between NIX and the peroxisomal markers catalase and PMP70 , with or without DFP treatment for 48 hours . Peroxisomes enriched for GFP - NIX or having minimal signal for GFP - NIX are depicted with white arrowheads or white arrows , respectively .

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

A-B. Representative confocal images of untreated (CTRL) and DFP-treated ARPE-19 cells (1mM, 48 hours), stably expressing GFP-WT NIX (green) and stained with anti-catalase antibody (A, red) or anti-PMP70 antibody (B, red). At right, quantification of Pearson correlation coefficients between NIX and the peroxisomal markers catalase and PMP70, with or without DFP treatment for 48 hours. Peroxisomes enriched for GFP-NIX or having minimal signal for GFP-NIX are depicted with white arrowheads or white arrows, respectively.


C . Representative confocal images of ARPE - 19 cells stably expressing GFP - WT NIX , GFP - NIXΔTM or GFP - NIX TM only ( green ) , treated with DFP ( 1mM ) for 48 hours and immunostained with anti - catalase antibody ( red ) . At right , quantification of Pearson correlation coefficients between the green and the red signal .

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

C. Representative confocal images of ARPE-19 cells stably expressing GFP-WT NIX, GFP-NIXΔTM or GFP-NIX TM only (green), treated with DFP (1mM) for 48 hours and immunostained with anti-catalase antibody (red). At right, quantification of Pearson correlation coefficients between the green and the red signal.


B . Representative immunoblots of the indicated proteins in lysates of WT or HA - Parkin ( PRKN ) - ARPE - 19 cells treated as in A .

O O O O O 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. Representative immunoblots of the indicated proteins in lysates of WT or HA-Parkin(PRKN)- ARPE-19 cells treated as in A.


B . Representative immunoblots of the indicated proteins in lysates of NIX KO ARPE - 19 cells ( Cl 31 ) stably expressing a pBabe Flag vector ( ∅ ) , a pBabe Flag - NIX vector ( NIX ) , a pBabe Flag - NIX W36A \ L39A vector ( W36A \ L39A ) or a pBabe Flag - NIX S34E \ S35E vector ( S34E \ S35E ) , and treated with 1 mM DFP for 48 hours prior lysis .

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

B. Representative immunoblots of the indicated proteins in lysates of NIX KO ARPE-19 cells (Cl 31) stably expressing a pBabe Flag vector (∅), a pBabe Flag-NIX vector (NIX), a pBabe Flag-NIX W36A\L39A vector (W36A\L39A) or a pBabe Flag-NIX S34E\S35E vector (S34E\S35E), and treated with 1 mM DFP for 48 hours prior lysis.


C - D . Representative confocal images of cells as in B , stably expressing the mito - QC reporter ( C ) or the pexo - QC reporter ( D ) and treated with 1 mM DFP for 24 hours ( C ) or 48 hours ( D ) ( left panel ) and flow cytometry analysis of the mCherry / GFP ratio ( right panel ) . Enlarged images of the area outlined in white are shown in the lower corners . Nuclei were stained in blue ( Hoechst )

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

C-D. Representative confocal images of cells as in B, stably expressing the mito-QC reporter (C) or the pexo-QC reporter (D) and treated with 1 mM DFP for 24 hours (C) or 48 hours (D) (left panel) and flow cytometry analysis of the mCherry/GFP ratio (right panel). Enlarged images of the area outlined in white are shown in the lower corners. Nuclei were stained in blue (Hoechst)


A . Representative confocal images of optical section from WT and NIX KO mouse retina immunostained with peroxisomal markers ( PMP70 and catalase ) or a lysosomal marker LAMP1 . Nuclei are coloured in white ( DAPI ) . RPE , retinal pigment epithelium ; ONL , outer nuclear layer ; OPL , outer plexiform layer ; INL ; inner nuclear layer ; IPL , inner plexiform layer ; RGC , retinal ganglion cells . B . Quantification of signal area in the ONL and INL from images as A . Each data point represents a single mouse ( n = 3 - 4 per group ) .

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

A. Representative confocal images of optical section from WT and NIX KO mouse retina immunostained with peroxisomal markers (PMP70 and catalase) or a lysosomal marker LAMP1. Nuclei are coloured in white (DAPI). RPE, retinal pigment epithelium; ONL, outer nuclear layer; OPL, outer plexiform layer; INL; inner nuclear layer; IPL, inner plexiform layer; RGC, retinal ganglion cells. B. Quantification of signal area in the ONL and INL from images as A. Each data point represents a single mouse (n=3-4 per group).


Dox - induced prolongation of Nanog expression in Nanogtg embryos up to E9 . 5 results in lack of blood ( left ) and downregulation of erythropoietic gene expression . The center and right panels show whole - mount in situ hybridization for Hbb - bh1 ( in embryos with intact yolk sacs ) and for the long non - coding RNA Redrum . Asterisks mark the aorta - gonad - mesonephros region ( AGM ) and arrows the tailbud . Embryos of the same genotype but not treated with dox were used as controls ( - dox ) . Scale bars , 500 µm .

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

Dox-induced prolongation of Nanog expression in Nanogtg embryos up to E9.5 results in lack of blood (left) and downregulation of erythropoietic gene expression. The center and right panels show whole-mount in situ hybridization for Hbb-bh1 (in embryos with intact yolk sacs) and for the long non-coding RNA Redrum. Asterisks mark the aorta-gonad-mesonephros region (AGM) and arrows the tailbud. Embryos of the same genotype but not treated with dox were used as controls (-dox). Scale bars, 500 µm.


Endomucin staining of vessels in control ( - dox ) or treated ( + dox ) E9 . 5 Nanogtg embryos . On the right , higher magnifications of the boxed areas . Scale bar , 500 µm .

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

Endomucin staining of vessels in control (-dox) or treated (+dox) E9.5 Nanogtg embryos. On the right, higher magnifications of the boxed areas. Scale bar, 500 µm.


Representative FACS plot of the distribution of the CD71 and Ter119 populations in dissected yolk sacs from untreated and dox - treated E9 . 5 Nanogtg embryos . Quantification of the CD71 + Ter119 + population in controls ( - dox , black dots ; n = 8 ) and Nanog expressing ( + dox , red dots ; n = 7 ) E9 . 5 yolk sacs . Each replicate contained a pool of 5 ( - dox ) or 8 ( + dox ) E9 . 5 Nanogtg embryos . * * * P . < 0 . 0005 ; Student ' s t - test . Horizontal line represents mean values and error bars standard deviation ( SD ) .

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

Representative FACS plot of the distribution of the CD71 and Ter119 populations in dissected yolk sacs from untreated and dox-treated E9.5 Nanogtg embryos. Quantification of the CD71+ Ter119+ population in controls (-dox, black dots; n=8) and Nanog expressing (+dox, red dots; n=7) E9.5 yolk sacs. Each replicate contained a pool of 5 (-dox) or 8 (+dox) E9.5 Nanogtg embryos. ***P. < 0.0005; Student's t-test. Horizontal line represents mean values and error bars standard deviation (SD).


Representative FACS plots showing the distribution of cKit and CD41 populations in yolk sacs from untreated controls ( - dox ) and Nanog expressing ( + dox ) E9 . 5 Nanogtg embryos . Quantification of different progenitor populations in yolk sacs from control ( - dox , black dots ; n = 8 ) and Nanog expressing ( + dox , red dots ; n = 7 ) E9 . 5 embryos . Each replicate contained a pool of 5 ( - dox ) or 8 ( + dox ) E9 . 5 Nanogtg embryos . Horizontal line represents mean values and error bars SD .

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

Representative FACS plots showing the distribution of cKit and CD41 populations in yolk sacs from untreated controls (-dox) and Nanog expressing (+dox) E9.5 Nanogtg embryos. Quantification of different progenitor populations in yolk sacs from control (-dox, black dots; n=8) and Nanog expressing (+dox, red dots; n=7) E9.5 embryos. Each replicate contained a pool of 5 (-dox) or 8 (+dox) E9.5 Nanogtg embryos. Horizontal line represents mean values and error bars SD.


Differences in the expression levels of Nanog and selected hematopoietic genes in the CD71 + Ter119 + population of control ( - dox ; n = 7 ) and Nanog - expressing ( + dox ; n = 4 ) E9 . 5 embryos . * * P < 0 . 005 , * * * P < 0 . 0005 ; Student ' s t - test . Horizontal line represents mean values and error bars SD .

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

Differences in the expression levels of Nanog and selected hematopoietic genes in the CD71+ Ter119+ population of control (-dox; n=7) and Nanog-expressing (+dox; n=4) E9.5 embryos. **P < 0.005, ***P < 0.0005; Student's t-test. Horizontal line represents mean values and error bars SD.


Freshly dissected dox - treated Nanogtg E10 . 5 embryos without ( control ) and with ( chimera ) contribution of wt - ESGFP cells ( left , brightfield ; right , GFP ) . Arrows mark the presence of blood in chimeric embryos that is absent from controls . Scale bar , 500 µm .

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

Freshly dissected dox-treated Nanogtg E10.5 embryos without (control) and with (chimera) contribution of wt-ESGFP cells (left, brightfield; right, GFP). Arrows mark the presence of blood in chimeric embryos that is absent from controls. Scale bar, 500 µm.


Representative FACS plots showing of red blood cell maturation as determined by CD71 / Ter119 staining in single dox - treated E10 . 5 control ( left ) and chimeric ( right ) embryos .

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

Representative FACS plots showing of red blood cell maturation as determined by CD71/Ter119 staining in single dox-treated E10.5 control (left) and chimeric (right) embryos.


Quantification of the CD71 + Ter119 + population in single dox - treated E10 . 5 embryos ( D ; control , n = 18 ; chimera , n = 10 ) , untreated embryos ( E ; control , n = 7 ; chimera , n = 12 ) , and in GFP - cells ( not derived from wild type ES cells ) from dox - treated embryos ( F ; control , n = 18 ; chimera , n = 10 ) . * * * P < 0 . 0005 ; Student ' s t - test . Horizontal line represents mean values and error bars SD .

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

Quantification of the CD71+ Ter119+ population in single dox-treated E10.5 embryos (D; control, n=18; chimera, n=10), untreated embryos (E; control, n=7; chimera, n=12), and in GFP- cells (not derived from wild type ES cells) from dox-treated embryos (F; control, n=18; chimera, n=10). ***P < 0.0005; Student's t-test. Horizontal line represents mean values and error bars SD.


Quantification of colony - forming units generated by wild type ( wt ) and knockout ( Nanog - / - ) ES cells after culture of EBs for 5 ( D5 ) , 6 ( D6 ) , or 7 ( D7 ) days and plating disaggregated cells in different hemogenic - promoting conditions ) . Panels on the left show representative images of mouse hematopoietic colonies obtained after 12 days of culture in specific media . CFU - GEMM , progenitors giving rise to granulocytes , erythrocytes , monocytes , and megakaryocytes ; BFU - E , burst forming units - erythroid ; Ery - P , colony forming primitive erythroid ; CFU - GM , granulocyte - monocyte precursors ; CFU - M , monocyte precursors ; CFU - G , granulocyte precursors . No CFU - GEMM are detected at D5 and no BFU - E at D7 . For both wt and knockout cells , n = 3 each with 3 technical replicates . * P < 0 . 05 , * * P < 0 . 005 , * * * P < 0 . 00005 ; Student ' s t - test . Horizontal line represents mean values and error bars SD .

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

Quantification of colony-forming units generated by wild type (wt) and knockout (Nanog-/-) ES cells after culture of EBs for 5 (D5), 6 (D6), or 7 (D7) days and plating disaggregated cells in different hemogenic-promoting conditions). Panels on the left show representative images of mouse hematopoietic colonies obtained after 12 days of culture in specific media. CFU-GEMM, progenitors giving rise to granulocytes, erythrocytes, monocytes, and megakaryocytes; BFU-E, burst forming units-erythroid; Ery-P, colony forming primitive erythroid; CFU-GM, granulocyte-monocyte precursors; CFU-M, monocyte precursors; CFU-G, granulocyte precursors. No CFU-GEMM are detected at D5 and no BFU-E at D7. For both wt and knockout cells, n=3 each with 3 technical replicates. *P < 0.05, **P < 0.005, ***P < 0.00005; Student's t-test. Horizontal line represents mean values and error bars SD.


RT - qPCR determination of the relative expression of Brachyury and selected hematopoietic genes in control ( wt , right ) and knockout ( Nanog - / - , left ) ES cells ( n = 3 ) during 10 days of EB differentiation in hematopoietic - cytokine - enriched medium . Black arrowheads indicate the peak of Brachyury expression and white arrowheads the time of maximum hematopoietic - gene expression .

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

RT-qPCR determination of the relative expression of Brachyury and selected hematopoietic genes in control (wt, right) and knockout (Nanog-/-, left) ES cells (n=3) during 10 days of EB differentiation in hematopoietic-cytokine-enriched medium. Black arrowheads indicate the peak of Brachyury expression and white arrowheads the time of maximum hematopoietic-gene expression.


Representative FACS plots showing the distribution of different populations distinguished by CD71 / Ter119 staining in whole bone marrow from untreated ( - dox ) or treated ( + dox ) adult mice . S0 ( double negative cell ) , S1 ( proerythroblast ) , S2 ( basophylic erythroblast ) , S3 ( polychromatic erythroblast ) , and S4 ( orthochromatic erythroblast ) are different stages of blood maturation . Quantification of the S1 - S4 erythroid populations ( - dox , n = 4 ; + dox , n = 5 ) . * P < 0 . 05 , * * P < 0 . 005 , * * * P < 0 . 0005 ; Student ' s t - test . Horizontal line represents mean values and error bars SD .

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

Representative FACS plots showing the distribution of different populations distinguished by CD71/Ter119 staining in whole bone marrow from untreated (-dox) or treated (+dox) adult mice. S0 (double negative cell), S1 (proerythroblast), S2 (basophylic erythroblast), S3 (polychromatic erythroblast), and S4 (orthochromatic erythroblast) are different stages of blood maturation. Quantification of the S1-S4 erythroid populations (-dox, n=4; +dox, n=5). *P < 0.05, **P < 0.005, ***P < 0.0005; Student's t-test. Horizontal line represents mean values and error bars SD.


Representative FACS plots showing the distribution of CD16 / 32 and CD34 hematopoietic precursors sorted from the cKit + Sca1 - LIN - bone marrow of untreated ( - dox ) or treated ( + dox ) adult Nanogtg mice .

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

Representative FACS plots showing the distribution of CD16/32 and CD34 hematopoietic precursors sorted from the cKit+Sca1-LIN- bone marrow of untreated (-dox) or treated (+dox) adult Nanogtg mice.


Quantification of precursor populations based on CD16 / 32 and CD34 sorting , as total number of cells per individual femur ( - dox , n = 5 ; + dox , n = 6 ) . * P < 0 . 05 , * * P < 0 . 005 ; Student ' s t - test . Horizontal line represents mean values and error bars SD .

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

Quantification of precursor populations based on CD16/32 and CD34 sorting, as total number of cells per individual femur (-dox, n=5; +dox, n=6). *P < 0.05, **P < 0.005; Student's t-test. Horizontal line represents mean values and error bars SD.


RT - qPCR quantification of the relative expression of hematopoietic genes in megakaryocyte - erythroid progenitors ( MEP ) ( - dox , n = 8 ; + dox , n = 5 ) . * P < 0 . 05 , * * * * P < 0 . 00005 ; Student ' s t - test . Horizontal line represents mean values and error bars SD .

RT-qPCR quantification of the relative expression of hematopoietic genes in megakaryocyte-erythroid progenitors (MEP) (-dox, n=8; +dox, n=5). *P < 0.05, ****P < 0.00005; Student's t-test. Horizontal line represents mean values and error bars SD.


Contribution of Nanogtg transplanted bone marrow cells to peripheral blood before ( left ) and after ( right ) dox treatment . Percentage of host derived cells ( CD45 . 1 + ) are shown in black , and of donor derived cells ( CD45 . 1 / CD45 . 2 double + ) in red . Individual mice are indicated on the x - axis ( n = 7 ) .

Contribution of Nanogtg transplanted bone marrow cells to peripheral blood before (left) and after (right) dox treatment. Percentage of host derived cells (CD45.1+) are shown in black, and of donor derived cells (CD45.1/CD45.2 double +) in red. Individual mice are indicated on the x-axis (n=7).


Contribution of Nanogtg transplanted cells to LSK , CMP , GMP and MEP populations purified from bone marrow . Percentage of host derived cells ( CD45 . 1 + ) are show in black , and of donor derived cells ( CD45 . 1 / CD45 . 2 double + ) in blue . Individual mice are indicated on the x - axis ( n = 7 ) .

Contribution of Nanogtg transplanted cells to LSK, CMP, GMP and MEP populations purified from bone marrow. Percentage of host derived cells (CD45.1+) are show in black, and of donor derived cells (CD45.1/CD45.2 double +) in blue. Individual mice are indicated on the x-axis (n=7).


Expected and observed number of mesodermal ( Flk1 + ) cells of the E7 . 0 mouse embryo expressing Nanog and selected mesodermal or hematopoietic gene expression , based on single cell RNA - seq data ( Scialdone et al . , 2016 ) . Statistical significance was calculated with a hypergeometric test .

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

Expected and observed number of mesodermal (Flk1+) cells of the E7.0 mouse embryo expressing Nanog and selected mesodermal or hematopoietic gene expression, based on single cell RNA-seq data (Scialdone et al., 2016). Statistical significance was calculated with a hypergeometric test.


PCA showing the distribution of Flk1 + E7 . 0 mesoderm cells expressing Nanog ( green ) or Tal1 ( red ) . The single cell expressing both genes is shown in yellow and indicated by an arrow .

PCA showing the distribution of Flk1+ E7.0 mesoderm cells expressing Nanog (green) or Tal1 (red). The single cell expressing both genes is shown in yellow and indicated by an arrow.


E6 . 5 Nanogtg embryos after 8 hours ex - utero culture in the presence ( + dox ) or absence ( - dox ) of doxycycline . Scale bar , 100 µm .

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

E6.5 Nanogtg embryos after 8 hours ex-utero culture in the presence (+dox) or absence (-dox) of doxycycline. Scale bar, 100 µm.


RT - qPCR quantification of the relative expression of Nanog , Tal1 , and Klf1 in individual untreated embryos ( - dox ) or treated embryos ( + dox ) ( n = 5 ) . * * P < 0 . 005 , * * * P < 0 . 0005 ; Student ' s t - test . Horizontal line represents mean values and error bars SD .

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

RT-qPCR quantification of the relative expression of Nanog, Tal1, and Klf1 in individual untreated embryos (-dox) or treated embryos (+dox) (n=5). **P < 0.005, ***P < 0.0005; Student's t-test. Horizontal line represents mean values and error bars SD.


Whole mount in situ hybridization of Tal1 in E7 . 5 untreated ( - dox ) or in utero treated ( + dox ) Nanogtg embryos . Scale bar , 100 µm .

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

Whole mount in situ hybridization of Tal1 in E7.5 untreated (-dox) or in utero treated (+dox) Nanogtg embryos. Scale bar, 100 µm.


RT - qPCR determination of relative expression in wild type and CRISPR - deleted embryos ( n = 5 ) of Tal1 ( wt , n = 19 ; deleted , n = 13 ) , Klf1 ( wt , n = 3 ; deleted , n = 6 ) , Gfi1b ( wt , n = 10 ; deleted , n = 8 ) , Runx1 ( wt , n = 13 ; deleted , n = 5 ) and Stil ( wt , n = 19 ; deleted , n = 13 ) . * * P < 0 . 005 , Student ' s t - test . Horizontal line represents mean values and error bars SD .

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

RT-qPCR determination of relative expression in wild type and CRISPR-deleted embryos (n=5) of Tal1 (wt, n=19; deleted, n=13), Klf1 (wt, n=3; deleted, n=6), Gfi1b (wt, n=10; deleted, n=8), Runx1 (wt, n=13; deleted, n=5) and Stil (wt, n=19; deleted, n=13). **P < 0.005, Student's t-test. Horizontal line represents mean values and error bars SD.


Experimental design for ES to EpiL cell differentiation of Nanogtg cells and two independent clones ( Nanogtg ; dTal1del # 1 and Nanogtg ; dTal1del # 2 ) where the binding site for NANOG distal to Tal1 has been deleted ( left ) . On the right , relative expression of Tal1 determined by RT - qPCR for each ES cell line ( ESC ; n = 9 for all three lines ) and EpiL cells without ( EpiLC ; Nanogtg and Nanogtg ; dTal1del # 1 , n = 8 ; Nanogtg ; dTal1del # 2 , n = 6 ) or with dox treatment ( EpiLC + dox ; n = 9 for all three lines ) . The genotype of the cell lines is indicated below . Values were normalized to Nanogtg ESC . * P < 0 . 05 , * * P < 0 . 01 , ns = not significant ; ANOVA with Fisher post - test . Horizontal line represents mean values and error bars standard error of the mean ( SEM ) .

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

Experimental design for ES to EpiL cell differentiation of Nanogtg cells and two independent clones (Nanogtg;dTal1del#1 and Nanogtg;dTal1del#2) where the binding site for NANOG distal to Tal1 has been deleted (left). On the right, relative expression of Tal1 determined by RT-qPCR for each ES cell line (ESC; n=9 for all three lines) and EpiL cells without (EpiLC; Nanogtg and Nanogtg;dTal1del#1, n=8; Nanogtg;dTal1del#2, n=6) or with dox treatment (EpiLC +dox; n=9 for all three lines). The genotype of the cell lines is indicated below. Values were normalized to Nanogtg ESC. *P < 0.05, **P < 0.01, ns = not significant; ANOVA with Fisher post-test. Horizontal line represents mean values and error bars standard error of the mean (SEM).


A ) Phase separation over time as monitored by turbidity of 50 μM MBP - N after addition of TEV protease in the presence of salt ( 183 mM NaCl ) and 0 . 3 mg / ml RNA at pH 7 . 4 ( 50 mM Tris ) , pH 6 . 1 , pH 5 . 5 , or pH 4 . 9 ( pH 6 . 1 and below in 20 mM MES ) . Error bars represent standard deviation of three replicates . For samples at B ) pH 7 . 4 or C ) pH 5 . 5 , DIC micrographs of 50 μM MBP - N in the presence of salt ( 183 mM NaCl ) without or with TEV protease ( to cleave MBP from N ) and without or with 0 . 3 mg / mL desalted total torula yeast RNA . Scale bars represent 50 μm .

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

A) Phase separation over time as monitored by turbidity of 50 μM MBP-N after addition of TEV protease in the presence of salt (183 mM NaCl) and 0.3 mg/ml RNA at pH 7.4 (50 mM Tris), pH 6.1, pH 5.5, or pH 4.9 (pH 6.1 and below in 20 mM MES). Error bars represent standard deviation of three replicates. For samples at B) pH 7.4 or C) pH 5.5, DIC micrographs of 50 μM MBP-N in the presence of salt (183 mM NaCl) without or with TEV protease (to cleave MBP from N) and without or with 0.3 mg/mL desalted total torula yeast RNA. Scale bars represent 50 μm.