Representative showing expression location of YAP in control LATS2 knockout hOSE . were collected at 4th passage YAP expression was examined by immunohistochemistry . YAP was visualized using an Alexa - 488 ( Green ) conjugated secondary antibody . Nuclei were stained with DAPI .

Representative images showing the expression and location of YAP in control and LATS2 knockout hOSE cells. Cells were collected at the 4th passage and YAP expression was examined by fluorescent immunohistochemistry. YAP protein was visualized using an Alexa-488 (Green) conjugated secondary antibody. Nuclei were stained with DAPI.


Representative showing YAP expression in 13th passage hOSE with or without LATS2 knockout . YAP was examined by immunohistochemistry visualized using an Alexa - 488 ( Green ) conjugated secondary antibody . Nuclei were stained with DAPI ( blue ) .

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Representative images showing YAP protein expression in the 13th passage hOSE Cells with or without LATS2 knockout. YAP protein was examined by fluorescent immunohistochemistry and visualized using an Alexa-488 (Green) conjugated secondary antibody. Nuclei were stained with DAPI (blue).


Representative showing morphological change ( E ) in 13th passage ( P13 ) hOSE with ( LATS2 KO ) or without ( CTRL ) LATS2 knockout .

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Representative images showing morphological change (E) in the 13th passage (P13) hOSE cells with (LATS2 KO) or without (CTRL) LATS2 knockout.


Representative showing SA - β - gal ( F ) in 13th passage ( P13 ) hOSE with ( LATS2 KO ) or without ( CTRL ) LATS2 knockout . Scale bar : 50 µm .

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Representative images showing SA-β-gal staining (F) in the 13th passage (P13) hOSE cells with (LATS2 KO) or without (CTRL) LATS2 knockout. Scale bar: 50 µm.


Quantitative data showing of SA - β - gal positive in control ( CTRL ) LATS2 - knockout ( LATS2KO ) P13 hOSE .

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 data showing the ratio of SA-β-gal positive cells in the control (CTRL) and LATS2-knockout (LATS2KO) P13 hOSE cells.


Representative blots showing expression of LATS1 / 2 , CCNE1 , CCND1 YAP in hOSE - MXIV , hOSE - YAP hOSE - YAPS127A . were collected at passage four ( P4 ) Passage seven ( P7 ) . Actin was used as internal control .

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Representative blots showing expression levels of LATS1/2, CCNE1, CCND1 and YAP in hOSE-MXIV, hOSE-YAP and hOSE-YAPS127A cells. Cells were collected at passage four (P4) and Passage seven (P7). Actin was used as internal control.


mRNA of LATS1 , LATS2 YAP in hOSE - MXIV , hOSE - YAP hOSE - YAPS127A . were collected at passage seven were analyzed using quantitative real - time PCR .

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mRNA levels of LATS1, LATS2 and YAP in hOSE-MXIV, hOSE-YAP and hOSE-YAPS127A cells. Cells were collected at passage seven and protein levels were analyzed using quantitative real-time PCR.


Representative gel showing mRNA of LATS1 , LATS2 , YAP in LATS2 - knockout hOSE - MXIV , hOSE - YAP hOSE - YAPS127A examined using semi - quantitative RT - PCR at passage seven .

Representative gel photographs showing mRNA levels of LATS1, LATS2, and YAP in the LATS2-knockout hOSE-MXIV, hOSE-YAP and hOSE-YAPS127A cells examined using semi-quantitative RT-PCR at passage seven.


Representative showing morphologic changes SA - β - gal in control LATS2 - knockout hOSE - MXIV , hOSE - YAP hOSE - YAPS127A .

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Representative images showing the morphologic changes and SA-β-gal staining in control and LATS2-knockout hOSE-MXIV, hOSE-YAP and hOSE-YAPS127A cells.


Quantitative data showing of SA - β - gal positive in control LATS2 - knockout hOSE - MXIV , hOSE - YAP hOSE - YAPS127A at seventh passage .

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Quantitative data showing the ratio of SA-β-gal positive cells in the control and LATS2-knockout hOSE-MXIV, hOSE-YAP and hOSE-YAPS127A cells at the seventh passage.


Growth curves of control LATS2 - knockout hOSE - MXIV , hOSE - YAP hOSE - YAPS127A .

Growth curves of the control and LATS2-knockout hOSE-MXIV, hOSE-YAP and hOSE-YAPS127A cells.


Representative showing with ectopic expression of LATS2 , which is labelled with GFP ( control is empty vectors ) .

Representative images showing cells with ectopic expression of LATS2, which is labelled with GFP (the control is empty vectors).


Relative mRNA of LATS1 , LATS2 , YAP in control LATS2 - expressing hOSEs at 4th passage . Relative mRNA were determined with RT - PCR .

Relative mRNA levels of LATS1, LATS2, and YAP in the control and LATS2-expressing hOSEs at the 4th passage. Relative mRNA levels were determined with RT-PCR.


Growth curves of hOSE at 4th passage with or without ectopic expression of LATS2 .

Growth curves of hOSE cells at the 4th passage with or without ectopic expression of LATS2.


Representative showing effects of LATS2 overexpression on growth of hOSE at 9th passage .

Representative images showing the effects of LATS2 overexpression on the morphology and growth of hOSE cells at the 9th passage.


Growth curves of control LATS2 - overexpressing hOSE .

Growth curves of control and LATS2-overexpressing hOSE cells.


Quantitative data showing of SA - β - gal positive hOSEs at their 4th passage with or without ectopic expression of LATS2 .

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Quantitative data showing the ratio of SA-β-gal positive hOSEs at their 4th passage with or without ectopic expression of LATS2.


Representative showing effect of LATS2 on β - galactosidase activities in cultured hOSE at 9th passage .

Representative images showing the effect of LATS2 on the morphology and β-galactosidase activities in cultured primary hOSE cells at the 9th passage.


Quantitative data showing of SA - β - gal positive hOSEs at their 9th passage with or without ectopic expression of LATS2 .

Quantitative data showing the ratio of SA-β-gal positive hOSEs at their 9th passage with or without ectopic expression of LATS2.


mRNA of major components of DREAM ( E2F4 , E2F5 , RBBP4 , TFDP2 ) in control LATS2 - overexpressing hOSEs at 9th passage . Relative mRNA were examined by real - time PCR .

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mRNA levels of major components of the DREAM complex (E2F4, E2F5, RBBP4, TFDP2) in the control and LATS2-overexpressing hOSEs at the 9th passage. Relative mRNA levels were examined by real-time PCR.


Growth curves of hOSE - shRB1 hOSE - shRB1 - LATS2 at their 16th passages .

Growth curves of the hOSE-shRB1 cells and hOSE-shRB1-LATS2 cells at their 16th passages.


Representative showing β - galactosidase activities in control LATS2 - overexpressing hOSEs ( K ) . Scale Bar : 50 µm . Right bar graph ( L ) showing quantitative results of SA - β - gal positive in hOSE - shRB1 hOSE - shRB1 - LATS2 .

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Representative images showing cellular morphology and β-galactosidase activities in control and LATS2-overexpressing hOSEs (K). Scale Bar: 50 µm. Right bar graph (L) showing quantitative results of SA-β-gal positive cells in the hOSE-shRB1 cells and hOSE-shRB1-LATS2 cells.


Representative showing colony formation in hOSE - MXIV , hOSE - YAP hOSE - YAPS127A with or without pRB knockdown or LATS2 knockout . hOSEs at 7th passage were incubated in growth medium in a soft agar culture system for 9 days before imaging .

O O O O O O O O O O O O O O O O O O O O O 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 images showing colony formation in hOSE-MXIV, hOSE-YAP and hOSE-YAPS127A cells with or without pRB knockdown or LATS2 knockout. hOSEs at the 7th passage were incubated in the growth medium in a soft agar culture system for 9 days before imaging.


Quantitative data showing colony numbers in hOSE - MXIV , hOSE - YAP hOSE - YAPS127A with or without pRB knockdown or LATS2 deletion . hOSE at 7th passage were incubated in growth medium in a soft agar culture system for 9 days before counting colonies under a microscope .

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Quantitative data showing colony numbers in hOSE-MXIV, hOSE-YAP and hOSE-YAPS127A cells with or without pRB knockdown or LATS2 deletion. hOSE cells at the 7th passage were incubated in the growth medium in a soft agar culture system for 9 days before counting colonies under a microscope.


Representative showing tumorigenesis ( red circle ) of hOSE - YAPS127A with or without pRB knockdown or LATS2 deletion . Tumors formed by hOSE - YAPS127A with RB1 knockdown or LATS2 deletion are shown in red circle . Right panel showing representative tumors formed by hOSE - YAPS127A - LATS2KO hOSE - YAPS127A - shRB1 . Please note that no xenografts formed in hOSE - YAPS127A alone 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

Representative images showing tumorigenesis (red circle area) of hOSE-YAPS127A cells with or without pRB knockdown or LATS2 deletion. Tumors formed by hOSE-YAPS127A cells with RB1 knockdown or LATS2 deletion are shown in red circle area. Right panel showing representative tumors formed by hOSE-YAPS127A-LATS2KO and hOSE-YAPS127A-shRB1 cells. Please note that no tumor xenografts formed in hOSE-YAPS127A alone group.


Representative showing expression of YAP , Ki67 LATS2 in hOSE - LATS2KO - YAPS127A - derived tumors examined by immunohistochemistry .

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Representative images showing expression of YAP, Ki67 and LATS2 in hOSE-LATS2KO-YAPS127A cell-derived tumors examined by immunohistochemistry.


( A ) Alcian blue ‐ stained sections of descending colons from control Atg5VC mice . between two black dashed lines indicates crypt base where epithelial progenitors nascent goblet reside . Bars = 200 μm .

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(A) Alcian blue‐stained sections of descending colons from control and Atg5VC mice. The area between the two black dashed lines indicates the crypt base where epithelial progenitors and nascent goblet cells reside. Bars=200 μm.


( B ) of average mucin / goblet in control Atg5VC mice ( n = 5 - 7 mice / group from 3 independent experiments ; 100 goblet measured / mouse ) . * * P0 . 01 as determined by Student ' s t ‐ . This was performed on upper crypt goblet ( above upper dashed 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 O O O O O O O O O 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 average mucin area/goblet cell in control and Atg5VC mice (n=5-7 mice/group from 3 independent experiments; 100 goblet cells measured/mouse). **P0.01 as determined by the Student's t‐test. This analysis was performed on upper crypt goblet cells (above the upper dashed line in A).


( C ) of average number of goblet per crypt ( n = 3 mice / group ; over 100 crypts measured / mouse ) .

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(C) Quantification of the average number of goblet cells per crypt (n=3 mice/group; over 100 crypts measured/mouse).


( D ) of upper crypt goblet from control Atg5VC transverse colons . yellow dashed lines outline mucin granules in apex of . Bars = 2 μm .

(D) TEM images of upper crypt goblet cells from control and Atg5VC transverse colons. The yellow dashed lines outline the mucin granules in the apex of the cells. Bars=2 μm.


( E , F ) of ( E ) average granule density ( F ) average number of mucin granules / goblet ( n = 3 mice / group 20 goblet evaluated / mouse ) . * * * P0 . 001 as determined by Student ' s t ‐ .

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Quantification of (E) the average granule density and (F) the average number of mucin granules/goblet cell (n=3 mice/group and 20 goblet cells evaluated/mouse). ***P0.001 as determined by the Student's t‐test.


( G , H ) Whole ‐ mount of mucosal surface from control Atg5VC transverse colons . Whole ‐ mount colonic samples were either stained with Ulex europaeus agglutinin ‐ 1 ( UEA ) that labelled mucus ( G ) or analysed by scanning electron microscopy ( H ) . Yellow arrows indicate crypt opening orifices . Green arrowheads indicate goblet protruding from surface epithelium of Atg5VC mice ( insets in H show higher power image of latter ) . 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Whole‐mount images of the mucosal surface from control and Atg5VC transverse colons. Whole‐mount colonic samples were either stained with Ulex europaeus agglutinin‐1 (UEA) that labelled mucus (G) or analysed by the scanning electron microscopy (H). Yellow arrows indicate the crypt opening orifices. Green arrowheads indicate goblet cells protruding from the surface epithelium of Atg5VC mice (insets in H show higher power image of the latter). Bars=100 μm.


( B ) Representative whole ‐ mount of single spheroids cultured in either 50 % conditioned media ( CM ) or 5 % CM + 5 μM DAPT stained for Muc2 ( green ) to visualize goblet . Bars = 20 μm .

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(B) Representative whole‐mount images of single spheroids cultured in either 50% conditioned media (CM) or 5% CM+5 μM DAPT and stained for Muc2 (green) to visualize goblet cells. Bars=20 μm.


( C , D ) Graph of relative expression ( as measured by qRT ‐ PCR ) of Atoh1 ( C ) Muc2 ( D ) mRNAs comparing spheroids grown in either 5 % CM or 5 % CM + DAPT to spheroids grown in 50 % CM ( n = 4 - 5 samples / group ) . Error bars indicate s . e . m . * P0 . 05 ; * * P0 . 01 as determined by a two ‐ Student ' s t ‐ .

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

(C, D) Graph of relative expression (as measured by qRT‐PCR) of Atoh1 (C) and Muc2 (D) mRNAs comparing spheroids grown in either 5% CM or 5% CM+ DAPT to spheroids grown in 50% CM (n=4-5 samples/group). Error bars indicate s.e.m. *P0.05; **P0.01 as determined by a two‐tailed Student's t‐test.


( E , F ) of colonic epithelial spheroids isolated from control Atg5VC mice grown in 5 % CM + DAPT stained for Muc2 ( green : E ) or with lectin , UEA ( red : F ) . Bars = 20 μm .

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(E, F) Images of colonic epithelial spheroids isolated from control and Atg5VC mice grown in 5% CM+DAPT and stained for Muc2 (green: E) or with the lectin, UEA (red: F). Bars=20 μm.


( G ) of average mucin / goblet . Error bars indicate s . e . m . ( n = 6 samples / group ; 100 were quantified / sample ; * * P0 . 01 as determined by a two ‐ Student ' s t ‐ ) .

(G) Quantification of average mucin area/goblet cell. Error bars indicate s.e.m. (n=6 samples/group; 100 cells were quantified/sample; **P0.01 as determined by a two‐tailed Student's t‐test).


( H ) Relative expression of Muc2 mRNA in differentiated spheroids from control Atg5VC mice as measured by qRT - PCR ( n = 4 - 5 samples / group ) . No statistically significant differences were found in Muc2 expression as determined by Student ' s t ‐ .

(H) Relative expression of Muc2 mRNA in differentiated spheroids from control and Atg5VC mice as measured by qRT-PCR (n=4-5 samples/group). No statistically significant differences were found in Muc2 expression as determined by the Student's t‐test.


( I , K ) of FIP200f / f ( I ) Atg14f / f ( K ) colonic epithelial spheroids treated with recombinant Tat ‐ Cre stained with UEA ( red ) .

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(I, K) Images of FIP200f/f (I) and Atg14f/f (K) colonic epithelial spheroids treated with recombinant Tat‐Cre and stained with UEA (red).


( J , L ) of average mucin / goblet . Error bars indicate s . e . m . ( n = 6 samples / group ; 100 were quantified / sample ; * * P0 . 01 * * * P0 . 001 as determined by a two ‐ Student ' s t ‐ ) .

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

(J, L) Quantification of average mucin area/goblet cell. Error bars indicate s.e.m. (n=6 samples/group; 100 cells were quantified/sample; **P0.01 and ***P0.001 as determined by a two‐tailed Student's t‐test).


( A ) of colonic sections stained for LC3β ( green ) from control Atg5VC mice . ‐ dashed lines indicate border between basal surface of crypt epitheliumand mesenchyme . asterisks denote non ‐ specific plasma from mouse monoclonal anti ‐ LC3β antibody . Bars = 50 μm .

(A) Images of colonic sections stained for LC3β (green) from control and Atg5VC mice. The white‐dashed lines indicate the border between the basal surface of the crypt epitheliumand the mesenchyme. White asterisks denote non‐specific plasma cell staining from the mouse monoclonal anti‐LC3β antibody. Bars=50 μm.


( B ) Immunogold transmission electron microscopic image for LC3β in a colonic goblet from a wild ‐ type mouse . inset is a low power image of entire goblet . black box within inset indicates shown in this panel . Note this is not associated with mucin granules . Bar = 500 nm .

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(B) Immunogold transmission electron microscopic image for LC3β in a colonic goblet cell from a wild‐type mouse. The inset is a low power image of the entire goblet cell. The black box within the inset indicates the area shown in this panel. Note this area is not associated with mucin granules. Bar=500 nm.


( C ) Enriched KEGG pathways for Atg5 ‐ deficient epithelial obtained from a microarray comparing control Atg5VC colonic crypt base epithelium . P ‐ values were determined by modified Fisher ' s exact .

O O O O O O O O O O O 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) Enriched KEGG pathways for Atg5‐deficient epithelial cells obtained from a microarray comparing control and Atg5VC colonic crypt base epithelium. P‐values were determined by modified Fisher's exact test.


( D - G ) Double label immunogold transmission electron microscopic from wild ‐ type mice for LC3β ( 12 nm particles ) EEA1 ( D ) , Rab7 ( E ) , Rab11 ( F ) , or Lamp1 ( G ) ( 18 nm particles ) . Bars = 200 nm .

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(D-G) Double label immunogold transmission electron microscopic images from wild‐type mice for LC3β (12 nm particles) and EEA1 (D), Rab7 (E), Rab11 (F), or Lamp1 (G) (18 nm particles). Bars=200 nm.


( B ) Colonic epithelial monolayers from wild ‐ type mice treated with either vehicle or 100 μM Dynasore stained for LC3β ( green ) EEA1 ( red ) . Bars = 5 μm .

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(B) Colonic epithelial monolayers from wild‐type mice treated with either vehicle or 100 μM Dynasore and stained for LC3β (green) and EEA1 (red). Bars=5 μm.


( C , D ) of average number of LC3β dots per ( C ) of LC3β dots that co ‐ localized with EEA1 per ( D ) ( n = 3 experiments / group ; 25 - 30 / 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

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


( E ) LC3 immunoblots of epithelial spheroids treated with 100 μM Dynasore 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 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 ) 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 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 ) of average mucin / goblet ( n = 6 samples / group ; 150 were quantified / sample ) . Error bars indicate s . e . m . * * * P0 . 001 as determined by Student ' s t ‐ .

O O O O O O O O O O O O O O O O O O O O O O O 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 p22 mut mouse stained with LC3β ( green ) EEA1 ( red ) . Bar = 5 μm .

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


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

(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 .

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(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 for LC3β ( 12μnm particles ) p22phox ( 18 nm particles ) showing a p22phox ‐ positive single vesicle ( D ) a p22phox ‐ , LC3β ‐ positive vacuole ( E ) . Bars = 200μnm .

(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 ) p22phox ( red ) of LC3β dots co ‐ localized with p22phox per , Bar = 5 μm .

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(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 p22 mut mice stained with TRITC ‐ UEA to label goblet cellmucin . Bars = 50 μm .

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


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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 spheroids from p22phox − / − , Atg5VC 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 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 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

(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 ) of 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

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


( C ) of average of 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 Student ' s t ‐ .

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

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

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(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 ) of 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 ‐

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

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(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 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 .

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(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 ) 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 ) of average mucin / goblet ( n = 6 sample / group ; 30 - 60 were quantified / sample ) corresponding to experiments performed using p22 mut ( E ) Dynasore ‐ treated wild ‐ type spheroids ( G )

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

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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 six fragments HeLa NE were performed . After nuclease P1 digestion , products were analyzed by 1D - TLC with pU pψ as controls .

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

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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 of 7SK obtained by either IVT or immunoprecipitation ( IP ) with indicated antibodies .

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


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

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

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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 ; N - Q . tagged WT or mutant 7SK was co - transfected with indicated Flag - tagged into HeLa . Anti - Flag or immunoprecipitates ( IP ) were analyzed by immunoblotting for indicated primer extension for bound 7SK .

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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 ; N - Q . tagged WT or mutant 7SK was co - transfected with indicated Flag - - tagged into HeLa . Anti - Flag immunoprecipitates ( IP ) were analyzed by immunoblotting for indicated primer extension for bound 7SK .

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 ; N - Q . tagged WT or mutant 7SK was co - transfected with indicated Flag - tagged into HeLa . Anti - Flag immunoprecipitates ( IP ) were analyzed by immunoblotting for indicated primer extension for bound 7SK .

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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 ; N - Q . tagged WT or mutant 7SK was co - transfected with indicated HA - tagged into HeLa . Anti - HA immunoprecipitates ( IP ) were analyzed by immunoblotting for indicated primer extension for bound 7SK .

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 . indicated 7SK were in vitro transcribed in presence of 32p - UTP subjected to in vitro modification reactions . nuclease P1 - digested products were analyzed by 1D - TLC with pU 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 expressing nothing ( - ) or V5 - DKC1 were analyzed by immunoblotting for V5 - DKC1 qRT - PCR for bound 7SK . error bars represent mean ± SD from three independent experiments .

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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 HeLa - derived F1C2 expressing CDK9 - F were analyzed by Western blotting ( WB ) for indicated primer extension ( PEx ) for 7SK .

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

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


D . 7SK in these two clones were detected by qRT - PCR before after DOX treatment normalized to GAPDH mRNA . 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 of Ψ250 in 7SK from inducible DKC1 KD clone 1 before after DOX treatment .

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


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

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 then transfected with Tat - Flag cDNA ( 20 ug / 150 - mm dish ) . Two days later , NE anti - FlagIP from NE were analyzed as in F .

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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 transfected with indicated reporter constructs , an empty vector ( - ) or Tat cDNA , either siDKC1 or a non - target siRNA ( siNT ) . For each reporter , activity in expressing siNT was set to 1 .

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

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

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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 , E . mRNA of Tat ( C E ) in expressing indicated siRNAs were determined by qRT - PCR normalized to GAPDH mRNA plotted .

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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 of DKC1 CDK9 in a Jurkat 2D10 - derived clone inducibly expressing shDKC1 upon exposure to DOX .

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


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

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

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

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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 . distributions for vesicles as analyzed in D .

C. Size distributions for vesicles as analyzed in D.


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

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


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

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 - deletion mutants of IQGAP1 tested in HEK293 .

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 .

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


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

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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 .

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


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

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


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

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

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 for IQGAP1 GSDMD in IQGAP1 FL restored ΔIQ restored .

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


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

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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 for exosomes concentrated from supernatants collected from LPS or LPS ATP treated as indicated .

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


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

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


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

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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 IQGAP1 in YAMC GSDMD KO YAMC . Yellow dots indicated co - localization signal .

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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 GSDMD in YAMC IQGAP1 KO YAMC .

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


D . Co - localization of CD63 with IQGAP1 - GSDMD , as measured by PLA , shown by yellow dots after LPS 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 pro - IL - 1β in YAMC IQGAP1 KO YAMC .

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


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

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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 distribution for exosomes collected from WT TSG101 knockdown supernatants with indicated treatment .

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


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

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 IQGAP1 KO by pulling down TSG101 under indicated treatments .

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


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

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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 for IQGAP1 CDC42 interaction in IQGAP1 FL restored ΔGRD restored . green dots showed interaction of IQGAP1 CDC42 .

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 ( shown by PLA signal ) in WT GSDMD KO under LPS treatment . was performed by counting PLA green dots .

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 of endogenous IQGAP1 with CDC42 in wild - type GSDMD KO upon LPS or LPS plus ATP treatment .

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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 of GTP - bound CDC42 in indicated following LPS treatment .

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


F . Co - immunoprecipitation of endogenous GSDMD with CDC42 in wild - type IQGAP1 KO 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 . were pretreated with without 10µM ML141 for 24 hours to fully block its GTPase activity , followed by LPS ATP treatment as indicated . H . Particle distribution for exosomes collected from with same treatment as panel G .

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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 of endogenous GSDMD with CDC42 in vehicle or ML141 ( 10µM for 24 hours ) treated following LPS or LPS plus ATP treatment .

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

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


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

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

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

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

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


( C ) of AQR position in adult WT , cst - 1 / or cst - 2 mutant . full between URXs end of nematode tail is divided into 10 blocks , of AQR that stopped within each block is indicated . * * * , P < 0 . 001 by Fisher ' s exact . 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

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

(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 ) time - lapse of F - actin , plasma histone during QR . ap migration in mig - 2 ( rh17 ) gain - of - function mutant . Merged , left ; inverted of F - actin , right ; arrows , migration direction ; asterisks , nucleus ; double headed arrow , migration . dotted lines indicate periphery . Time is presented in minutes . Scale bar , 5 μm . ( B - C ) of QR . ap migration angle ( B ) ( 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

(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 . peptide was identified from 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

(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 of CST - 1 / 2 to MIG - 2 . Bacterially expressed His - tagged MIG - 2 or His - tagged MIG - 2S139A were incubated with GST - tagged kinase domain of GST - CST - 1 / 2 ( 1 - 318 aa ) . kinase may use ATP - γ - S as a phosphodonor to thiophosphorylate its substrate ; thus , phosphorylation reaction was performed with ATP - γ - S , anti - thiophosphate ester antibody was used to detect substrate phosphorylation ( Allen et al , 2005 ; Allen et al , 2007 ) . 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

(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 human Rac1 polypeptide which indicate position of T135 ( S139 in MIG - 2 ) located between 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

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

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

O O O O O O O O O O O O O O O O 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 ) of QR . ap migration angle ( C D ) , ( E ) speed ( F ) in mig - 2S139A / E mutant . Each in ( C E ) represents 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ( up ) schematics ( down ) of QR . ap in WT , cst - 1 ; cst - 2 double or mig - 2 mutant . plasma histones were labeled with mCherry . Scale bar , 5 μm . ( H ) between nucleus in WT mutant . Error bars indicate SD . N = 17 - 22 . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 by Student ' s t .

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

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) ( left ) quantifications ( right ) of GFP - tagged CST - 1 mCherry tagged plasma histone in WT mig - 2 null mutant . Blue lines , sites of measurements ; dotted lines , periphery ; Green lines ; CST - 1 ; red lines , mCherry . Scale bars , 5 μm . ( G ) of of GFP : : CST - 1 mCherry - between leading edge lagging edge of AQR in WT mig - 2 null mutant . Error bars indicate SD . N = 20 . * * * , P < 0 . 001 by Student ' s t .

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

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

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 of Pc stx double mutant stxd77 ; ; pcXT109 sleep profile compared with controls . Quantitative RT - PCR of Octβ2R in adult head of stxd77 w1118 control . ( Data represent mean ± : 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

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

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

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

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

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 knock down of Octβ2R results in sleep rebound increase . In response to OA , ellipsoid knock down of Octβ2R rescues control phenotype . Data represent mean ± : 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 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

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 PcXT109 mutant flies , treatment of OA results in sleep decrease , n = 16 ( G ) . Quantifications of decreasing amount showed that PcXT109 mutant fly loss significantly more sleep that control flies ( From left to right mean ± : 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 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

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 control flies in video - method ( From left to right mean ± : 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

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 with representative blot including lamin A / C , NLRP3 , caspase 1 , actin in skin fibroblats from with HGPS , n = 2 controls 2 . Positive control correspond to THP1 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 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 ) nuclei ( blue ) in skin fibroblasts from a representative 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

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


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

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

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

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

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(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 Caspase 1 transcript expression were determined in heart liver tissues by real - time quantitative RT - PCR . n = 5 for Zmpste24 + / + 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 .

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

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(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 with representative blots including lamin A / C , NLRP3 , IL - 1β actin in skin fibroblats from control HGPS after 48 hr of vehicle MCC950 treatment .

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

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(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 maximum lifespan in WT mice compared with Zmpste24 - / - mice . N = 7 per group .

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


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

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


( H ) 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 .

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(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 quantifications ( below ) of indicated in lysates of ARPE - 19 treated with 1 mM DFP for indicated time ( h , hours ) .

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

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

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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 indicated in lysates of WT or HIF1α KO U2OS , treated with 1mM DFP for 48 hours .

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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 of WT or HIF1α KO U2OS stably expressing pexo - QC reporter treated with 1 mM DFP for 48 hours ( left panel ) flow cytometry of mCherry / GFP ( right panel ) .

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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 ) ( right panel ) of indicated in lysates of WT or two NIX KO ARPE - 19 clones ( Cl ) , treated with 1 mM DFP for indicated time ( h , hours ) .

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

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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 ) ( right panel ) of indicated . NIX KO ARPE - 19 ( 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 immunoblot .

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

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

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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 indicated in lysates of WT or HA - Parkin ( PRKN ) - ARPE - 19 treated as in A .

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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 indicated in lysates of NIX KO ARPE - 19 ( 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 ) , treated with 1 mM DFP for 48 hours prior lysis .

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

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

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

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

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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 distribution of CD71 Ter119 populations in dissected yolk sacs from untreated dox - treated E9 . 5 Nanogtg . of CD71 + Ter119 + population in controls ( - dox , black dots ; n = 8 ) 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 . * * * P . < 0 . 0005 ; Student ' s t - . Horizontal represents mean values error bars standard deviation ( SD ) .

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

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

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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 without ( control ) with ( chimera ) contribution of wt - ESGFP ( left , brightfield ; right , GFP ) . Arrows mark presence of blood in chimeric that is absent from controls . Scale bar , 500 µm .

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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 maturation as determined by CD71 / Ter119 in single dox - treated E10 . 5 control ( left ) chimeric ( right ) .

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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.


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

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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.