( C , D ) Atg7 + / + and Atg7 - / - mice were intranasally injected with 3 x107 CFU of WT , ΔS , ΔTY or ΔSTY P . aeruginosa . After 24 hours , lungs ( C ) , spleens and livers ( D ) were harvested , homogenized and diluted for CFU counting . Each data point represents bacterial count ( Log10 CFU / g ) from individual mouse . The horizontal lines represent the mean value from each group ( 6 mice each group ) , error bars represent standard deviation . The significance of differences between different drug treatment was determined using One - way ANOVA and Dunn ' s multiple comparisons test . , NS : not significant ; * p ≤ 0 .

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(C,D) Atg7+/+ and Atg7-/- mice were intranasally injected with 3 x107 CFU of WT, ΔS, ΔTY or ΔSTY P. aeruginosa. After 24 hours, lungs (C), spleens and livers (D) were harvested, homogenized and diluted for CFU counting. Each data point represents bacterial count (Log10 CFU/g) from individual mouse. The horizontal lines represent the mean value from each group (6 mice each group), error bars represent standard deviation. The significance of differences between different drug treatment was determined using One-way ANOVA and Dunn's multiple comparisons test., NS: not significant; *p ≤ 0.05


( E , F ) Atg7 + / + and Atg7 - / - mice were intranasally injected with PBS with or without 1 x109 CFU of ΔTY ( E ) ΔSTY ( F ) . Mice survival was monitored for 10 days post infection . Kaplan - Meyer survival curves were made based on the observation of 12 - 18 mice / group from two independent experiments . The P - values were calculated using the log - rank test ( ΔTYAtg7 + / + vs ΔTYAtg7 - / - , P = 0 . 2186 ; ΔSTYAtg7 + / + vs ΔSTYAtg7 - / - , P = 0 . 0145 ) .

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(E,F) Atg7+/+ and Atg7-/- mice were intranasally injected with PBS with or without 1 x109 CFU of ΔTY (E) ΔSTY (F). Mice survival was monitored for 10 days post infection. Kaplan-Meyer survival curves were made based on the observation of 12-18 mice/group from two independent experiments. The P-values were calculated using the log-rank test (ΔTYAtg7+/+ vs ΔTYAtg7-/-, P = 0.2186; ΔSTYAtg7+/+ vs ΔSTYAtg7-/-, P=0.0145).


( A A549 cells were infected for 4 hours with ExoS - deficient P . aeruginosa ( ΔS ) , ExoS containing P . aeruginosa ( SWT ) , P . aeruginosa containing ExoS with loss - of - function mutations in the GTPase activating domain ( SG - A + ) , in the ADP - ribosylation domain ( SG + A - ) , or in both domains ( SG - A - ) . Cell lysates were evaluated by immunoblotting .

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(A A549 cells were infected for 4 hours with ExoS-deficient P. aeruginosa (ΔS), ExoS containing P. aeruginosa (SWT), P. aeruginosa containing ExoS with loss-of-function mutations in the GTPase activating domain (SG-A+), in the ADP-ribosylation domain (SG+A-), or in both domains (SG-A-). Cell lysates were evaluated by immunoblotting.


( B ) A549 cells were transfected , for 24hours , with a plasmid expressing empty vector ( EV ) , WT GFP - ExoS , a GFP - ExoS mutant for ADPRT domain ( SG + A - ) or a GFP - ExoS mutant deficient in both GAP and ADPRT domains ( SG - A - ) . Cell lysates were evaluated by immunoblotting .

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(B) A549 cells were transfected, for 24hours, with a plasmid expressing empty vector (EV), WT GFP-ExoS, a GFP-ExoS mutant for ADPRT domain (SG+A-) or a GFP-ExoS mutant deficient in both GAP and ADPRT domains (SG-A-). Cell lysates were evaluated by immunoblotting.


( C ) A549 cells were treated for 24 hours with 100 µM of the ADPRT activity inhibitor MIBG and then left uninfected ( NI ) or infected with P . aeruginosa containing ExoS with ADPRT activity only ( SG - A + ) . Cell lysates were evaluated by immunoblotting .

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(C) A549 cells were treated for 24 hours with 100 µM of the ADPRT activity inhibitor MIBG and then left uninfected (NI) or infected with P. aeruginosa containing ExoS with ADPRT activity only (SG-A+). Cell lysates were evaluated by immunoblotting.


A549 cells were infected for 4 hours with WT or P . aeruginosa mutants ( ΔpscD Cell lysates were evaluated by immunoblotting .

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A549 cells were infected for 4 hours with WT or P. aeruginosa mutants (ΔpscD Cell lysates were evaluated by immunoblotting.


A549 cells were infected for 4 hours with WT or P . aeruginosa mutants STY , ΔS and ΔTY ) . Cell lysates were evaluated by immunoblotting .

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A549 cells were infected for 4 hours with WT or P. aeruginosa mutants STY, ΔS and ΔTY). Cell lysates were evaluated by immunoblotting.


F ) A549 cells were infected for 4 hours with ExoS - deficient P . aeruginosa ( ΔS ) , ExoS containing P . aeruginosa ( SWT ) , P . aeruginosa containing ExoS with loss - of - function mutations in the GTPase activating domain ( SG - A + ) , in the ADP - ribosylation domain ( SG + A - ) , or in both domains ( SG - A - ) . Cell lysates were evaluated by immunoblotting .

F) A549 cells were infected for 4 hours with ExoS-deficient P. aeruginosa (ΔS), ExoS containing P. aeruginosa (SWT), P. aeruginosa containing ExoS with loss-of-function mutations in the GTPase activating domain (SG-A+), in the ADP-ribosylation domain (SG+A-), or in both domains (SG-A-). Cell lysates were evaluated by immunoblotting.


( A ) A549 cells were transfected with empty vector ( VE ) and vector contains Ras - G12V for 24 hours , followed by a 4 - hours infection with P . aeruginosa ΔSTY or ΔTY . Cell lysates were evaluated by immunoblotting .

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(A) A549 cells were transfected with empty vector (VE) and vector contains Ras-G12V for 24 hours, followed by a 4-hours infection with P. aeruginosa ΔSTY or ΔTY. Cell lysates were evaluated by immunoblotting.


( B ) A549 cells were transfected , for 24 hours , with empty vector ( VE ) and vectors contain Ras - G12V or Ras - G12V & R41K followed by a 4 - hours infection with P . aeruginosa ΔSTY or ΔTY . Cell lysates were evaluated by immunoblotting .

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(B) A549 cells were transfected, for 24 hours, with empty vector (VE) and vectors contain Ras-G12V or Ras-G12V&R41K followed by a 4-hours infection with P. aeruginosa ΔSTY or ΔTY. Cell lysates were evaluated by immunoblotting.


( A ) A549 cells were transfected , for 48 hours , with a mCherry - DFCP1 plasmid and then infected , for 4 hours , with P . aeruginosa ( WT , ΔSTY , ΔS , ΔTY , SG - A - , SG - A + or SG + A - ) and the mCherry puncta number per cell was analyzed by fluorescence microscopy . Representative immunofluorescence images were showed , and scale bars represent 10 µm . ( B ) Quantitative analysis of mCherry - DFCP1 puncta by visually count more than 100 cells for each sample . Each bar represents mean value of from three independent experiments and error bars represent standard deviation . The significance of differences between assays was determined using ANOVA with Dunnett multiple - comparison posttest , NS : not significant ; * p ≤ 0 . 05 ; * * p ≤ 0 . 01 .

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(A) A549 cells were transfected, for 48 hours, with a mCherry-DFCP1 plasmid and then infected, for 4 hours, with P. aeruginosa (WT, ΔSTY, ΔS, ΔTY, SG-A-, SG-A+ or SG+A-) and the mCherry puncta number per cell was analyzed by fluorescence microscopy. Representative immunofluorescence images were showed, and scale bars represent 10 µm. (B) Quantitative analysis of mCherry-DFCP1 puncta by visually count more than 100 cells for each sample. Each bar represents mean value of from three independent experiments and error bars represent standard deviation. The significance of differences between assays was determined using ANOVA with Dunnett multiple-comparison posttest, NS: not significant; *p ≤ 0.05; **p ≤ 0.01.


( A A549 cells , transfected for 48 hours with vector encoding Flag - Atg14L , were infected with P . aeruginosa ( WT , ΔSTY , ΔS , ΔTY Flag - Atg14L - Vps34 complex was immunoprecipitated ( IP ) by Flag - antibody magnetic beads and evaluated by immunoblotting .

(A A549 cells, transfected for 48 hours with vector encoding Flag-Atg14L, were infected with P. aeruginosa (WT, ΔSTY, ΔS, ΔTY Flag-Atg14L-Vps34 complex was immunoprecipitated (IP) by Flag-antibody magnetic beads and evaluated by immunoblotting.


( B , The IP complexes were subjected to in vitro kinase assay using phosphatidylinositol as substrate . The products radiolabeled PI3P were separated by TLC and detected by autoradiography . Data are mean ± SD of three independent experiments . The significance of differences between different assays was determined using One - way ANOVA analysis followed Dunn ' s multiple - comparison posttest , NS : not significant ; * p ≤ 0 . 05 .

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(B, The IP complexes were subjected to in vitro kinase assay using phosphatidylinositol as substrate. The products radiolabeled PI3P were separated by TLC and detected by autoradiography. Data are mean ± SD of three independent experiments. The significance of differences between different assays was determined using One-way ANOVA analysis followed Dunn's multiple-comparison posttest, NS: not significant; *p ≤ 0.05.


C ) A549 cells , transfected for 48 hours with vector encoding Flag - Atg14L , were infected with P . aeruginosa , ΔSTY SG - A - , SG - A + or SG + A - ) . Flag - Atg14L - Vps34 complex was immunoprecipitated ( IP ) by Flag - antibody magnetic beads and evaluated by immunoblotting .

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C) A549 cells, transfected for 48 hours with vector encoding Flag-Atg14L, were infected with P. aeruginosa , ΔSTY SG-A-, SG-A+ or SG+A-). Flag-Atg14L-Vps34 complex was immunoprecipitated (IP) by Flag-antibody magnetic beads and evaluated by immunoblotting.


D ) The IP complexes were subjected to in vitro kinase assay using phosphatidylinositol as substrate . The products radiolabeled PI3P were separated by TLC and detected by autoradiography . Data are mean ± SD of three independent experiments . The significance of differences between different assays was determined using One - way ANOVA analysis followed Dunn ' s multiple - comparison posttest , NS : not significant ; * p ≤ 0 . 05 .

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D) The IP complexes were subjected to in vitro kinase assay using phosphatidylinositol as substrate. The products radiolabeled PI3P were separated by TLC and detected by autoradiography. Data are mean ± SD of three independent experiments. The significance of differences between different assays was determined using One-way ANOVA analysis followed Dunn's multiple-comparison posttest, NS: not significant; *p ≤ 0.05.


A . Representative photomicrographs of mouse liver sections following a two - thirds PH of Ki67 positive hepatocytes ( brown ) visualized by IHC . Images are representative of 3 independent animals per time point .

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A. Representative photomicrographs of mouse liver sections following a two-thirds PH of Ki67 positive hepatocytes (brown) visualized by IHC. Images are representative of 3 independent animals per time point.


B . Western blot detection of p - MST1 , MST1 , p - LATS1 , LATS1 from proteins isolated from homogenised liver tissue at several time points . TBP was used as a loading control . Results of a single experiment with n = 3 animals per group . Each band is from an independent animal . Protein levels were normalized to TBP and mean value of the ratio of p - MST to total MST and p - LATS to total LATS is plotted .

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B. Western blot detection of p-MST1, MST1, p-LATS1, LATS1 from proteins isolated from homogenised liver tissue at several time points. TBP was used as a loading control. Results of a single experiment with n = 3 animals per group. Each band is from an independent animal. Protein levels were normalized to TBP and mean value of the ratio of p-MST to total MST and p-LATS to total LATS is plotted.


C . Nuclear enriched protein fractions obtained from sham - operated livers and liver 48 h post PH were analyzed by Western blot for YAP expression . Sp1 was used as a loading control . Image is representative of a single experiment with n = 3 animals per group .

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C. Nuclear enriched protein fractions obtained from sham-operated livers and liver 48 h post PH were analyzed by Western blot for YAP expression. Sp1 was used as a loading control. Image is representative of a single experiment with n = 3 animals per group.


D . Photomicrograph of YAP1 expression in sham and regenerating liver 48 h post PH visualized by immunoflourescence microscopy . Images are representative of 3 independent animals per condition .

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D. Photomicrograph of YAP1 expression in sham and regenerating liver 48 h post PH visualized by immunoflourescence microscopy. Images are representative of 3 independent animals per condition.


E . Livers from sham and 48 h following PH were digested to isolate the hepatocyte population . Protein extracts were analyzed by Western blot for YAP and TAZ expression . β - actin was used as a loading control . Results of a single experiment with n = 2 animals per group . Each band is representative of an independent animal .

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E. Livers from sham and 48 h following PH were digested to isolate the hepatocyte population. Protein extracts were analyzed by Western blot for YAP and TAZ expression. β-actin was used as a loading control. Results of a single experiment with n = 2 animals per group. Each band is representative of an independent animal.


F . RT - PCR analysis of RNA isolated from mouse liver for Birc5 , FoxM1B and cyclin B1 . Data are presented as log2 fold change and were calculated using non - operated mouse liver tissue as a control . Unpaired , two - tailed Student t - test was used to determine the significance between the log2 values of livers 6 , 24 and 48 h post PH compared to the sham operated controls . Log2 values are plotted on a linear scale as mean ± SD .

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F. RT-PCR analysis of RNA isolated from mouse liver for Birc5, FoxM1B and cyclin B1. Data are presented as log2 fold change and were calculated using non-operated mouse liver tissue as a control. Unpaired, two-tailed Student t-test was used to determine the significance between the log2 values of livers 6, 24 and 48 h post PH compared to the sham operated controls. Log2 values are plotted on a linear scale as mean ± SD.


A - C . Representative photomicrograph of young ( 6 - 8 weeks ) and aged ( > 12 months ) livers 40 h post PH stained with Ki67 and visualized by IHC and H E for histology . Sections were scanned using Histech Pannoramic MIDI Scanner and Ki67 positive cells were quantitated with Quant Center 2 . 0 software . A proliferation index ( p . i . ) was calculated based on the number of Ki67 positive hepatocytes to total hepatocytes and livers were classified as ( + ) regenerating , p . i . 10 % or ( - ) non - regenerating , p . i . 10 % . D . Number of young ( 6 - 8 weeks ) and aged ( 12 months ) mice with ( - ) non - regenerating and ( + ) regenerating or livers based on p . i . , 40 h post PH . Animals deemed non - surviving for ethical reasons were euthanized 24 hours post PH . Representative results of a single experiment with n = 9 aged and n = 6 young animals per group .

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A-C. Representative photomicrograph of young (6-8 weeks) and aged (>12 months) livers 40 h post PH stained with Ki67 and visualized by IHC and H E for histology. Sections were scanned using Histech Pannoramic MIDI Scanner and Ki67 positive cells were quantitated with Quant Center 2.0 software. A proliferation index (p.i.) was calculated based on the number of Ki67 positive hepatocytes to total hepatocytes and livers were classified as (+) regenerating, p.i.10% or (-) non-regenerating, p.i.10%.D. Number of young (6-8 weeks) and aged (12 months) mice with (-) non-regenerating and (+) regenerating or livers based on p.i., 40 h post PH. Animals deemed non-surviving for ethical reasons were euthanized 24 hours post PH. Representative results of a single experiment with n=9 aged and n=6 young animals per group.


A . Western blot detection of p - MST1 , MST , p - YAP , YAP and TAZ in control and 40 h following PH in young and aged mice . TBP was used as a loading control . Representative results of a single experiment with n = 6 independent animals . Each band is from an independent animal . Western blot quantification ratios are graphically depicted . Proteins levels were normalized to TBP and mean value of the group is plotted . Unpaired , two - tailed Student t - test was used to calculate the significance between 0h ( resected liver ) and 40h following PH . Error bars represent mean ±

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A. Western blot detection of p-MST1, MST, p-YAP, YAP and TAZ in control and 40 h following PH in young and aged mice. TBP was used as a loading control. Representative results of a single experiment with n = 6 independent animals. Each band is from an independent animal. Western blot quantification ratios are graphically depicted. Proteins levels were normalized to TBP and mean value of the group is plotted. Unpaired, two-tailed Student t-test was used to calculate the significance between 0h (resected liver) and 40h following PH. Error bars represent mean ± SD


B . RT - PCR analysis of RNA isolated from mouse liver for FoxM1B , Birc5 and cyclin B1 , Cyclin A2 , Cyclin D1 and p21 . RT - PCR data are presented as log2 fold change and each value post PH was calculated for each mouse by comparison to its resected liver lobe . Circle , with red outline marks the aged non - regenerating livers shown in Figure 2B . Each dot is representing an independent animal .

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B. RT-PCR analysis of RNA isolated from mouse liver for FoxM1B, Birc5 and cyclin B1, Cyclin A2, Cyclin D1 and p21. RT-PCR data are presented as log2 fold change and each value post PH was calculated for each mouse by comparison to its resected liver lobe. Circle, with red outline marks the aged non-regenerating livers shown in Figure 2B. Each dot is representing an independent animal.


A . Eight weeks old mice were injected with liposomes coupled to scrambled siRNA ( siScr ) or sequences targeting MST1 MST2 ( siMST ) . Each dot is representing an independent animal . The % of Mst1 Mst2 mRNA remaining in the liver was tested by RT - PCR , 1 , 3 and 6 days post injection and calculated compared to control non - transfected livers . Unpaired , two - tailed Student t - test was used to calculate the significance of mRNA remaining in comparison to non - transfected control livers at each time point .

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A. Eight weeks old mice were injected with liposomes coupled to scrambled siRNA (siScr) or sequences targeting MST1 MST2 (siMST). Each dot is representing an independent animal. The % of Mst1 Mst2 mRNA remaining in the liver was tested by RT-PCR, 1, 3 and 6 days post injection and calculated compared to control non-transfected livers. Unpaired, two-tailed Student t-test was used to calculate the significance of mRNA remaining in comparison to non-transfected control livers at each time point.


B . Western blot detection of MST1 , p - LATS1 and β - actin 1 , 3 and 6 days post siRNA injection . A representative animal from each time point is shown .

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B. Western blot detection of MST1, p-LATS1 and β-actin 1, 3 and 6 days post siRNA injection. A representative animal from each time point is shown.


C . Western blot detection of YAP1 , p - YAP1 , H3 and p - H3 , 3 days post siRNA injection . Two representative animals from each group are shown . All samples were run on the same blot . The blot is split due to non - consecutive loading of the samples .

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C. Western blot detection of YAP1, p-YAP1, H3 and p-H3, 3 days post siRNA injection. Two representative animals from each group are shown. All samples were run on the same blot. The blot is split due to non-consecutive loading of the samples.


D . Quantitative RT - PCR analysis of RNA isolated from mouse liver for FoxM1B , Birc5 and cyclin B1 , 1 , 3 and 6 days post injection . Log2 fold change was calculated using non - transfected mouse liver as control . Unpaired two tailed Student t - test was used to calculate the significance of fold change of in comparison to a panel non - transfected control livers .

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D. Quantitative RT-PCR analysis of RNA isolated from mouse liver for FoxM1B, Birc5 and cyclin B1, 1, 3 and 6 days post injection. Log2 fold change was calculated using non-transfected mouse liver as control. Unpaired two tailed Student t-test was used to calculate the significance of fold change of in comparison to a panel non-transfected control livers.


E . HNF4α and Ki67 were detected by IHC and cells positive for both were classified as proliferating hepatocytes . Ki67 positive hepatocytes are indicated with an asterisk and Ki67 negative bile ducts / biliary cells with an arrow in tissue 3 days post siRNA delivery . The percentages of positive hepatocytes at day 3 and day 6 - post injection are graphed . Two - tailed Student t - test was used to calculate the significance of percentage of positive hepatocytes in comparison to non - transfected control livers .

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E. HNF4α and Ki67 were detected by IHC and cells positive for both were classified as proliferating hepatocytes. Ki67 positive hepatocytes are indicated with an asterisk and Ki67 negative bile ducts/biliary cells with an arrow in tissue 3 days post siRNA delivery. The percentages of positive hepatocytes at day 3 and day 6-post injection are graphed. Two-tailed Student t-test was used to calculate the significance of percentage of positive hepatocytes in comparison to non-transfected control livers.


A . Aged mice ( > 12 months ) were injected i . v . with siScr or siMST . Twenty - four h after siRNA injection a 70 % PH was performed and remnant / regenerating liver tissue was harvested 40 h later . p . i . of the livers was calculated by IHC staining of Ki67 positive hepatocytes and based on the percentage of positive cells liver were classified ( - ) non regenerating or ( + ) regenerating . Non - surviving animals were sacrificed before the 40 h end time point . " n " indicates the number of animals per group . B . Ki67 immunostaining of the regenerating aged mice livers , 40 h post PH . Sections were scanned using Histotech Pannoramic MIDI Scanner and quantitated with Quant Center 2 . 0 software . p . i . values are provided on the image .

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A. Aged mice (>12 months) were injected i.v. with siScr or siMST. Twenty-four h after siRNA injection a 70% PH was performed and remnant/regenerating liver tissue was harvested 40 h later. p.i. of the livers was calculated by IHC staining of Ki67 positive hepatocytes and based on the percentage of positive cells liver were classified (-) non regenerating or (+) regenerating. Non-surviving animals were sacrificed before the 40 h end time point. "n" indicates the number of animals per group.B. Ki67 immunostaining of the regenerating aged mice livers, 40 h post PH. Sections were scanned using Histotech Pannoramic MIDI Scanner and quantitated with Quant Center 2.0 software. p.i. values are provided on the image.


C . Representative photomicrograph of an aged liver treated with siMST and stained with H & amp ; amp ; E and by IHC for CK19 and Ki67 . Arrows are indicating ductal regions with no signs of reaction or oval cell expansion .

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C. Representative photomicrograph of an aged liver treated with siMST and stained with H&amp;E and by IHC for CK19 and Ki67. Arrows are indicating ductal regions with no signs of reaction or oval cell expansion.


D . Quantitative RT - PCR analysis of RNA isolated from mouse liver for FoxM1B , Birc5 and cyclin B1 at 0 and 40 h post PH . Log2 fold change was calculated using non - transfected / non - resected mouse liver as a control . Representative results of a single experiment with n = 6 animals per group are shown . Paired , two - tailed Student t - test was used to calculate the significant change in signals between liver tissues at the time of resection ( 0h ) compared to 40 h post PH .

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D. Quantitative RT-PCR analysis of RNA isolated from mouse liver for FoxM1B, Birc5 and cyclin B1 at 0 and 40 h post PH. Log2 fold change was calculated using non-transfected/non-resected mouse liver as a control. Representative results of a single experiment with n = 6 animals per group are shown. Paired, two-tailed Student t-test was used to calculate the significant change in signals between liver tissues at the time of resection (0h) compared to 40 h post PH.


E . Percentage of Ki67 positive hepatocytes in aged and aged + siMST 40 hours post resection . Representative results of a single experiment with n = 6 independent aged animals for group are shown . Two - tailed Student t - test was used to calculate the significance of percentage of positive hepatocytes of siMST treated animals compared to non - treated controls .

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E. Percentage of Ki67 positive hepatocytes in aged and aged + siMST 40 hours post resection. Representative results of a single experiment with n = 6 independent aged animals for group are shown. Two-tailed Student t-test was used to calculate the significance of percentage of positive hepatocytes of siMST treated animals compared to non-treated controls.


F . Endpoint liver weight was taken of the remnant lobe and expressed as a percentage to total body weight . Representative results of 1experiment with n = 6 independent animals for group expect for control group were n = 6 are shown . Estimated liver - to - body weight ratio before PH in mice = 3 . 85 ± 0 . 05 ( SD ) . Unpaired , two - tailed Student t - test was used to calculate the significance of each aged group in comparison to the control young group .

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F. Endpoint liver weight was taken of the remnant lobe and expressed as a percentage to total body weight. Representative results of 1experiment with n = 6 independent animals for group expect for control group were n = 6 are shown. Estimated liver-to-body weight ratio before PH in mice = 3.85 ± 0.05 (SD). Unpaired, two-tailed Student t-test was used to calculate the significance of each aged group in comparison to the control young group.


G . Hepatocyte geometric diameter was calculated ( see material and methods ) . Representative results of 1 experiment with n = 3 independent animals per group . Unpaired , two - tailed Student t - test was used to calculate the significant change in hepatocyte size in liver tissue with and without siMST 40 h post PH .

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G. Hepatocyte geometric diameter was calculated (see material and methods). Representative results of 1 experiment with n = 3 independent animals per group. Unpaired, two-tailed Student t-test was used to calculate the significant change in hepatocyte size in liver tissue with and without siMST 40 h post PH.


Representative Western blot showing CARD14 protein levels in skin and liver lysates of E18 . 5 pups . Actin is shown as a loading control .

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Representative Western blot showing CARD14 protein levels in skin and liver lysates of E18.5 pups. Actin is shown as a loading control.


Representative images of K5creTg / + CARD14E138A + / + and K5creTg / + CARD14E138ATg / + pups at E18 . 5 .

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Representative images of K5creTg/+ CARD14E138A+/+ and K5creTg/+ CARD14E138ATg/+ pups at E18.5.


Representative image of 6 month - old Malt1fl / fl K5cretg / + CARD14E138A + / + and Malt1fl / fl K5cretg / + CARD14E138Atg / + mice .

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Representative image of 6 month-old Malt1fl/fl K5cretg/+ CARD14E138A+/+ and Malt1fl/fl K5cretg/+ CARD14E138Atg/+ mice.


Body weight and ear thickness of untreated WT and Malt1fl / fl K5creTg / + CARD14E138ATg / + male and female mice ( between 4 and 7 months old ) . Each symbol represents one mouse ; the line represents the mean value ( n ≥ 4 mice per group ) . WT = Malt1fl / fl K5cre + / + CARD14E138A + / + , Malt1fl / fl K5cre + / + CARD14E138ATg / + and Malt1fl / fl K5creTg / + CARD14E138A + / + mice . Statistical difference between two groups was determined using a Mann - Whitney U - test ( ns : p - value > 0 . 05 )

Body weight and ear thickness of untreated WT and Malt1fl/fl K5creTg/+ CARD14E138ATg/+ male and female mice (between 4 and 7 months old). Each symbol represents one mouse; the line represents the mean value (n≥4 mice per group). WT = Malt1fl/fl K5cre+/+ CARD14E138A+/+, Malt1fl/fl K5cre+/+ CARD14E138ATg/+ and Malt1fl/fl K5creTg/+ CARD14E138A+/+ mice. Statistical difference between two groups was determined using a Mann-Whitney U-test (ns: p-value>0.05)


Representative H & E - stained histological sections of ear and skin tissue of Malt1fl / fl K5cre + / + CARD14E138A + / + and Malt1fl / fl K5creTg / + CARD14E138ATg / + mice ( scale bar represents 100 μm ) .

Representative H&E-stained histological sections of ear and skin tissue of Malt1fl/fl K5cre+/+ CARD14E138A+/+ and Malt1fl/fl K5creTg/+ CARD14E138ATg/+ mice (scale bar represents 100 μm).


Changes in relative body weight and ear thickness upon CARD14E138A induction with tamoxifen . Malt1EKO = Malt1fl / fl . The combined results of five independent experiments are shown ( Malt1 + / + WT n = 17 , Malt1fl / fl WT n = 8 , Malt1 + / + ieCARD14E138A n = 18 , Malt1fl / fl ieCARD14E138A n = 12 ) .

Changes in relative body weight and ear thickness upon CARD14E138A induction with tamoxifen. Malt1EKO = Malt1fl/fl. The combined results of five independent experiments are shown (Malt1+/+ WT n=17, Malt1fl/fl WT n=8, Malt1+/+ ieCARD14E138A n=18, Malt1fl/fl ieCARD14E138A n=12).


Representative H & E - stained histological sections of ear tissue of tamoxifen - treated mice ( Scale bar represents 200 μm ) . Arrowheads indicate infiltrates of immune cells .

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Representative H&E-stained histological sections of ear tissue of tamoxifen-treated mice (Scale bar represents 200 μm). Arrowheads indicate infiltrates of immune cells.


Epidermal thickness measured on ear sections . Each symbol represents the mean of at least ten measurements for each ear ; the line represents the mean value . The combined results of three independent experiments are shown .

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Epidermal thickness measured on ear sections. Each symbol represents the mean of at least ten measurements for each ear; the line represents the mean value. The combined results of three independent experiments are shown.


Analysis of infiltrating immune cells in the ears of tamoxifen - treated mice using flow cytometry . Cell count of neutrophils ( CD45 + CD3 / CD19 - CD64 - CD11b + Ly6G + ) , eosinophils ( CD45 + CD3 / CD19 - CD64 - Ly6G - SiglecF + ) , T cells ( CD45 + CD3 / CD19 + , MHCII - ) , DCs ( CD45 + CD3 / CD19 - CD64 - MHCII + CD11c + ) in single cell suspensions of the ear ( n ≥ 4 ) .

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Analysis of infiltrating immune cells in the ears of tamoxifen-treated mice using flow cytometry. Cell count of neutrophils (CD45+ CD3/CD19- CD64- CD11b+ Ly6G+), eosinophils (CD45+ CD3/CD19- CD64- Ly6G- SiglecF+), T cells (CD45+ CD3/CD19+, MHCII-), DCs (CD45+ CD3/CD19- CD64- MHCII+CD11c+) in single cell suspensions of the ear (n≥4).


mRNA expression levels of the indicated genes in ears relative to reference genes ( Hprt1 , Rpl13a and Tbp1 ) . Each symbol represents one mouse ; the line represents the mean value ( n ≥ 3 ) .

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mRNA expression levels of the indicated genes in ears relative to reference genes (Hprt1, Rpl13a and Tbp1). Each symbol represents one mouse; the line represents the mean value (n≥3).


Cleavage of MALT1 substrates CYLD and BCL10 as analyzed by western blotting of lysates of ear tissue of mice four days after treatment with tamoxifen and MALT1 inhibitor or vehicle . Actin is shown as a loading control . Each lane represents one mouse .

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Cleavage of MALT1 substrates CYLD and BCL10 as analyzed by western blotting of lysates of ear tissue of mice four days after treatment with tamoxifen and MALT1 inhibitor or vehicle. Actin is shown as a loading control. Each lane represents one mouse.


Changes in relative ear thickness and body weight in vehicle - and MALT1 inhibitor - treated mice upon CARD14E138A induction . M1 inh = MALT1 inhibitor , WT = K14creERTg / + Rosa26 + / + , ieCARD14E138A = K14creERTg / + Rosa26LSL - CARD14 - E138A / + . Combined results of two independent experiments are shown ( WT + vehicle : n = 9 , WT + M1 inh : n = 8 , ieCARD14E138A + vehicle : n = 9 , ieCARD14E138A + M1 inh : n = 10 ) .

Changes in relative ear thickness and body weight in vehicle- and MALT1 inhibitor-treated mice upon CARD14E138A induction. M1 inh= MALT1 inhibitor, WT = K14creERTg/+ Rosa26+/+, ieCARD14E138A = K14creERTg/+ Rosa26LSL-CARD14-E138A/+. Combined results of two independent experiments are shown (WT + vehicle: n=9, WT + M1 inh: n=8, ieCARD14E138A + vehicle: n=9, ieCARD14E138A + M1 inh: n=10).


Representative histological sections of ear tissue stained with hematoxylin and eosin


measurements of epidermal thickness of tamoxifen - treated mice . Combined results of two independent experiments are shown . Each symbol represents the mean of at least ten epidermal thickness measurements for each ear ; the line represents the mean value .

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measurements of epidermal thickness of tamoxifen-treated mice. Combined results of two independent experiments are shown. Each symbol represents the mean of at least ten epidermal thickness measurements for each ear; the line represents the mean value.


IL - 6 and IL - 17a production by ear draining lymphocytes upon ex vivo restimulation with anti - CD3 / anti - CD28 for 3 days ( n ≥ 3 biological replicates ) .

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IL-6 and IL-17a production by ear draining lymphocytes upon ex vivo restimulation with anti-CD3/anti-CD28 for 3 days (n≥3 biological replicates).


Heatmap showing relative mRNA expression levels of the indicated genes in ear tissue normalized to reference genes . Values represent median of each group ( n ≥ 3 ) .

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Heatmap showing relative mRNA expression levels of the indicated genes in ear tissue normalized to reference genes. Values represent median of each group (n≥3).


Proliferation curves of parental and AGO2KO HeLaS3 cells are plotted ( n = 6 experimental replicates ) .

Proliferation curves of parental and AGO2KO HeLaS3 cells are plotted (n=6 experimental replicates).


Colony - forming activity of HeLaS3 and AGO2KO cells was determined by colony - formation assay ( n = 3 experimental replicates ) .

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Colony-forming activity of HeLaS3 and AGO2KO cells was determined by colony-formation assay (n=3 experimental replicates).


Tumour growth curves of xenografts derived from HeLaS3 or AGO2KO cells are shown as tumour diameter at different time points ( n = 5 mice for each group ) .

Tumour growth curves of xenografts derived from HeLaS3 or AGO2KO cells are shown as tumour diameter at different time points (n=5 mice for each group).


Xenograft tumour weights were measured 30 days post - injection ( n = 5 mice for each group ) .

Xenograft tumour weights were measured 30 days post- injection (n=5 mice for each group).


HeLaS3 cells were transduced with a lentiviral vector coding for human TERC ( HeLaS3 _ TERC ) or for GFP , as a control ( HeLaS3 _ GFP ) . The relative expression of TERC was measured by RT - qPCR . HPRT1 was used as internal control ( n = 3 experimental replicates ) .

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HeLaS3 cells were transduced with a lentiviral vector coding for human TERC (HeLaS3_TERC) or for GFP, as a control (HeLaS3_GFP). The relative expression of TERC was measured by RT-qPCR. HPRT1 was used as internal control (n = 3 experimental replicates).


The relative expression of terc - sRNA was measured in HeLaS3 _ TERC and HeLaS3 _ GFP by RT - qPCR . RNU44 was used as internal control ( n = 3 experimental replicates ) .

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The relative expression of terc-sRNA was measured in HeLaS3_TERC and HeLaS3_GFP by RT-qPCR. RNU44 was used as internal control (n = 3 experimental replicates).


Average telomere length was measured from genomic DNA of parental and AGO2KO HeLaS3 , by qPCR amplification of telomere repeats ( T ) and multicopy gene Alu ( R ) , as a reference . The relative telomere length ( T / R ) was plotted ( n = 3 experimental replicates )

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Average telomere length was measured from genomic DNA of parental and AGO2KO HeLaS3, by qPCR amplification of telomere repeats (T) and multicopy gene Alu (R), as a reference. The relative telomere length (T/R) was plotted (n=3 experimental replicates)


Representative images of metaphase spreads obtained from HeLaS3 and AGO2KO cells stained for telomeric sequences and centromere 2 alphoid DNA .

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Representative images of metaphase spreads obtained from HeLaS3 and AGO2KO cells stained for telomeric sequences and centromere 2 alphoid DNA.


Representative telomere length distributions in parental and AGO2KO HeLaS3 . Average telomere length and the standard deviation , as well as the total number of telomeres analyzed , are indicated . Notice the marked shift towards short telomeres in AGO2KO cells compared with the parental HeLa S3 cell line .

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Representative telomere length distributions in parental and AGO2KO HeLaS3. Average telomere length and the standard deviation, as well as the total number of telomeres analyzed, are indicated. Notice the marked shift towards short telomeres in AGO2KO cells compared with the parental HeLa S3 cell line.


Telomerase activity was detected by TRAP in parental HeLaS3 and AGO2KO cells . 293T cells were used as a positive control . As negative controls , telomerase was heat - inactivated in cell extracts , in the last lane no cells were added in lysis buffer . Intensity of the telomerase products ( 6 bp - ladder ) was determined by ImageLab Software ( Biorad ) and normalized with the intensity of the Internal Control ( IC ) ( n = 3 experimental replicates ) .

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Telomerase activity was detected by TRAP in parental HeLaS3 and AGO2KO cells. 293T cells were used as a positive control. As negative controls, telomerase was heat- inactivated in cell extracts, in the last lane no cells were added in lysis buffer. Intensity of the telomerase products (6 bp-ladder) was determined by ImageLab Software (Biorad) and normalized with the intensity of the Internal Control (IC) (n=3 experimental replicates).


AGO2KO cells were transduced with a lentiviral vector coding for FLAG - HA - tagged AGO2 ( AGO2KO _ FH AGO2 ) or for GFP as a control ( AGO2KO _ GFP ) . Quantification of telomerase activity in AGO2KO _ FH AGO2 and AGO2KO _ GFP , as assessed by TRAP , was plotted ( n = 3 experimental replicates ) .

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AGO2KO cells were transduced with a lentiviral vector coding for FLAG-HA-tagged AGO2 (AGO2KO_FH AGO2) or for GFP as a control (AGO2KO_GFP). Quantification of telomerase activity in AGO2KO_FH AGO2 and AGO2KO_GFP, as assessed by TRAP, was plotted (n=3 experimental replicates).


HeLaS3 cells were transduced with a lentiviral vector coding for FLAG - HA - tagged AGO2 ( HeLaS3 _ FH AGO2 ) or for GFP as a control ( HeLaS3 _ GFP ) . Quantification of telomerase activity in HeLaS3 _ FH AGO2 and HeLaS3 _ GFP , as assessed by TRAP , was plotted ( n = 3 experimental replicates ) .

HeLaS3 cells were transduced with a lentiviral vector coding for FLAG-HA-tagged AGO2 (HeLaS3_FH AGO2) or for GFP as a control (HeLaS3_GFP). Quantification of telomerase activity in HeLaS3_FH AGO2 and HeLaS3_GFP, as assessed by TRAP, was plotted (n=3 experimental replicates).


The relative expression of TERC was measured by RT - qPCR from total RNA of HeLaS3 and AGO2KO cells . HPRT1 was used as internal control ( n = 3 experimental replicates ) . qPCR of 3 ' - extended TERC transcript in cDNA from HeLaS3 and AGO2KO cells generated using random hexamers . HPRT1 was used for normalization ( n = 3 experimental replicates ) . qPCR of TERC transcript in cDNA from HeLaS3 and AGO2KO cells generated using oligo ( dT ) 10 priming . HPRT1 was used for normalization ( n = 3 experimental replicates ) .

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The relative expression of TERC was measured by RT-qPCR from total RNA of HeLaS3 and AGO2KO cells. HPRT1 was used as internal control (n = 3 experimental replicates). qPCR of 3'-extended TERC transcript in cDNA from HeLaS3 and AGO2KO cells generated using random hexamers. HPRT1 was used for normalization (n=3 experimental replicates). qPCR of TERC transcript in cDNA from HeLaS3 and AGO2KO cells generated using oligo(dT)10 priming. HPRT1 was used for normalization (n=3 experimental replicates).


Whole cell extract of HeLaS3 and AGO2KO was analyzed by western blot for the presence of TERT protein . GAPDH was used as loading control . Data are representative of 4 independent experiments . TERT protein level was quantified by ImageLab Software ( Biorad ) . GAPDH was use as internal control ( n = 4 experimental replicates )

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Whole cell extract of HeLaS3 and AGO2KO was analyzed by western blot for the presence of TERT protein. GAPDH was used as loading control. Data are representative of 4 independent experiments. TERT protein level was quantified by ImageLab Software (Biorad). GAPDH was use as internal control (n=4 experimental replicates)


HeLaS3 and AGO2KO cell extracts ( n = 3 ) , were immunoprecipitated using an anti - TERT antibody or IgG as mock IP . TERC and HOTAIR ( as a negative control ) abundance was assessed by RT - qPCR . HPRT1 was used for normalization and enrichment in TERT - RIP as compared to IgG RIP was plotted .

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HeLaS3 and AGO2KO cell extracts (n=3), were immunoprecipitated using an anti-TERT antibody or IgG as mock IP. TERC and HOTAIR (as a negative control) abundance was assessed by RT-qPCR. HPRT1 was used for normalization and enrichment in TERT-RIP as compared to IgG RIP was plotted.


AGO2KO _ FH AGO2 and AGO2KO _ GFP cell extracts ( n = 3 experimental replicates ) were immunoprecipitated using an anti - TERT antibody or IgG as mock IP . TERC and HOTAIR ( as a negative control ) abundance was assessed by RT - qPCR . HPRT1 was used for normalization and enrichment in TERT - RIP as compared to IgG RIP was plotted .

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AGO2KO_FH AGO2 and AGO2KO_GFP cell extracts (n=3 experimental replicates) were immunoprecipitated using an anti-TERT antibody or IgG as mock IP. TERC and HOTAIR (as a negative control) abundance was assessed by RT-qPCR. HPRT1 was used for normalization and enrichment in TERT-RIP as compared to IgG RIP was plotted.


HeLaS3 whole - cell extract was immunoprecipitated using anti - AGO2 antibody or IgG , as mock IP . Whole cell lysates ( input ) and immunoprecipitates were analyzed by western blot with anti - TERT antibody . As a positive control , the presence in immunoprecipitates of GW182 , a known AGO2 - interacting protein , was assessed . Data are representative of three independent experiments .

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HeLaS3 whole-cell extract was immunoprecipitated using anti- AGO2 antibody or IgG, as mock IP. Whole cell lysates (input) and immunoprecipitates were analyzed by western blot with anti-TERT antibody. As a positive control, the presence in immunoprecipitates of GW182, a known AGO2-interacting protein, was assessed. Data are representative of three independent experiments.


HeLaS3 whole - cell extract was immunoprecipitated using anti - AGO2 antibody or IgG , as mock IP . TERC RNA enrichment in AGO2 RIP as compared to IgG RIP was assessed by RT - qPCR . 7SK RNA was used for normalization ( n = 4 experimental replicates ) .

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HeLaS3 whole-cell extract was immunoprecipitated using anti- AGO2 antibody or IgG, as mock IP. TERC RNA enrichment in AGO2 RIP as compared to IgG RIP was assessed by RT-qPCR . 7SK RNA was used for normalization (n=4 experimental replicates).


RIP assay was performed from HeLaS3 _ FH AGO2 and HeLaS3 _ GFP cell extract using anti - HA antibody or IgG , as negative control , followed by TERC detection by RT - qPCR . 7SK RNA was used for normalization ( n = 3 experimental replicates ) .

RIP assay was performed from HeLaS3_FH AGO2 and HeLaS3_GFP cell extract using anti-HA antibody or IgG, as negative control, followed by TERC detection by RT-qPCR. 7SK RNA was used for normalization (n=3 experimental replicates).


HeLaS3 cells were transduced with a lentiviral vector coding for wild - type TERC ( HeLaS3 _ TERCwt ) , TERC mutated in position 313 - 340 ( HeLaS3 _ TERC ( 313 - 340mut ) ) or TERC delated in position 12 - 31 ( HeLaS3 _ TERC ( Δ 12 - 31 ) ) . Whole - cell lysates from HeLaS3 _ TERCwt , HeLaS3 _ TERC ( 313 - 340mut ) and HeLaS3 _ TERC ( Δ 12 - 31 ) was immunoprecipitated using an anti - AGO2 antibody in order to assessed the impact of TERC mutations on AGO2 binding . For each experiment the enrichment of ectoTERC as compared to 7SK RNA in RIP samples was normalized on the amount of ectoTERC and 7SK RNA in input samples . Association of AGO2 with TERC ( 313 - 340mut and TERC ( Δ 12 - 31 ) was compared to the association with TERCwt ( n = 3 experimental replicates ) .

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HeLaS3 cells were transduced with a lentiviral vector coding for wild-type TERC (HeLaS3_TERCwt), TERC mutated in position 313-340 (HeLaS3_TERC (313-340mut)) or TERC delated in position 12-31 (HeLaS3_TERC (Δ 12-31)). Whole-cell lysates from HeLaS3_TERCwt, HeLaS3_TERC(313-340mut) and HeLaS3_TERC (Δ 12-31) was immunoprecipitated using an anti-AGO2 antibody in order to assessed the impact of TERC mutations on AGO2 binding. For each experiment the enrichment of ectoTERC as compared to 7SK RNA in RIP samples was normalized on the amount of ectoTERC and 7SK RNA in input samples. Association of AGO2 with TERC(313-340mut and TERC (Δ 12-31) was compared to the association with TERCwt (n=3 experimental replicates).


HeLaS3 and AGO2KO cells were transfected with a synthetic terc - sRNA or an siRNA with no target in humans ( siCtl ) , as a control . Four days after transfection , telomerase activity in 100 - and 50 cell - lysate was assessed by TRAP . Intensity of the telomerase products ( 6 bp - ladder ) was determined by ImageLab Software ( Biorad ) and normalized with the intensity of the Internal Control ( IC ) , ( n = 4 experimental replicates )

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HeLaS3 and AGO2KO cells were transfected with a synthetic terc-sRNA or an siRNA with no target in humans (siCtl), as a control. Four days after transfection, telomerase activity in 100- and 50 cell-lysate was assessed by TRAP. Intensity of the telomerase products (6 bp-ladder) was determined by ImageLab Software (Biorad) and normalized with the intensity of the Internal Control (IC), (n=4 experimental replicates)


C - E NF2 and WWC1 - 3 regulate the kinase activity of LATS1 , but not MST1 or MAP4K4 . LATS1 , MST1 or MAP4K4 was immunoprecipitated from WT or KO HEK293A cells and subjected to in vitro kinase assay using purified GST - YAP or GST - LATS2 as substrates . Phosphorylation - specific signals of YAP reflect the activity of LATS1 , and phosphorylation - specific signals of LATS2 reflect the activity of MST1 and MAP4K4 .

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C-E NF2 and WWC1-3 regulate the kinase activity of LATS1, but not MST1 or MAP4K4. LATS1, MST1 or MAP4K4 was immunoprecipitated from WT or KO HEK293A cells and subjected to in vitro kinase assay using purified GST-YAP or GST-LATS2 as substrates. Phosphorylation-specific signals of YAP reflect the activity of LATS1, and phosphorylation-specific signals of LATS2 reflect the activity of MST1 and MAP4K4.


H NF2 and WWC2 interact with LATS1 as indicated by BioID labeling . HEK293A cells stably expressing BirA - NF2 , WWC2 , MOB1A , or SAV1 were labeled with biotin overnight , and endogenous kinases were immunoprecipitated and subjected to immunoblotting using Streptavidin - HRP . No biotinylation of MAP4K4 was detected .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 NF2 and WWC2 interact with LATS1 as indicated by BioID labeling. HEK293A cells stably expressing BirA-NF2, WWC2, MOB1A, or SAV1 were labeled with biotin overnight, and endogenous kinases were immunoprecipitated and subjected to immunoblotting using Streptavidin-HRP. No biotinylation of MAP4K4 was detected.


B - D NF2 is required for the activation of LATS1 / 2 by RAP2A . WT and NF2 KO HEK293A cells were transfected with or without HA - RAP2A . Immunoprecipitated LATS1 ( B ) , MAP4K4 ( C ) or MST1 ( D ) was then subjected to in vitro kinase assays .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-D NF2 is required for the activation of LATS1/2 by RAP2A. WT and NF2 KO HEK293A cells were transfected with or without HA-RAP2A. Immunoprecipitated LATS1 (B), MAP4K4 (C) or MST1(D) was then subjected to in vitro kinase assays.


E , F NF2 is required for the activation of LATS1 / 2 by MAP4K kinases . MST1 / 2 dKO or MST1 / 2 ; NF2 3KO cells were transfected with siRNAs against STRIP1 / 2 . Immunoprecipitated MAP4K4 ( E ) or LATS1 ( F ) was then subjected to in vitro kinase assays .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 NF2 is required for the activation of LATS1/2 by MAP4K kinases. MST1/2 dKO or MST1/2;NF2 3KO cells were transfected with siRNAs against STRIP1/2. Immunoprecipitated MAP4K4 (E) or LATS1 (F) was then subjected to in vitro kinase assays.


B Both HPO1 and HPO2 are required for LATS1 / 2 activation by serum deprivation . Control or KO HEK293A cells were serum starved for 0 , 1 or 2 hours , and cell lysates were subjected to immunoblotting to analyze LATS1 and YAP phosphorylation ( phostag gel ) . Genotypes ( in B and C ) labeled in blue , orange , or red indicate deficiencies in HPO1 , HPO2 , or both , respectively .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Both HPO1 and HPO2 are required for LATS1/2 activation by serum deprivation. Control or KO HEK293A cells were serum starved for 0, 1 or 2 hours, and cell lysates were subjected to immunoblotting to analyze LATS1 and YAP phosphorylation (phostag gel). Genotypes (in B and C) labeled in blue, orange, or red indicate deficiencies in HPO1, HPO2, or both, respectively.


C Both HPO1 and HPO2 are required for LATS1 / 2 activation by actin disrupting agent LatB . Control or KO HEK293A cells were treated with 0 . 2 μg / ml LatB for 30 min , and cell lysates were subjected to immunoblotting to analyze phosphorylation of LATS1 , YAP , NF2 , and MOB1 . Asterisk indicates S13 phosphorylated NF2 .

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 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

C Both HPO1 and HPO2 are required for LATS1/2 activation by actin disrupting agent LatB. Control or KO HEK293A cells were treated with 0.2 μg/ml LatB for 30 min, and cell lysates were subjected to immunoblotting to analyze phosphorylation of LATS1, YAP, NF2, and MOB1. Asterisk indicates S13 phosphorylated NF2.


A Survival curves showing life expectancies of mice with genetic deletions of HPO1 and / or HPO2 genes . 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

A Survival curves showing life expectancies of mice with genetic deletions of HPO1 and/or HPO2 genes. n≥3.


B Concurrent deletion of Nf2 and Wwc1 / 2 at developmental stage driven by Alb - Cre results in liver dysfunction and perinatal death . Gross liver images , H & E , IHC , and IF images of 3 - day - old livers from WT and Nf2 ; Wwc1 / 2 compound KO mice were shown . Scale bars : 1 cm ( gross liver image ) , 50 μm ( H & E and IHC ) , 20 μm ( IF ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Concurrent deletion of Nf2 and Wwc1/2 at developmental stage driven by Alb-Cre results in liver dysfunction and perinatal death. Gross liver images, H&E, IHC, and IF images of 3-day-old livers from WT and Nf2;Wwc1/2 compound KO mice were shown. Scale bars: 1 cm (gross liver image), 50 μm (H&E and IHC), 20 μm (IF).


E , F Activities of Hippo pathway kinases in Nf2 ; Wwc1 / 2 deficient livers . LATS1 , MST2 , and MAP4K4 were immunoprecipitated from control or Nf2F / F ; Wwc1F / F ; Wwc2F / F ( Ad - Cre ) mouse livers ( 4 each ) , and kinase assays were performed ( E ) and quantified ( F ) . Four biological replicates were shown in F , error bars indicate SEM .

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

E, F Activities of Hippo pathway kinases in Nf2;Wwc1/2 deficient livers. LATS1, MST2, and MAP4K4 were immunoprecipitated from control or Nf2F/F;Wwc1F/F;Wwc2F/F (Ad-Cre) mouse livers (4 each), and kinase assays were performed (E) and quantified (F). Four biological replicates were shown in F, error bars indicate SEM.


Phenotypes of 1 - week - old livers with HPO1 and / or HPO2 gene deletions . liver / body weight ratio ( B ) of WT , Nf2 - / - , Sav1 - / - , Wwc1 - / - ; Wwc2 - / - , Mst1 - / - ; Mst2Δ / Δ and compound KO mice . Scale bar in A : 1cm . Each data point in B stands for one mouse , data shown are mean + SEM .

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

Phenotypes of 1-week-old livers with HPO1 and/or HPO2 gene deletions. liver/body weight ratio (B) of WT, Nf2-/-, Sav1-/-, Wwc1-/-;Wwc2-/-, Mst1-/-;Mst2Δ/Δ and compound KO mice. Scale bar in A: 1cm. Each data point in B stands for one mouse, data shown are mean + SEM.


C HPO1 and HPO2 inactivation results in rapid iCCA . H & E and CK19 staining for Nf2 - / - ( 8 - month - old ) , Nf2 - / - ; Sav1 - / - ( 1 - week - old ) and Nf2 - / - ; Wwc1 - / - ; Wwc2 + / - ( 1 - week - old ) livers . Nf2 - / - liver displayed bile duct hyperplasia at 8 months , while Nf2 - / - ; Sav1 - / - and Nf2 - / - ; Wwc1 - / - ; Wwc2 + / - livers at 1 week exhibited massive expansion of cholangiocytes with enlarged nuclei , irregular shape , and invasive behaviors . Nf2 - / - ; Sav1 - / - also exhibited cholestasis , as indicated by asterisk . Scale bar : 20 μm ( H & E and IHC ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 HPO1 and HPO2 inactivation results in rapid iCCA. H&E and CK19 staining for Nf2-/- (8-month-old), Nf2-/-;Sav1-/- (1-week-old) and Nf2-/-;Wwc1-/-;Wwc2+/- (1-week-old) livers. Nf2-/- liver displayed bile duct hyperplasia at 8 months, while Nf2-/-;Sav1-/- and Nf2-/-;Wwc1-/-;Wwc2+/- livers at 1 week exhibited massive expansion of cholangiocytes with enlarged nuclei, irregular shape, and invasive behaviors. Nf2-/-;Sav1-/- also exhibited cholestasis, as indicated by asterisk. Scale bar: 20 μm (H&E and IHC).


E Hippo pathway signature score of 1 - week - old livers .

E Hippo pathway signature score of 1-week-old livers.


B Phenotypes of 3 - week - old livers . Gross images , H & E staining and IF images of livers from mice with HPO1 or HPO2 inactivation . Mice were euthanized at 3 weeks old . IF : green for CK19 , red for YAP / TAZ , Ki67 or HNF4α , blue for nuclei . Scale bars : 1 cm ( gross liver image ) , 50 μm ( H & E ) , 50 μm ( IF ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Phenotypes of 3-week-old livers. Gross images, H&E staining and IF images of livers from mice with HPO1 or HPO2 inactivation. Mice were euthanized at 3 weeks old. IF: green for CK19, red for YAP/TAZ, Ki67 or HNF4α, blue for nuclei. Scale bars: 1 cm (gross liver image), 50 μm (H&E), 50 μm (IF).


F Temporal analysis of expression of Hippo pathway components . Mouse liver protein samples were obtained from wild - type mice at different postnatal days ( P1 to P28 ) and probed with antibodies against different Hippo pathway components .

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

F Temporal analysis of expression of Hippo pathway components. Mouse liver protein samples were obtained from wild-type mice at different postnatal days (P1 to P28) and probed with antibodies against different Hippo pathway components.


A Tumor formation in mouse livers with long term HPO1 or HPO2 inactivation . Gross images ( black arrow heads indicate HCC lesions ) , H & E staining and IF images of livers with HPO1 or HPO2 inactivation at indicated ages . IF : green for CK19 , red for YAP / TAZ , Ki67 or HNF4α , blue for nuclei . Scale bars : 1 cm ( gross liver image ) , 50 μm ( H & E ) , 50 μm ( IF ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Tumor formation in mouse livers with long term HPO1 or HPO2 inactivation. Gross images (black arrow heads indicate HCC lesions), H&E staining and IF images of livers with HPO1 or HPO2 inactivation at indicated ages. IF: green for CK19, red for YAP/TAZ, Ki67 or HNF4α, blue for nuclei. Scale bars: 1 cm (gross liver image), 50 μm (H&E), 50 μm (IF).


qRT - PCR detection of the fold induction of IFNB1 mRNA relative to unstimulated condition in different cell types . Cells were stimulated with ecGAMP ( 5 μg / ml ) for 4 h .

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

qRT-PCR detection of the fold induction of IFNB1 mRNA relative to unstimulated condition in different cell types. Cells were stimulated with ecGAMP (5 μg/ml) for 4 h.


qRT - PCR detection of Ifnb1 mRNA abundance in mBMDMs treated with different eCDNs ( 5 μg / ml ) for 4 h .

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

qRT-PCR detection of Ifnb1 mRNA abundance in mBMDMs treated with different eCDNs (5 μg/ml) for 4 h.


ELISA detection of IFNβ release by mBMDMs treated for 4 h or 24 h with extracellular 2 ' 3 ' - cGAMP at indicated concentrations .

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

ELISA detection of IFNβ release by mBMDMs treated for 4 h or 24 h with extracellular 2'3'-cGAMP at indicated concentrations.


ELISA detection of IFNβ release by mBMDMs treated for 4 h or 24 h with extracellular c - di - AMP at indicated concentrations .

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

ELISA detection of IFNβ release by mBMDMs treated for 4 h or 24 h with extracellular c-di-AMP at indicated concentrations.


qRT - PCR detection of IFNB1 mRNA in THP - 1 cells stimulated with indicated eCDNs ( 5 μg / ml ) for 4 h .

qRT-PCR detection of IFNB1 mRNA in THP-1 cells stimulated with indicated eCDNs (5 μg/ml) for 4 h.


ELISA detection of IFNβ in supernatants of THP - 1 cells stimulated with indicated eCDNs at indicated concentrations for 4 h .

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

ELISA detection of IFNβ in supernatants of THP-1 cells stimulated with indicated eCDNs at indicated concentrations for 4 h.


qRT - PCR detection of the fold induction of IFNB1 mRNA relative to unstimulated condition in human CD14 + monocytes derived from PBMC stimulated with indicated eCDNs ( 5 μg / ml ) for 4 h and 8 h . Each symbol represents one individual donor . Data are means + SD averaged from 10 healthy donors .

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

qRT-PCR detection of the fold induction of IFNB1 mRNA relative to unstimulated condition in human CD14+ monocytes derived from PBMC stimulated with indicated eCDNs (5 μg/ml) for 4 h and 8 h. Each symbol represents one individual donor. Data are means+SD averaged from 10 healthy donors.


ELISA detection of IFNβ in supernatants of human CD14 + monocytes derived from PBMC stimulated with indicated eCDNs ( 5 μg / ml ) for 4 h . Each symbol represents result from one individual donor . Data are means + SD averaged from 10 healthy donors .

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

ELISA detection of IFNβ in supernatants of human CD14+ monocytes derived from PBMC stimulated with indicated eCDNs (5 μg/ml) for 4 h. Each symbol represents result from one individual donor. Data are means+SD averaged from 10 healthy donors.


qRT - PCR detection of IFNB1 mRNA abundance in THP - 1 cells treated with ecGAMP and icGAMP at indicated concentrations for 4 h .

qRT-PCR detection of IFNB1 mRNA abundance in THP-1 cells treated with ecGAMP and icGAMP at indicated concentrations for 4 h.


qRT - PCR detection of IFNB1 mRNA abundance in THP - 1 cells treated with ec - di - AMP and ic - di - AMP ( B ) at indicated concentrations for 4 h .

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

qRT-PCR detection of IFNB1 mRNA abundance in THP-1 cells treated with ec-di-AMP and ic-di-AMP (B) at indicated concentrations for 4 h.


ELISA detection of IFNβ release from THP - 1 cells stimulated with ecGAMP and icGAMP ( C ) at indicated concentrations for 4 h .

ELISA detection of IFNβ release from THP-1 cells stimulated with ecGAMP and icGAMP (C) at indicated concentrations for 4 h.


ELISA detection of IFNβ release from THP - 1 cells stimulated with ec - di - AMP and ic - di - AMP ( D ) at indicated concentrations for 4 h .

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

ELISA detection of IFNβ release from THP-1 cells stimulated with ec-di-AMP and ic-di-AMP (D) at indicated concentrations for 4 h.


qRT - PCR detection of Ifnb1 mRNA abundance in mBMDMs treated with ecGAMP and icGAMP ( E ) at indicated concentrations for 4 h .

qRT-PCR detection of Ifnb1 mRNA abundance in mBMDMs treated with ecGAMP and icGAMP (E) at indicated concentrations for 4 h.


qRT - PCR detection of Ifnb1 mRNA abundance in mBMDMs treated with ec - di - AMP and ic - di - AMP ( F ) at indicated concentrations for 4 h .

qRT-PCR detection of Ifnb1 mRNA abundance in mBMDMs treated with ec-di-AMP and ic-di-AMP (F) at indicated concentrations for 4 h.


ELISA detection of IFNβ release from mBMDMs stimulated with ecGAMP and icGAMP at indicated concentrations for 4 h .

ELISA detection of IFNβ release from mBMDMs stimulated with ecGAMP and icGAMP at indicated concentrations for 4 h.


ELISA detection of IFNβ release from mBMDMs stimulated with ec - di - AMP and ic - di - AMP ( H ) at indicated concentrations for 4 h .

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

ELISA detection of IFNβ release from mBMDMs stimulated with ec-di-AMP and ic-di-AMP (H) at indicated concentrations for 4 h.


Frequencies of FITC + THP - 1 cells stimulated with FITC - ecGAMP for 1 h in presence of DMSO or indicated inhibitors including Dynasore ( 10 μM ) , chlorpromazine ( CPZ , 10 μM ) , dimethylamiloride ( DMA , 100 μM ) , polyinosinic acid ( Poly I , 50 μg / ml ) or mannans from Sacharomyces cerevesiae ( Mannans , 1 mg / ml ) .

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

Frequencies of FITC+ THP-1 cells stimulated with FITC-ecGAMP for 1 h in presence of DMSO or indicated inhibitors including Dynasore (10 μM), chlorpromazine (CPZ, 10 μM), dimethylamiloride (DMA, 100 μM), polyinosinic acid (Poly I, 50 μg/ml) or mannans from Sacharomyces cerevesiae (Mannans, 1 mg/ml).


qRT - PCR detection of IFNB1 ( B ) and IL6 ( C ) mRNA in THP - 1 cells stimulated with ecGAMP ( 5 μg / ml ) or intracellular 2 ′ 3 ′ - cGAMP ( icGAMP ) ( 0 . 1 μg / ml ) for 4 h in presence of DMSO or dynasore ( 10 μM ) . qRT - PCR detection of Ifnb1 ( D ) and Il6 ( E ) mRNA in mBMDMs stimulated with ecGAMP ( 5 μg / ml ) or icGAMP ( 0 . 1 μg / ml ) for 4 h in presence of DMSO or dynasore ( 10 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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR 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 B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O

qRT-PCR detection of IFNB1 (B) and IL6 (C) mRNA in THP-1 cells stimulated with ecGAMP (5 μg/ml) or intracellular 2′3′-cGAMP (icGAMP) (0.1 μg/ml) for 4 h in presence of DMSO or dynasore (10 μM). qRT-PCR detection of Ifnb1 (D) and Il6 (E) mRNA in mBMDMs stimulated with ecGAMP (5 μg/ml) or icGAMP (0.1 μg/ml) for 4 h in presence of DMSO or dynasore (10 μM).


Immunostaining for EEA1 ( red ) ( F ) and LAMP2 ( red ) ( G ) in THP - 1 cells stimulated with FITC - ecGAMP ( 5 μg / ml , green ) for 30 min , nucleus in blue ( DAPI ) . Data are representative of 3 independent experiments . Scale bar , 10 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

Immunostaining for EEA1 (red) (F) and LAMP2 (red) (G) in THP-1 cells stimulated with FITC-ecGAMP (5 μg/ml, green) for 30 min, nucleus in blue (DAPI). Data are representative of 3 independent experiments. Scale bar, 10 μm.


qRT - PCR detection of IFNB1 and IL6 mRNA in THP - 1 cells ( H , I ) and mBMDMs ( J , K ) stimulated with ecGAMP ( 5 μg / ml ) and icGAMP ( 0 . 1 μg / ml ) , respectively , for 4 h in presence of DMSO or bafilomycin A1 ( BafA ) ( 1 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 B-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O

qRT-PCR detection of IFNB1 and IL6 mRNA in THP-1 cells (H, I) and mBMDMs (J, K) stimulated with ecGAMP (5 μg/ml) and icGAMP (0.1 μg/ml), respectively, for 4 h in presence of DMSO or bafilomycin A1 (BafA) (1 μM).


qRT - PCR detection of IFNB1 mRNA in scrambled ( Scramble ) or STING shRNA stably transfected ( STING KD ) THP - 1 cells stimulated with ec - di - AMP ( 5 μg / ml ) and ecGAMP ( 5 μg / ml ) or transfected with ISD or poly ( I : C ) .

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

qRT-PCR detection of IFNB1 mRNA in scrambled (Scramble) or STING shRNA stably transfected (STING KD) THP-1 cells stimulated with ec-di-AMP (5 μg/ml) and ecGAMP (5 μg/ml) or transfected with ISD or poly(I:C).


ELISA detection of IFNβ protein in supernatants of Scramble or STING KD THP - 1 cells stimulated with indicated eCDNs ( 5 μg / ml ) or transfected with ISD or poly ( I : C ) .

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

ELISA detection of IFNβ protein in supernatants of Scramble or STING KD THP-1 cells stimulated with indicated eCDNs (5 μg/ml) or transfected with ISD or poly(I:C).


qRT - PCR detection of Ifnb1 mRNA in WT and Sting - / - ( Sting KO ) mBMDMs stimulated with indicated eCDNs ( 5 μg / ml ) or transfected with ISD .

O O O O O O O O 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

qRT-PCR detection of Ifnb1 mRNA in WT and Sting-/- (Sting KO) mBMDMs stimulated with indicated eCDNs (5 μg/ml) or transfected with ISD.


ELISA detection of IFNβ protein secretion in supernatants of mBMDMs stimulated with indicated eCDNs ( 5 μg / ml ) or transfected with ISD or poly ( I : C ) .

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

ELISA detection of IFNβ protein secretion in supernatants of mBMDMs stimulated with indicated eCDNs (5 μg/ml) or transfected with ISD or poly (I:C).


qRT - PCR detection of Ifnb1 mRNA in WT , Sting KO or Sting KO complemented with mouse STING ( Sting KO + mSTING ) RAW264 . 7 cells stimulated with indicated eCDNs ( 5 μg / ml ) or transfected with ISD .

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

qRT-PCR detection of Ifnb1 mRNA in WT, Sting KO or Sting KO complemented with mouse STING (Sting KO+mSTING) RAW264.7 cells stimulated with indicated eCDNs (5 μg/ml) or transfected with ISD.


Western blot detection of indicated proteins in lysates of WT and Sting - / - ( KO ) mBMDMs stimulated with ecGAMP ( 5 μg / ml ) or transfected with ISD . Data are representative of 3 independent experiments .

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

Western blot detection of indicated proteins in lysates of WT and Sting-/- (KO) mBMDMs stimulated with ecGAMP (5 μg/ml) or transfected with ISD. Data are representative of 3 independent experiments.


qRT - PCR detection of IFNB1 mRNA levels in STING stable HEK293T cells ( HA - STING - HEK293T ) stimulated with ecGAMP or icGAMP at 5 μg / ml for indicated times .

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

qRT-PCR detection of IFNB1 mRNA levels in STING stable HEK293T cells (HA-STING-HEK293T) stimulated with ecGAMP or icGAMP at 5 μg/ml for indicated times.


Western blot detection of indicated proteins in HA - STING - HEK293T cells stimulated with increasing amounts of ecGAMP and icGAMP at 5 μg / ml for 24 h . Data are representative of 3 independent experiments .

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

Western blot detection of indicated proteins in HA-STING-HEK293T cells stimulated with increasing amounts of ecGAMP and icGAMP at 5 μg/ml for 24 h. Data are representative of 3 independent experiments.


qRT - PCR detection of the induction of IFNB1 mRNA in HEK293T cells stably transfected with pcDNA3 . 1 - HA ( HA ) , HA - STING and HA - STING + HA - cGAS stimulated with ecGAMP ( 5 μg / ml ) , icGAMP ( 0 . 1 μg / ml ) or transfected with ISD .

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qRT-PCR detection of the induction of IFNB1 mRNA in HEK293T cells stably transfected with pcDNA3.1-HA (HA), HA-STING and HA-STING+HA-cGAS stimulated with ecGAMP (5 μg/ml), icGAMP (0.1 μg/ml) or transfected with ISD.


qRT - PCR detection of IFNB1 mRNA in WT and CGAS KO THP - 1 cells stimulated with indicated eCDNs ( 5 μg / ml ) or transfected with ISD or poly ( I : C ) for 4 h .

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qRT-PCR detection of IFNB1 mRNA in WT and CGAS KO THP-1 cells stimulated with indicated eCDNs (5 μg/ml) or transfected with ISD or poly (I:C) for 4 h.


ELISA detection of IFNβ in supernatants of WT and CGAS KO THP - 1 cells stimulated with indicated eCDNs ( 5 μg / ml ) or transfected with ISD or poly ( I : C ) for 4 h .

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ELISA detection of IFNβ in supernatants of WT and CGAS KO THP-1 cells stimulated with indicated eCDNs (5 μg/ml) or transfected with ISD or poly(I:C) for 4 h.


qRT - PCR detection of Ifnb1 mRNA in mBMDMs from WT and Cgas - / - ( Cgas KO ) mice stimulated with indicated eCDNs ( 5 μg / ml ) or transfected with ISD or poly ( I : C ) for 4 h .

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qRT-PCR detection of Ifnb1 mRNA in mBMDMs from WT and Cgas-/- (Cgas KO) mice stimulated with indicated eCDNs (5 μg/ml) or transfected with ISD or poly(I:C) for 4 h.


ELISA detection of Ifnb1 in supernatants of mBMDMs from WT and Cgas KO mice stimulated with indicated eCDNs ( 5 μg / ml ) or transfected with ISD or poly ( I : C ) for 4 h .

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ELISA detection of Ifnb1 in supernatants of mBMDMs from WT and Cgas KO mice stimulated with indicated eCDNs (5 μg/ml) or transfected with ISD or poly(I:C) for 4 h.


qRT - PCR detection of IFNB1 mRNA in WT and CGAS KO THP - 1 cells stimulated with ecGAMP ( 5 μg / ml ) or icGMAP ( 0 . 1 μg / ml ) or transfected with ISD .

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qRT-PCR detection of IFNB1 mRNA in WT and CGAS KO THP-1 cells stimulated with ecGAMP (5 μg/ml) or icGMAP (0.1 μg/ml) or transfected with ISD.


qRT - PCR detection of Ifnb1 mRNA in mBMDMs from WT and Cgas KO stimulated with ecGAMP ( 5 μg / ml ) or icGMAP ( 0 . 1 μg / ml ) or transfected with ISD .

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qRT-PCR detection of Ifnb1 mRNA in mBMDMs from WT and Cgas KO stimulated with ecGAMP (5 μg/ml) or icGMAP (0.1 μg/ml) or transfected with ISD.


Western blot detection of indicated proteins in lysates of mBMDMs from WT and Cgas KO mice stimulated with ecGAMP ( 5 μg / ml ) , icGAMP ( 0 . 1 μg / ml ) or transfected with ISD . Data are representative of 3 independent experiments .

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Western blot detection of indicated proteins in lysates of mBMDMs from WT and Cgas KO mice stimulated with ecGAMP (5 μg/ml), icGAMP (0.1 μg/ml) or transfected with ISD. Data are representative of 3 independent experiments.


qRT - PCR detection of IFNB1 mRNA of WT , CGAS KO , CGAS KO complemented with cGAS ( CGAS KO + cGAS ) or CGAS KO stably transfected with the empty vector ( CGAS KO + Vector ) THP - 1 cells stimulated with ecGAMP ( 5 μg / ml ) .

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qRT-PCR detection of IFNB1 mRNA of WT, CGAS KO, CGAS KO complemented with cGAS (CGAS KO+cGAS) or CGAS KO stably transfected with the empty vector (CGAS KO+Vector) THP-1 cells stimulated with ecGAMP (5 μg/ml).


ELISA detection of IFNβ in supernatants ( I ) of WT , CGAS KO , CGAS KO complemented with cGAS ( CGAS KO + cGAS ) or CGAS KO stably transfected with the empty vector ( CGAS KO + Vector ) THP - 1 cells stimulated with ecGAMP ( 5 μg / ml ) .

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ELISA detection of IFNβ in supernatants (I) of WT, CGAS KO, CGAS KO complemented with cGAS (CGAS KO+cGAS) or CGAS KO stably transfected with the empty vector (CGAS KO+Vector) THP-1 cells stimulated with ecGAMP (5 μg/ml).


Purified h - cGAS protein was precipitated with Ctrl beads or cGAMP beads and then immunoblotted .

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Purified h-cGAS protein was precipitated with Ctrl beads or cGAMP beads and then immunoblotted.


THP - 1 cells were stimulated with biotin - cGAMP for indicated times and then whole cell lysates ( WCL ) were precipitated with streptavidin beads followed by immunoblotting .

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THP-1 cells were stimulated with biotin-cGAMP for indicated times and then whole cell lysates (WCL) were precipitated with streptavidin beads followed by immunoblotting.


Immunofluorescent staining of cGAS ( red ) in THP - 1 cells treated with FITC - ecGAMP ( 5 μg / ml ) ( green ) or FITC - icGAMP ( 0 . 1 μg / ml ) ( green ) for 4 h , nucleus in blue ( DAPI ) . Scale bar , 10 μm .

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Immunofluorescent staining of cGAS (red) in THP-1 cells treated with FITC-ecGAMP (5 μg/ml) (green) or FITC-icGAMP (0.1 μg/ml) (green) for 4 h, nucleus in blue (DAPI). Scale bar, 10 μm.


Purified h - cGAS protein was precipitated with beads coupled with cGAMP , c - di - GMP or c - di - AMP followed by immunoblotting .

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Purified h-cGAS protein was precipitated with beads coupled with cGAMP, c-di-GMP or c-di-AMP followed by immunoblotting.


Binding curves of surface - immobilized 2 ' 3 ' - cGAMP with His - cGAS at indicated concentrations . Vertical lines mark the start of association and dissociation phases of the binding events . The dashed lines are global fits to a Langmuir reaction model ; global fitting parameters are listed in the table below the plot . ( n = 4 independent experiment ) .

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Binding curves of surface-immobilized 2'3'-cGAMP with His-cGAS at indicated concentrations. Vertical lines mark the start of association and dissociation phases of the binding events. The dashed lines are global fits to a Langmuir reaction model; global fitting parameters are listed in the table below the plot. (n=4 independent experiment).


The small - angle X - ray scattering analysis of the full - length cGAS with cGAMP . The EOM fit of the measured SAXS data . The goodness - of - the fit χ2 = 1 . 1 .

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The small-angle X-ray scattering analysis of the full-length cGAS with cGAMP. The EOM fit of the measured SAXS data. The goodness-of-the fit χ2 = 1.1.


Immunostaining of STING ( anti - STING , red ) in THP - 1 cells stimulated with FITC - ecGAMP ( 5 μg / ml , green ) for 2 h , nucleus in blue ( DAPI ) . Data are representative of 3 independent experiments . Scale bar , 10 μm .

Immunostaining of STING (anti-STING, red) in THP-1 cells stimulated with FITC-ecGAMP (5 μg/ml, green) for 2 h, nucleus in blue (DAPI). Data are representative of 3 independent experiments. Scale bar, 10 μm.


Cellular localization of ecGAMP in WT and CGAS KO THP - 1 cells stimulated with FITC - ecGAMP ( 5 μg / ml , green ) for 2 h , nucleus in blue ( DAPI ) . Data are presentative of 5 independent experiments . Scale bar , 10 μm .

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Cellular localization of ecGAMP in WT and CGAS KO THP-1 cells stimulated with FITC-ecGAMP (5 μg/ml, green) for 2 h, nucleus in blue (DAPI). Data are presentative of 5 independent experiments. Scale bar, 10 μm.


Frequency of perinuclear accumulation of FITC - ecGAMP in WT and CGAS KO THP - 1 cells stimulated with FITC - ecGAMP ( 5 μg / ml ) for 2 h . Data are means + SD averaged from 5 independent experiments and approximately 100 cells were imaged and counted in each experiment . Each symbol represents the percentage of THP - 1 cells with perinuclear cGAMP aggregates in every independent experiment . Mann - Whitney U test was used for statistical analysis . * * p < 0 . 01 .

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Frequency of perinuclear accumulation of FITC-ecGAMP in WT and CGAS KO THP-1 cells stimulated with FITC-ecGAMP (5 μg/ml) for 2 h. Data are means+SD averaged from 5 independent experiments and approximately 100 cells were imaged and counted in each experiment. Each symbol represents the percentage of THP-1 cells with perinuclear cGAMP aggregates in every independent experiment. Mann-Whitney U test was used for statistical analysis. **p<0.01.


Western blot detection of the presence of GST - cGAS and His - STING in the immunoprecipitates of cGAMP agarose . Purified GST - cGAS and His - STING were incubated separately or together with cGAMP agarose . The input and immunoprecipitates were immunoblotted .

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Western blot detection of the presence of GST-cGAS and His-STING in the immunoprecipitates of cGAMP agarose. Purified GST-cGAS and His-STING were incubated separately or together with cGAMP agarose. The input and immunoprecipitates were immunoblotted.


Western blot detection of the presence of His - STING in the immunoprecipitates of Glutathione Sepharose . Purified GST - cGAS and His - STING were incubated together in IP lysis buffer overnight at 4 ° C .

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Western blot detection of the presence of His-STING in the immunoprecipitates of Glutathione Sepharose. Purified GST-cGAS and His-STING were incubated together in IP lysis buffer overnight at 4°C.


Western blot detection of the presence of GST - cGAS and His - STING in the immunoprecipitates of Glutathione Sepharose . Purified GST - cGAS and His - STING were incubated together in the absence or presence of increasing 2 ′ 3 ′ - cGAMP .

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Western blot detection of the presence of GST-cGAS and His-STING in the immunoprecipitates of Glutathione Sepharose. Purified GST-cGAS and His-STING were incubated together in the absence or presence of increasing 2′3′-cGAMP.


Western blot detection of the presence of cGAS and STING in the immunoprecipitates of streptavidin beads . THP - 1 cells were stimulated with biotin - ecGAMP ( 5 μg / ml ) or biotin - icGAMP ( 1 μg / ml ) for indicated time and cell lysates were harvested for IP with streptavidin beads . Data ( A - B , D - G ) are representative of 3 independent experiments .

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Western blot detection of the presence of cGAS and STING in the immunoprecipitates of streptavidin beads. THP-1 cells were stimulated with biotin-ecGAMP (5 μg/ml) or biotin-icGAMP (1 μg/ml) for indicated time and cell lysates were harvested for IP with streptavidin beads. Data (A-B, D-G) are representative of 3 independent experiments.


qRT - PCR detection of Ifnb1 mRNA ( A ) of mBMDMs infected with HSV - 1 at indicated MOI together with stimulation with ecGAMP at indicated concentrations for 4 h . Data are means + SD averaged from 3 independent experiments performed with technical triplicates . Each symbol represents the mean of technical triplicates . Two - way ANOVA followed by Bonferroni ' s post hoc test was used for statistical analysis . * , p < 0 . 05 ; * * p < 0 . 01 ; * * * , p < 0 . 001 .

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qRT-PCR detection of Ifnb1 mRNA (A) of mBMDMs infected with HSV-1 at indicated MOI together with stimulation with ecGAMP at indicated concentrations for 4 h. Data are means+SD averaged from 3 independent experiments performed with technical triplicates. Each symbol represents the mean of technical triplicates. Two-way ANOVA followed by Bonferroni's post hoc test was used for statistical analysis. *, p<0.05; **p<0.01; ***, p<0.001.


ELISA detection of IFNβ in supernatants ( B ) of mBMDMs infected with HSV - 1 at indicated MOI together with stimulation with ecGAMP at indicated concentrations for 4 h . Data are means + SD averaged from 3 independent experiments performed with technical triplicates . Each symbol represents the mean of technical triplicates . Two - way ANOVA followed by Bonferroni ' s post hoc test was used for statistical analysis . * , p < 0 . 05 ; * * p < 0 . 01 ; * * * , p < 0 . 001 .

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ELISA detection of IFNβ in supernatants (B) of mBMDMs infected with HSV-1 at indicated MOI together with stimulation with ecGAMP at indicated concentrations for 4 h. Data are means+SD averaged from 3 independent experiments performed with technical triplicates. Each symbol represents the mean of technical triplicates. Two-way ANOVA followed by Bonferroni's post hoc test was used for statistical analysis. *, p<0.05; **p<0.01; ***, p<0.001.


Western blot detection of indicated proteins in lysates of mBMDMs infected with HSV - 1 at indicated MOI together with stimulation with ecGAMP at indicated concentrations for 4 h . Data are representative of 3 independent experiments .

Western blot detection of indicated proteins in lysates of mBMDMs infected with HSV-1 at indicated MOI together with stimulation with ecGAMP at indicated concentrations for 4 h. Data are representative of 3 independent experiments.


A Peptide dotblot array probed with anti - H3K122succ AB # 2 . Peptides with indicated modifications were spotted on membranes .

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A Peptide dotblot array probed with anti-H3K122succ AB #2. Peptides with indicated modifications were spotted on membranes.


B Immunoblot analysis with anti - H3K122succ AB # 2 on indicated histone octamers . Octamers with H3K122succ are specifically recognized .

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B Immunoblot analysis with anti-H3K122succ AB #2 on indicated histone octamers. Octamers with H3K122succ are specifically recognized.


C Pairwise scatterplots of H3K122succ read counts within peaks from ChIP experiments using all three H3K122succ antibodies ( AB # 1 , AB # 2 and AB # 3 ) .

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C Pairwise scatterplots of H3K122succ read counts within peaks from ChIP experiments using all three H3K122succ antibodies (AB #1, AB #2 and AB #3).


A IGV browser snapshots showing the distribution of reads around the TSS of the actively transcribed PPM1D gene ( chr17 : 60 , 587 , 313 - 60 , 643 , 064 ; top panel ) and of the inactive CD40 gene ( chr20 : 46 , 114 , 180 - 46 , 129 , 721 ; bottom panel ) for the indicated histone modifications , input and RNAseq .

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A IGV browser snapshots showing the distribution of reads around the TSS of the actively transcribed PPM1D gene (chr17:60,587,313-60,643,064; top panel) and of the inactive CD40 gene (chr20:46,114,180-46,129,721; bottom panel) for the indicated histone modifications, input and RNAseq.


F H3K122succ occupancy profile over a meta gene as a function of expression level ( based on RNA - seq quartiles : Q1 - least expressed , Q4 - most expressed ) . All genes were normalized to the same length .

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F H3K122succ occupancy profile over a meta gene as a function of expression level (based on RNA-seq quartiles: Q1 - least expressed, Q4 - most expressed). All genes were normalized to the same length.


G Boxplots visualizing expression values of genes ( reads per kb ; RPK ) with peaks for indicated different combinations of histone PTMs at their TSS . Histone modifications are ( from left to right ) : none ( no PTM , black ) ; only H3K122succ ( green ) ; H3K122succ plus one tail modification ( H3K4me3 , or H3K9ac , or H3K27ac , green and light orange ) ; H3K122succ plus two tail modifications ( green , yellow and light orange ) ; H3K4me3 and H3K9ac and H3K27ac without H3K122succ ( yellow , light orange and dark orange ) ; H3K122succ and all three tail modifications ( green , yellow , light orange , and dark orange ) . The number of genes within the groups is indicated below each boxplot . Boxes indicate the range between the first and third quartile , the central line depicts the median , and the whiskers span the range of the data while extending no further than 1 . 5 times the interquartile range . Each distribution was compared to the " no PTM " distribution .

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G Boxplots visualizing expression values of genes (reads per kb; RPK) with peaks for indicated different combinations of histone PTMs at their TSS. Histone modifications are (from left to right): none (no PTM, black); only H3K122succ (green); H3K122succ plus one tail modification (H3K4me3, or H3K9ac, or H3K27ac, green and light orange); H3K122succ plus two tail modifications (green, yellow and light orange); H3K4me3 and H3K9ac and H3K27ac without H3K122succ (yellow, light orange and dark orange); H3K122succ and all three tail modifications (green, yellow, light orange, and dark orange). The number of genes within the groups is indicated below each boxplot. Boxes indicate the range between the first and third quartile, the central line depicts the median, and the whiskers span the range of the data while extending no further than 1.5 times the interquartile range. Each distribution was compared to the "no PTM" distribution.


A H3K122succ levels ( detected by immunoblot ) , relative to the scramble siRNA control , upon depletion of indicated HAT enzyme ( s ) ( GCN5 , pCAF , p300 and / or CBP ) from MCF7 cells by siRNA . The bar graph shows a representative

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A H3K122succ levels (detected by immunoblot), relative to the scramble siRNA control, upon depletion of indicated HAT enzyme(s) (GCN5, pCAF, p300 and/or CBP) from MCF7 cells by siRNA. The bar graph shows a representative experiment


B In vitro succinyltransferase assay on recombinant histone octamers as substrate . After incubation with p300 and suc - CoA , the H3K122succ levels were assessed by immunoblot . Ponceau staining of membrane is shown as a loading control .

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B In vitro succinyltransferase assay on recombinant histone octamers as substrate. After incubation with p300 and suc-CoA, the H3K122succ levels were assessed by immunoblot. Ponceau staining of membrane is shown as a loading control.