Figure 4Immunofluorescence images ( B ) for RPE1 cells transfected with control or ENKD1 siRNAs , cultured in serum - free medium , and stained with antibodies against CP110 and acetylated α - tubulin and DAPI . quantification of the percentage of cells with one CP110 dot ( C , n = 3 independent experiments ) for RPE1 cells transfected with control or ENKD1 siRNAs To quantify the percentage of cells with one CP110 dot ( C ) , > 120 cells were analyzed for each experiment . ( D , E ) Immunofluorescence images ( D ) and quantification of the percentage of cells with one CEP97 dot ( E , n = 3 independent experiments ) for RPE1 cells transfected with control or ENKD1 siRNAs , cultured in serum - free medium , and stained with antibodies against CEP97 and acetylated α - tubulin and DAPI . To quantify the percentage of cells with one CEP97 dot ( E ) , > 150 cells were analyzed for each experiment . Scale bar , 1 µm . ( F - H ) Immunoblot analysis ( F ) , immunofluorescence images ( G ) , and quantification of the percentage of cells with one CP110 dot ( H , n = 3 independent experiments ) for RPE1 cells transfected with control or ENKD1 siRNAs , cultured in serum - free medium for 120 h , and stained with antibodies against CP110 and acetylated α - tubulin and DAPI . To quantify the percentage of cells with one CP110 dot ( C ) , > 80 cells were analyzed for each experiment . Scale bar , 1 µm .

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

Figure 4Immunofluorescence images (B) for RPE1 cells transfected with control or ENKD1 siRNAs, cultured in serum-free medium, and stained with antibodies against CP110 and acetylated α-tubulin and DAPI.quantification of the percentage of cells with one CP110 dot (C, n = 3 independent experiments) for RPE1 cells transfected with control or ENKD1 siRNAs To quantify the percentage of cells with one CP110 dot (C), >120 cells were analyzed for each experiment.(D, E) Immunofluorescence images (D) and quantification of the percentage of cells with one CEP97 dot (E, n = 3 independent experiments) for RPE1 cells transfected with control or ENKD1 siRNAs, cultured in serum-free medium, and stained with antibodies against CEP97 and acetylated α-tubulin and DAPI. To quantify the percentage of cells with one CEP97 dot (E), >150 cells were analyzed for each experiment. Scale bar, 1 µm.(F-H) Immunoblot analysis (F), immunofluorescence images (G), and quantification of the percentage of cells with one CP110 dot (H, n = 3 independent experiments) for RPE1 cells transfected with control or ENKD1 siRNAs, cultured in serum-free medium for 120 h, and stained with antibodies against CP110 and acetylated α-tubulin and DAPI. To quantify the percentage of cells with one CP110 dot (C), >80 cells were analyzed for each experiment. Scale bar, 1 µm.


Figure 5 ( A - C ) Immunoblot analysis ( A ) , immunofluorescence images ( B ) and quantification of the percentage of cells with one CP110 dot ( C , n = 3 independent experiments ) for RPE1 cells transfected with the indicated siRNAs , cultured in serum - free medium for 48 h , and stained with antibodies against CP110 and acetylated α - tubulin and DAPI . To quantify the percentage of cells with one CP110 dot ( C ) , > 180 cells were analyzed for each experiment . Scale bar , 1 µm . ( D ) Immunoblot analysis of CP110 and β - actin in RPE1 cells transfected with control or CP110 siRNAs and cultured in serum - free medium for 48 h . ( E , F ) Immunofluorescence images ( E ) and quantification of the percentage of ciliated cells ( F , n = 3 independent experiments ) for RPE1 cells transfected with the indicated siRNAs , cultured in a serum - starved condition , and stained with antibodies against γ - tubulin and acetylated α - tubulin and DAPI . To quantify the percentage of ciliated cells ( F ) , > 120 cells were analyzed for each experiment . Scale bar , 10 µm . ( G , H ) Immunoprecipitation and immunoblotting ( G ) and quantification ( H , n = 3 mice ) showing the CP110 - CEP97 interaction in the retinal tissues of wild - type and Enkd1 knockout mice . The intensity of each CEP97 band was normalized to that of the β - actin band . ( I , J ) Immunoprecipitation and immunoblotting ( I ) and quantification ( J , n = 3 independent experiments ) showing the CP110 - CEP97 interaction in serum - starved RPE1 cells transfected with control or ENKD1 siRNAs . The intensity of each CEP97 band was normalized to that of the β - actin band .

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

Figure 5(A-C) Immunoblot analysis (A), immunofluorescence images (B) and quantification of the percentage of cells with one CP110 dot (C, n = 3 independent experiments) for RPE1 cells transfected with the indicated siRNAs, cultured in serum-free medium for 48 h, and stained with antibodies against CP110 and acetylated α-tubulin and DAPI. To quantify the percentage of cells with one CP110 dot (C), >180 cells were analyzed for each experiment. Scale bar, 1 µm.(D) Immunoblot analysis of CP110 and β-actin in RPE1 cells transfected with control or CP110 siRNAs and cultured in serum-free medium for 48 h.(E, F) Immunofluorescence images (E) and quantification of the percentage of ciliated cells (F, n = 3 independent experiments) for RPE1 cells transfected with the indicated siRNAs, cultured in a serum-starved condition, and stained with antibodies against γ-tubulin and acetylated α-tubulin and DAPI. To quantify the percentage of ciliated cells (F), >120 cells were analyzed for each experiment. Scale bar, 10 µm.(G, H) Immunoprecipitation and immunoblotting (G) and quantification (H, n = 3 mice) showing the CP110-CEP97 interaction in the retinal tissues of wild-type and Enkd1 knockout mice. The intensity of each CEP97 band was normalized to that of the β-actin band.(I, J) Immunoprecipitation and immunoblotting (I) and quantification (J, n = 3 independent experiments) showing the CP110-CEP97 interaction in serum-starved RPE1 cells transfected with control or ENKD1 siRNAs. The intensity of each CEP97 band was normalized to that of the β-actin band.


Figure 6 ( A ) Immunoprecipitation and immunoblotting showing the interaction between endogenous ENKD1 and CP110 in mouse retinal tissues . Immunoprecipitation was performed with the anti - ENKD1 ( upper panel ) or anti - CP110 ( lower panel ) antibody or the corresponding control IgG . ( B - D ) Immunoprecipitation and immunoblotting showing the interaction of GFP - ENKD1 with Flag - CP110 ( B and upper panel of D ) or HA - CP110 ( C and bottom panel of D ) in HEK293T cells . Immunoprecipitation was performed with antibodies against GFP ( B and C ) , Flag ( upper panel of D ) , or HA ( bottom panel of D ) . ( E ) Identification of the domains of ENKD1 mediating its interaction with CP110 . ( F ) Identification of the domains of CP110 mediating its interaction with ENKD1 . ( H ) GST pull - down showing the interaction of purified GST - CP110 - N with purified His - ENKD1 - M . ( I , J ) Immunoprecipitation and immunoblotting ( I ) and quantification ( J , n = 3 independent experiments ) showing the interaction of HA - CP110 with Flag - CEP97 in HEK293T cells transfected with the indicated plasmids . The intensity of each Flag - CEP97 band was normalized to that of the β - actin band .

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

Figure 6(A) Immunoprecipitation and immunoblotting showing the interaction between endogenous ENKD1 and CP110 in mouse retinal tissues. Immunoprecipitation was performed with the anti-ENKD1 (upper panel) or anti-CP110 (lower panel) antibody or the corresponding control IgG.(B-D) Immunoprecipitation and immunoblotting showing the interaction of GFP-ENKD1 with Flag-CP110 (B and upper panel of D) or HA-CP110 (C and bottom panel of D) in HEK293T cells. Immunoprecipitation was performed with antibodies against GFP (B and C), Flag (upper panel of D), or HA (bottom panel of D).(E) Identification of the domains of ENKD1 mediating its interaction with CP110.(F) Identification of the domains of CP110 mediating its interaction with ENKD1.(H) GST pull-down showing the interaction of purified GST-CP110-N with purified His-ENKD1-M.(I, J) Immunoprecipitation and immunoblotting (I) and quantification (J, n = 3 independent experiments) showing the interaction of HA-CP110 with Flag-CEP97 in HEK293T cells transfected with the indicated plasmids. The intensity of each Flag-CEP97 band was normalized to that of the β-actin band.


Figure 1A - D Mesenteric blood ( PECAM1 , red ) and lymphatic vessels ( LYVE1 , green ; PROX1 , gray ) . Asterisks indicate lymphatic valves , arrow indicates a lymphatic vessel stub , and arrowhead indicates an isolated lymphatic vessel fragmentE - H Blood vessels ( PECAM1 , green ) and lymphatic vessels ( VEGFR - 3 , red ) in the small intestinal wall . I , J Detection of chylous ascites ( arrows ) in the VCiΔR26 mice at P6 . K , L LYVE1 staining of intestinal lymphatic vessels at P6 . M - T LYVE1 staining of lymphatic vessels in the intestinal wall in adult mice . Genotypes and deletion lengths are indicated in ( U ) . U Quantification of LYVE1 areas in ( M - T ) . Length of the i ∆ Vegfc gene deletion is indicated in months ( mo ) , and Vegfd indicates the VEGF - D genotype . Data are represented as mean ± SEM . Significant differences were determined using one - way ANOVA and Bonferroni post hoc analysis compared to WT intestine represented in ( M ) . * P = 0 . 003 , * * P = 0 . 001 , * * * P = 0 . 0008 , ♯ P = 0 . 0002 , § P = 0 . 0001 .

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

Figure 1A-D Mesenteric blood (PECAM1, red) and lymphatic vessels (LYVE1, green; PROX1, gray). Asterisks indicate lymphatic valves, arrow indicates a lymphatic vessel stub, and arrowhead indicates an isolated lymphatic vessel fragmentE-H Blood vessels (PECAM1, green) and lymphatic vessels (VEGFR-3, red) in the small intestinal wall.I, J Detection of chylous ascites (arrows) in the VCiΔR26 mice at P6.K, L LYVE1 staining of intestinal lymphatic vessels at P6.M-T LYVE1 staining of lymphatic vessels in the intestinal wall in adult mice. Genotypes and deletion lengths are indicated in (U).U Quantification of LYVE1 areas in (M-T). Length of the i∆Vegfc gene deletion is indicated in months (mo), and Vegfd indicates the VEGF-D genotype. Data are represented as mean ± SEM. Significant differences were determined using one-way ANOVA and Bonferroni post hoc analysis compared to WT intestine represented in (M). *P = 0.003, **P = 0.001, ***P = 0.0008, ♯P = 0.0002, §P = 0.0001.


Figure 2Overview of the small intestine cross section stained with nuclear red and highlighting the location of higher magnification images in ( B - F ) ; ( I ) for the entire villus and ( II ) for the villus base . β - Gal staining pattern of the villus in wild - type ( Ctrl ) , Vegfc / LacZ ( VC ) , Vegfr3 / LacZ ( VR - 3 ) , and Vegfr2 / LacZ ( VR - 2 ) mice . Higher magnification images representing β - Gal staining of the villus base in Vegfc / LacZmice . Data information : Arrows indicate the VEGF - C expression in arterialSMC , arrowheads indicate the VEGF - C expression in SMCfibers in the villus , and asterisks highlight the VEGF - C expression in circular smooth muscle cell layer of the intestinal wall . Scale bars : 50 μm , except ( C ) inset 25 μm . Vegfc / LacZβ - Gal staining reaction with smooth muscle actin ( SMA ) peroxidase staining . Data information : Arrows indicate the VEGF - C expression in arterialSMC , arrowheads indicate the VEGF - C expression in SMCfibers in the villus , and asterisks highlight the VEGF - C expression in circular smooth muscle cell layer of the intestinal wall . Scale bars : 50 μm , except ( C ) inset 25 μm . Vegfc / LacZβ - Gal staining and LYVE1 peroxidase staining . Data information : Arrows indicate the VEGF - C expression in arterialSMC , arrowheads indicate the VEGF - C expression in SMCfibers in the villus , and asterisks highlight the VEGF - C expression in circular smooth muscle cell layer of the intestinal wall . Scale bars : 50 μm , except ( C ) inset 25 μm . Surface image of Vegfc / LacZβ - Gal - stained intestine ( left ) and cross section counterstaining with PECAM1 ( right ) . Data information : Arrows indicate the VEGF - C expression in arterialSMC , arrowheads indicate the VEGF - C expression in SMCfibers in the villus , and asterisks highlight the VEGF - C expression in circular smooth muscle cell layer of the intestinal wall . Scale bars : 50 μm , except ( C ) inset 25 μm . Immunofluorescence staining of lacteal lymphatic vessel ( LYVE1 ) , blood capillaries ( PECAM1 ) , and smooth muscle cells ( SMA ) . Data information : Arrows indicate the VEGF - C expression in arterialSMC , arrowheads indicate the VEGF - C expression in SMCfibers in the villus , and asterisks highlight the VEGF - C expression in circular smooth muscle cell layer of the intestinal wall . Scale bars : 50 μm , except ( C ) inset 25 μm .

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

Figure 2Overview of the small intestine cross section stained with nuclear red and highlighting the location of higher magnification images in (B-F); (I) for the entire villus and (II) for the villus base. β-Gal staining pattern of the villus in wild-type (Ctrl), Vegfc/LacZ (VC), Vegfr3/LacZ (VR-3), and Vegfr2/LacZ (VR-2) mice.Higher magnification images representing β-Gal staining of the villus base in Vegfc/LacZmice.Data information: Arrows indicate the VEGF-C expression in arterialSMC, arrowheads indicate the VEGF-C expression in SMCfibers in the villus, and asterisks highlight the VEGF-C expression in circular smooth muscle cell layer of the intestinal wall. Scale bars: 50 μm, except (C) inset 25 μm.Vegfc/LacZβ-Gal staining reaction with smooth muscle actin (SMA) peroxidase staining.Data information: Arrows indicate the VEGF-C expression in arterialSMC, arrowheads indicate the VEGF-C expression in SMCfibers in the villus, and asterisks highlight the VEGF-C expression in circular smooth muscle cell layer of the intestinal wall. Scale bars: 50 μm, except (C) inset 25 μm.Vegfc/LacZβ-Gal staining and LYVE1 peroxidase staining.Data information: Arrows indicate the VEGF-C expression in arterialSMC, arrowheads indicate the VEGF-C expression in SMCfibers in the villus, and asterisks highlight the VEGF-C expression in circular smooth muscle cell layer of the intestinal wall. Scale bars: 50 μm, except (C) inset 25 μm.Surface image of Vegfc/LacZβ-Gal-stained intestine (left) and cross section counterstaining with PECAM1 (right).Data information: Arrows indicate the VEGF-C expression in arterialSMC, arrowheads indicate the VEGF-C expression in SMCfibers in the villus, and asterisks highlight the VEGF-C expression in circular smooth muscle cell layer of the intestinal wall. Scale bars: 50 μm, except (C) inset 25 μm.Immunofluorescence staining of lacteal lymphatic vessel (LYVE1), blood capillaries (PECAM1), and smooth muscle cells (SMA).Data information: Arrows indicate the VEGF-C expression in arterialSMC, arrowheads indicate the VEGF-C expression in SMCfibers in the villus, and asterisks highlight the VEGF-C expression in circular smooth muscle cell layer of the intestinal wall. Scale bars: 50 μm, except (C) inset 25 μm.


Figure 3Two - month - old mice received tamoxifen and were fed on high - fat diet ( HFD ) for seven weeks before analysis . Body weight change during seven weeks of HFD , expressed as average fold change in comparison with the starting weight . n = 16 , WT ; n = 6 , Vegfd − / − ; n = 16 , VCiΔR26 ; n = 5 , Vegfd − / − ; VCiΔR26 . Body weight comparisons at seven weeks of HFD . Significant differences were determined using one - way ANOVA and Bonferroni post hoc analysis compared to WT . * P = 0 . 004 ; * * P = 0 . 003 . n = 16 , WT ; n = 6 , Vegfd − / − ; n = 16 , VCiΔR26 ; n = 5 , Vegfd − / − ; VCiΔR26 . Two - month - old mice received tamoxifen and were fed on high - fat diet ( HFD ) for seven weeks before analysis . Glucose tolerance test ( GTT ) after six weeks of HFD . Significant differences were determined using unpaired two - tailed t - test . * P = 0 . 014 ; * * P = 0 . 041 . n = 5 , WT ; n = 6 , VCiΔR26 . Two - month - old mice received tamoxifen and were fed on high - fat diet ( HFD ) for seven weeks before analysis . Total fatweight , fat percentage from body composition measurements after six weeks of HFD , and weights of visceral fat ( VF ) and subcutaneous fat ( SF ) at the time of necropsy . Significant differences were determined using unpaired two - tailed t - test . * P = 0 . 006 ; * * P = 0 . 001 ; # P = 0 . 008 ; § P = 0 . 006 . n = 4 in each group . Two - month - old mice received tamoxifen and were fed on high - fat diet ( HFD ) for seven weeks before analysis . Food consumption during the fifth week of HFD . n = 9 , WT ; n = 10 , VCiΔR26 . Two - month - old mice received tamoxifen and were fed on high - fat diet ( HFD ) for seven weeks before analysis . Whole - mount immunofluorescence staining of blood ( PECAM1 , red ) and lymphatic vessels ( LYVE1 , green ) in intestinal villi and intestinal wall . Quantification of the lacteal and villus length ( solid and striped color bars , respectively ) and the intestinal wallLYVE1 + area percentage from images represented in ( F ) . Significant differences were determined using unpaired two - tailed t - test . * P = 0 . 0002 ; * * P = 0 . 00007 . n = 5 , WT ; n = 6 , VCiΔR26 . Two - month - old mice received tamoxifen and were fed on high - fat diet ( HFD ) for seven weeks before analysis . Free fatty acid ( FFA ) and cholesterol measurements from the feces after six weeks of HFD . Significant differences were determined using unpaired two - tailed t - test . * P = 0 . 001 ; * * P = 0 . 007 . n = 5 , WT ; n = 6 , VCiΔR26 .

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

Figure 3Two-month-old mice received tamoxifen and were fed on high-fat diet (HFD) for seven weeks before analysis.Body weight change during seven weeks of HFD, expressed as average fold change in comparison with the starting weight. n = 16, WT; n = 6, Vegfd−/−; n = 16, VCiΔR26; n = 5, Vegfd−/−; VCiΔR26.Body weight comparisons at seven weeks of HFD. Significant differences were determined using one-way ANOVA and Bonferroni post hoc analysis compared to WT. *P = 0.004; **P = 0.003. n = 16, WT; n = 6, Vegfd−/−; n = 16, VCiΔR26; n = 5, Vegfd−/−; VCiΔR26.Two-month-old mice received tamoxifen and were fed on high-fat diet (HFD) for seven weeks before analysis. Glucose tolerance test (GTT) after six weeks of HFD. Significant differences were determined using unpaired two-tailed t-test. *P = 0.014; **P = 0.041. n = 5, WT; n = 6, VCiΔR26.Two-month-old mice received tamoxifen and were fed on high-fat diet (HFD) for seven weeks before analysis. Total fatweight, fat percentage from body composition measurements after six weeks of HFD, and weights of visceral fat (VF) and subcutaneous fat (SF) at the time of necropsy. Significant differences were determined using unpaired two-tailed t-test. *P = 0.006; **P = 0.001; #P = 0.008; §P = 0.006. n = 4 in each group.Two-month-old mice received tamoxifen and were fed on high-fat diet (HFD) for seven weeks before analysis. Food consumption during the fifth week of HFD. n = 9, WT; n = 10, VCiΔR26.Two-month-old mice received tamoxifen and were fed on high-fat diet (HFD) for seven weeks before analysis.Whole-mount immunofluorescence staining of blood (PECAM1, red) and lymphatic vessels (LYVE1, green) in intestinal villi and intestinal wall.Quantification of the lacteal and villus length (solid and striped color bars, respectively) and the intestinal wallLYVE1+ area percentage from images represented in (F). Significant differences were determined using unpaired two-tailed t-test. *P = 0.0002; **P = 0.00007. n = 5, WT; n = 6, VCiΔR26.Two-month-old mice received tamoxifen and were fed on high-fat diet (HFD) for seven weeks before analysis. Free fatty acid (FFA) and cholesterol measurements from the feces after six weeks of HFD. Significant differences were determined using unpaired two-tailed t-test. *P = 0.001; **P = 0.007. n = 5, WT; n = 6, VCiΔR26.


Figure 1A . Relative antiviral activity of IFNα ( circles ) or IFNλ ( triangles ) . AEC cultures were stimulated for 4hrs with stated IFN at specified concentrations ( ng / ml ) and induction of indicated ISGs was assessed by qPCR ( data shown is representative of four independent experiments , n = 3 - 4 ) . B , C . Mice were pretreated with equivalent doses of IFNα ( 1 . 45μg / 50μl ) or IFNλ ( 2 . 6μg / 50μl ) or Veh Ctrl ( squares , 50μl PBS ) 24hrs prior to infection with PR8 ; weight loss and survival was assessed throughout infection ( B ) , and viral load ( C ) assessed at 4dpi ( data shown is representative of two independent experiments , n = 8 - 10 ( B ) , n = 3 ( C ) ) . D , E . Mice were infected with PR8 and treated with equivalent doses of IFNα or IFNλ or Veh Ctrl at days 2 , 4 and 5 post infection ; survival and weight loss was monitored ( D , data pooled from 4 independent experiments , n = 12 - 29 ) ( data representative of two independent experiments , n = 3 - 5 ) . D , E . Mice were infected with PR8 and treated with equivalent doses of IFNα or IFNλ or Veh Ctrl at days 2 , 4 and 5 post infection ; viral load assessed at 4dpi ( E ) ( data representative of two independent experiments , n = 3 - 5 ) .

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Figure 1A. Relative antiviral activity of IFNα (circles) or IFNλ (triangles). AEC cultures were stimulated for 4hrs with stated IFN at specified concentrations (ng/ml) and induction of indicated ISGs was assessed by qPCR (data shown is representative of four independent experiments, n=3-4).B, C. Mice were pretreated with equivalent doses of IFNα (1.45μg/50μl) or IFNλ (2.6μg/50μl) or Veh Ctrl (squares, 50μl PBS) 24hrs prior to infection with PR8; weight loss and survival was assessed throughout infection (B), and viral load (C) assessed at 4dpi (data shown is representative of two independent experiments, n=8-10 (B), n=3 (C)).D, E. Mice were infected with PR8 and treated with equivalent doses of IFNα or IFNλ or Veh Ctrl at days 2, 4 and 5 post infection; survival and weight loss was monitored (D, data pooled from 4 independent experiments, n=12-29) (data representative of two independent experiments, n=3-5).D, E. Mice were infected with PR8 and treated with equivalent doses of IFNα or IFNλ or Veh Ctrl at days 2, 4 and 5 post infection; viral load assessed at 4dpi (E) (data representative of two independent experiments, n=3-5).


Figure 2A , B . Mice were infected with PR8 and treated with IFNα ( circles , 1 . 45μg / 50μl ) , IFNλ ( triangles , 2 . 6μg / 50μl ) or Veh Ctrl ( squares ) as previously stated . Concentrations of stated proinflammatory cytokines in BAL fluid was measured by multiplex cytokine assay ( A ) ( data shown is representative of two independent experiments , n = 2 - 6 ) . A , B . Mice were infected with PR8 and treated with IFNα ( circles , 1 . 45μg / 50μl ) , IFNλ ( triangles , 2 . 6μg / 50μl ) or Veh Ctrl ( squares ) as previously stated . Concentrations of stated proinflammatory cytokines in BAL fluid was measured by flow cytometry quantification of pDCs and Inflammatory Monocytes in the lung was performed ( B ) ( data shown is representative of two independent experiments , n = 2 - 6 ) . C , D . Lung sections from control and infected mice treated as indicated , were stained by TUNEL for apoptotic cells at 6dpi . Quantification of TUNEL + cells in whole lung slides by Icy Spot Detector ( ICY - R3M2Y2 ) ( C ) ( data shown is pooled from three independent experiments , n = 3 - 8 ) . Red arrowheads indicate TUNEL signal ( D ) . Scale bar , 200 μM ( data shown is representative of two independent experiments , n = 3 - 4 ) .

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Figure 2A, B. Mice were infected with PR8 and treated with IFNα (circles, 1.45μg/50μl), IFNλ (triangles, 2.6μg/50μl) or Veh Ctrl (squares) as previously stated. Concentrations of stated proinflammatory cytokines in BAL fluid was measured by multiplex cytokine assay (A) (data shown is representative of two independent experiments, n=2-6).A, B. Mice were infected with PR8 and treated with IFNα (circles, 1.45μg/50μl), IFNλ (triangles, 2.6μg/50μl) or Veh Ctrl (squares) as previously stated. Concentrations of stated proinflammatory cytokines in BAL fluid was measured by flow cytometry quantification of pDCs and Inflammatory Monocytes in the lung was performed (B) (data shown is representative of two independent experiments, n=2-6).C, D. Lung sections from control and infected mice treated as indicated, were stained by TUNEL for apoptotic cells at 6dpi. Quantification of TUNEL+ cells in whole lung slides by Icy Spot Detector (ICY-R3M2Y2) (C) (data shown is pooled from three independent experiments, n=3-8). Red arrowheads indicate TUNEL signal (D). Scale bar, 200 μM (data shown is representative of two independent experiments, n=3-4).


Figure 3A , B . IL - 6 , IP - 10 and MCP - 1 concentrations were measured by multiplex cytokine assay in AEC culture supernatants ( A ) at 24hrs post stimulation with IFNα4 ( 0 . 725ng / ml ) or IFNλ2 ( 1 . 3ng / ml ) or LPS ( AEC only ) ( data shown is representative of two independent experiments , n = 3 - 6 ) . A , B . IL - 6 , IP - 10 and MCP - 1 concentrations were measured by multiplex cytokine assay in Macrophage , pDC and cDC culture supernatants ( B ) at 24hrs post stimulation with IFNα4 ( 0 . 725ng / ml ) or IFNλ2 ( 1 . 3ng / ml ) ( data shown is representative of two independent experiments , n = 3 - 6 ) . C . BAL samples taken from mice treated with IFNα , IFNλ or Veh Ctrl at specified time points ( data shown is representative of two independent experiments , n = 5 - 6 ) .

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Figure 3A, B. IL-6, IP-10 and MCP-1 concentrations were measured by multiplex cytokine assay in AEC culture supernatants (A) at 24hrs post stimulation with IFNα4 (0.725ng/ml) or IFNλ2 (1.3ng/ml) or LPS (AEC only) (data shown is representative of two independent experiments, n=3-6).A, B. IL-6, IP-10 and MCP-1 concentrations were measured by multiplex cytokine assay in Macrophage, pDC and cDC culture supernatants (B) at 24hrs post stimulation with IFNα4 (0.725ng/ml) or IFNλ2 (1.3ng/ml) (data shown is representative of two independent experiments, n=3-6).C. BAL samples taken from mice treated with IFNα, IFNλ or Veh Ctrl at specified time points (data shown is representative of two independent experiments, n=5-6).


Figure 4Mice were treated with IFNα ( 1 . 45μg / 50μl ) , IFNλ ( 2 . 6μg / 50μl ) or Veh Ctrl ( 50μl PBS ) , and whole lungs were taken at 18hrs post treatment for global analysis by Illumina . SingleColor . Mouse WG - 6 _ V2 _ 0 _ R0 _ 1127 microarrays . Samples ( n = 5 ) were normalized to the median of the vehicle control group and filtered for a fold change of 1 . 5 , yielding 553 genes differently regulated between treatments ( One way ANOVA , P < 0 . 01 , Benjamini - Hochberg multiple test correction ) , of which 429 genes are upregulated . K means clustering revealed six gene clusters , one of which encompassed genes primarily induced by IFNα4 ( A ) , while the remaining clusters contained genes upregulated by both IFNα4 and IFNλ2 ( B ) . The two clusters of genes were analysed by Ingenuity Pathway Analysis ( IPA ) ( C , D ) .

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Figure 4Mice were treated with IFNα (1.45μg/50μl), IFNλ (2.6μg/50μl) or Veh Ctrl (50μl PBS), and whole lungs were taken at 18hrs post treatment for global analysis by Illumina.SingleColor.Mouse WG-6_V2_0_R0_1127 microarrays. Samples (n=5) were normalized to the median of the vehicle control group and filtered for a fold change of 1.5, yielding 553 genes differently regulated between treatments (One way ANOVA, P<0.01, Benjamini-Hochberg multiple test correction), of which 429 genes are upregulated. K means clustering revealed six gene clusters, one of which encompassed genes primarily induced by IFNα4 (A), while the remaining clusters contained genes upregulated by both IFNα4 and IFNλ2 (B). The two clusters of genes were analysed by Ingenuity Pathway Analysis (IPA) (C,D).


Figure 5A . HumanAEC cultures were stimulated for 4hrs with IFNα ( circles ) or IFNλ ( triangles ) at specified concentrations then assessed for stated ISG induction by qPCR ( data is representative of 2 independent experiments , n = 3 ) . B , C . ISG induction in humanPBMCs was assessed at 4 and 24hrs post IFNα ( 21 U / ml ) or IFNλ ( 1 . 2 ng / ml ) stimulation ( B ) ( data shown is pooled from 6 independent donors ) . B , C . PBMC proinflammatory cytokine secretion was measured by multiplex cytokine assay at 4 and 24hrs post stimulation with IFNα or IFNλ ( C ) ( data shown is pooled from 6 independent donors ) .

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Figure 5A. HumanAEC cultures were stimulated for 4hrs with IFNα (circles) or IFNλ (triangles) at specified concentrations then assessed for stated ISG induction by qPCR (data is representative of 2 independent experiments, n=3).B, C. ISG induction in humanPBMCs was assessed at 4 and 24hrs post IFNα (21 U/ml) or IFNλ (1.2 ng/ml) stimulation (B) (data shown is pooled from 6 independent donors).B, C. PBMC proinflammatory cytokine secretion was measured by multiplex cytokine assay at 4 and 24hrs post stimulation with IFNα or IFNλ (C) (data shown is pooled from 6 independent donors).


Figure 1B Box plots of the Em / Ad expression ratios of GATA6 ( left ) and NKX2 - 1 ( right ) in FFPE lung tissue sections from controls ( Ctrl , n = 61 ) or lung cancer ( LC , n = 51 ) patients . Isoform - specific expression of the indicated genes was analyzed by qRT - PCR after total RNA isolation from tissue samples . Each point represents one sample . C Box plots of Em / Ad of GATA6 ( top ) or NKX2 - 1 ( bottom ) show that high Em / Ad ratios in LC samples are maintained among ethnic groups ( left ) and gender ( right ) . GER , samples collected in Germany ; MEX , samples collected in Mexico . D Box plots of Em / Ad of GATA6 ( left ) or NKX2 - 1 ( right ) in FFPE lung tissue sections from controls or LC patients . Samples were staged according to the TNM Classification ( UICC , 7th edition ) .

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Figure 1B Box plots of the Em/Ad expression ratios of GATA6 (left) and NKX2-1 (right) in FFPE lung tissue sections from controls (Ctrl, n=61) or lung cancer (LC, n=51) patients. Isoform-specific expression of the indicated genes was analyzed by qRT-PCR after total RNA isolation from tissue samples. Each point represents one sample.C Box plots of Em/Ad of GATA6 (top) or NKX2-1 (bottom) show that high Em/Ad ratios in LC samples are maintained among ethnic groups (left) and gender (right). GER, samples collected in Germany; MEX, samples collected in Mexico.D Box plots of Em/Ad of GATA6 (left) or NKX2-1 (right) in FFPE lung tissue sections from controls or LC patients. Samples were staged according to the TNM Classification (UICC, 7th edition).


Figure 2A Box plots of the Em / Ad expression ratios of GATA6 ( left ) and NKX2 - 1 ( right ) in EBCs from controls ( Ctrl , n = 65 ) or lung cancer ( LC , n = 48 ) patients ( training set ) . Each point represents one sample .

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Figure 2A Box plots of the Em/Ad expression ratios of GATA6 (left) and NKX2-1 (right) in EBCs from controls (Ctrl, n=65) or lung cancer (LC, n=48) patients (training set). Each point represents one sample.


Figure 2 ( B ) Volcano plots showing differentially expressed genes between untreated and DLL4 & PDGFBB - treated mMuSCs , human myoblasts and hiMPs . Red dots represent genes which display a positive fold - change in expression upon treatment with DLL4 & PDGF - BB whilst violet dots represent genes which are significantly downregulated . Differentially expressed genes required a P value of ≤ 0 . 05 to be considered significant . ( C ) Heatmaps showing changes in expression of key myogenic ( MYOD , MYOGENIN ) , perivascular ( PDGFRB , NG2 , CD146 , ALPL ) and NOTCH target ( HEY1 , HES1 ) genes upon treatment with DLL4 & PDGF - BB in mMuSC - derived myoblasts ( left ) , human myoblasts ( middle ) and hiMPs ( right ) . Clustering was performed by genes / probes with Pearson correlation . Colour scale based on z - scores : red regions indicate high expression whilst blue regions indicate low expression . Dendrograms indicate the similarity of clusters as well as the orders in which clusters were assembled .

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Figure 2(B) Volcano plots showing differentially expressed genes between untreated and DLL4 & PDGFBB-treated mMuSCs, human myoblasts and hiMPs. Red dots represent genes which display a positive fold-change in expression upon treatment with DLL4 & PDGF-BB whilst violet dots represent genes which are significantly downregulated. Differentially expressed genes required a P value of ≤ 0.05 to be considered significant.(C) Heatmaps showing changes in expression of key myogenic (MYOD, MYOGENIN), perivascular (PDGFRB, NG2, CD146, ALPL) and NOTCH target (HEY1, HES1) genes upon treatment with DLL4 & PDGF-BB in mMuSC-derived myoblasts (left), human myoblasts (middle) and hiMPs (right). Clustering was performed by genes/probes with Pearson correlation. Colour scale based on z-scores: red regions indicate high expression whilst blue regions indicate low expression. Dendrograms indicate the similarity of clusters as well as the orders in which clusters were assembled.


Figure 3 ( B ) Phase contrast images displaying morphology of untreated and treated hiMPs . Scale bar : 25 μm . ( C ) Scatter dot plot showing morphometric analysis of treated and untreated hiMPs . Morphology was quantified as circularity ratio , where 1 = perfect circle and 0 = line ( experimental replicates = 3 ) . Statistical analysis ( paired t test ) was performed on the first quartile to enhance detection of morphological changes ( error bars : SD ) . ( E ) Immunofluorescence images of untreated and treated hiMPs incubated with EdU for 2 hours . Scale bar : 75 μm . ( F ) Bar graphs quantifying EdU experiment shown in ( E ) ( experimental replicates = 3 ; error bars : SEM ) . Statistical analysis based on an unpaired t - test . ( H ) Immunofluorescence images of hiMPs expanded in control or treated conditions for 1 week , induced to differentiate for 4 days in the presence or absence of γ - secretase inhibitor L685458 and immunostained for myosin heavy chain ( MyHC ) . ( I ) Bar graph quantifying the average percentage of nuclei within MyHC positive myotubes ( experimental replicates = 3 ; error bars : SEM ) . Statistical significance based on one - way ANOVA with Tukey ' s multiple comparison . ( J ) Immunofluorescence panels showing human specific LAMIN A / C + ( nuclei ) and SPECTRIN ( sarcolemma ) staining in tibialis anterior muscles of immunodeficient mice ( N = 3 ) transplanted with treated ( n = 3 muscles ) and untreated ( n = 3 muscles ) N5 hiMPs . ( K ) Quantification of LAMIN A / C + grafted human cells across each muscle . Data information : full gene list for heatmaps in ( A ) and ( D ) available in Appendix Table S4 .

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Figure 3(B) Phase contrast images displaying morphology of untreated and treated hiMPs. Scale bar: 25 μm. (C) Scatter dot plot showing morphometric analysis of treated and untreated hiMPs. Morphology was quantified as circularity ratio, where 1 = perfect circle and 0 = line (experimental replicates = 3). Statistical analysis (paired t test) was performed on the first quartile to enhance detection of morphological changes (error bars: SD).(E) Immunofluorescence images of untreated and treated hiMPs incubated with EdU for 2 hours. Scale bar: 75 μm. (F) Bar graphs quantifying EdU experiment shown in (E) (experimental replicates = 3; error bars: SEM). Statistical analysis based on an unpaired t-test.(H) Immunofluorescence images of hiMPs expanded in control or treated conditions for 1 week, induced to differentiate for 4 days in the presence or absence of γ-secretase inhibitor L685458 and immunostained for myosin heavy chain (MyHC). (I) Bar graph quantifying the average percentage of nuclei within MyHC positive myotubes (experimental replicates = 3; error bars: SEM). Statistical significance based on one-way ANOVA with Tukey's multiple comparison.(J) Immunofluorescence panels showing human specific LAMIN A/C+ (nuclei) and SPECTRIN (sarcolemma) staining in tibialis anterior muscles of immunodeficient mice (N = 3) transplanted with treated (n = 3 muscles) and untreated (n = 3 muscles) N5 hiMPs. (K) Quantification of LAMIN A/C+ grafted human cells across each muscle. Data information: full gene list for heatmaps in (A) and (D) available in Appendix Table S4.


Figure 5 ( F ) Representative fluorescence images of CMFDA - stained untreated and DLL4 & PDGF - BB - treated hiMPs within the top perfusion channel , 15 minutes after delivery and kept on the OrganoFlow ® . ( G ) Bar graph quantifying adhesion images in ( E ) . Statistical significance was calculated based on a paired t - test ( experimental replicates = 3 ) . Each point on the plot represents the number of adhered cells after 15 minutes within a single chip . ( H ) Assessment of DLL4 & PDGF - BB - treated WT and genetically corrected DMD hiMP migration through a layer of endothelial cells . Representative images showing the lower side of the trans - well membrane on which treated and untreated hiMPs ( stained with the transient dye CFDA , in green ) are simultaneously seeded on HUVECs for 8 hours . Bar graphs quantifying the average number of CFDA - positive cells / mm2 , that have migrated through the endothelial layer in each considered condition . ( experimental replicates = 3 ) . A minimum of 10 ( 1 . 5 mm2 ) fields per condition was quantified ( error bars : SEM ) . ( I ) Bar graph showing fold - change in trans - endothelial migration ( error bars : SEM ) . Statistical significance based on one - way ANOVA with Bonferroni ' s multiple comparison .

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Figure 5(F) Representative fluorescence images of CMFDA-stained untreated and DLL4 & PDGF-BB-treated hiMPs within the top perfusion channel, 15 minutes after delivery and kept on the OrganoFlow®. (G) Bar graph quantifying adhesion images in (E). Statistical significance was calculated based on a paired t-test (experimental replicates = 3). Each point on the plot represents the number of adhered cells after 15 minutes within a single chip.(H) Assessment of DLL4 & PDGF-BB-treated WT and genetically corrected DMD hiMP migration through a layer of endothelial cells. Representative images showing the lower side of the trans-well membrane on which treated and untreated hiMPs (stained with the transient dye CFDA, in green) are simultaneously seeded on HUVECs for 8 hours. Bar graphs quantifying the average number of CFDA-positive cells/ mm2, that have migrated through the endothelial layer in each considered condition. (experimental replicates = 3). A minimum of 10 (1.5 mm2) fields per condition was quantified (error bars: SEM). (I) Bar graph showing fold-change in trans-endothelial migration (error bars: SEM). Statistical significance based on one-way ANOVA with Bonferroni's multiple comparison.


Figure 1 ( C ) HeLa cells were harvested at indicated time points and processed for western blotting probed for GBP1 and 5 protein levels , α - Tubulin ( Tubulin ) as loading control .

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Figure 1(C) HeLa cells were harvested at indicated time points and processed for western blotting probed for GBP1 and 5 protein levels, α-Tubulin (Tubulin) as loading control.


Figure 2 ( B ) Top : Experimental outline of CRISPRa targeting of GBP1 and ASCL1 ( control ) by transient transfection with plasmids containing dCAS9 - SAM . Bottom : HeLa cells were transfected with dCas9 - SAM technology with gRNA for indicated genes , either alone or in combination with a 24h exposure to IFNγ . RNA was isolated and GBP1 mRNA expression was measured by RT - qPCR after 48h of transfection and normalized to ACTB expression . Statistical significance was determined using two - way ANOVA . Data are shown as mean . Error bars , SD ; n = 3 biological replicates . ( C ) Top : Experimental outline of CRISPRa targeting of GBP1 and ASCL1 ( control ) by transient transfection with plasmids containing dCAS9 - SAM , followed by induction with IFNγ . Bottom : RNA was isolated , GBP1 mRNA level was determined as indicted in experimental outline in top , by RT - qPCR and normalized to ACTB mRNA level . Statistical significance was determined using Ordinary one - way ANOVA . Data are shown as mean . Error bars , SD ; n = 3 biological replicates . ( D ) Experiment as in C but with addition of IFNγ during priming . Statistical significance was determined using Ordinary one - way ANOVA . Data are shown as mean . Error bars , SD ; n = 3 biological replicates .

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Figure 2(B) Top: Experimental outline of CRISPRa targeting of GBP1 and ASCL1 (control) by transient transfection with plasmids containing dCAS9-SAM. Bottom: HeLa cells were transfected with dCas9-SAM technology with gRNA for indicated genes, either alone or in combination with a 24h exposure to IFNγ. RNA was isolated and GBP1 mRNA expression was measured by RT-qPCR after 48h of transfection and normalized to ACTB expression. Statistical significance was determined using two-way ANOVA. Data are shown as mean. Error bars, SD; n=3 biological replicates.(C) Top: Experimental outline of CRISPRa targeting of GBP1 and ASCL1 (control) by transient transfection with plasmids containing dCAS9-SAM, followed by induction with IFNγ. Bottom: RNA was isolated, GBP1 mRNA level was determined as indicted in experimental outline in top, by RT-qPCR and normalized to ACTB mRNA level. Statistical significance was determined using Ordinary one-way ANOVA. Data are shown as mean. Error bars, SD; n=3 biological replicates.(D) Experiment as in C but with addition of IFNγ during priming. Statistical significance was determined using Ordinary one-way ANOVA. Data are shown as mean. Error bars, SD; n=3 biological replicates.


Figure 3 ( B ) Stable CRISPR knockouts were generated for indicated genes in HeLa cells . Knockout ( KO ) cells or their parental controls ( WT ) were induced with IFNγ for 24 hours or left untreated and prepared for SDS - PAGE and immunoblotting . Blots incubated with GBP5 antibody assess gene expression . α - Tubulin ( Tubulin ) was used as a loading control . Note that GBP5 and Tubulin blot of parental cells is as in 1C . HeLa cells were primed with IFNγ for 24 hours , followed with IFNγ washout . After 48h , naïve and primed cells were induced by IFNγ for 1h and 3h . Cells were harvested at indicated time points and processed for CUT & RUN . Representation of processed data of CUT & RUN for STAT1 ( D ) and IRF1 ( E ) occupancy at GBP5 and GBP4 genes . Sequenced reads were mapped to the human genome ( hg38 ) , coverage data are displayed as reads per million ( RPM ) at equal scaling .

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Figure 3(B) Stable CRISPR knockouts were generated for indicated genes in HeLa cells. Knockout (KO) cells or their parental controls (WT) were induced with IFNγ for 24 hours or left untreated and prepared for SDS-PAGE and immunoblotting. Blots incubated with GBP5 antibody assess gene expression. α-Tubulin (Tubulin) was used as a loading control. Note that GBP5 and Tubulin blot of parental cells is as in 1C.HeLa cells were primed with IFNγ for 24 hours, followed with IFNγ washout. After 48h, naïve and primed cells were induced by IFNγ for 1h and 3h. Cells were harvested at indicated time points and processed for CUT&RUN. Representation of processed data of CUT&RUN for STAT1 (D) and IRF1 (E) occupancy at GBP5 and GBP4 genes. Sequenced reads were mapped to the human genome (hg38), coverage data are displayed as reads per million (RPM) at equal scaling.


Figure 5 ( A ) Single - cell RNA - seq from HeLa cells for STAT1 Each dot represents STAT1 expression in one cell in naïve ( n = 91 ) , induction ( n = 90 ) and reinduction ( n = 92 ) . Error bars , SEM . ( C ) Blot probing for STAT1 before and after IFNγ induction in STAT1 knockout ( STAT1KO ) , STAT1 rescued ( STAT1 , constitutive ) and parental control ( WT ) , to confirm knockout and rescue status . α - Tubulin ( Tubulin ) was used as a loading control . ( D ) STAT1 rescue cells and their parental control were subjected to IFNγ induction and reinduction regime as outlined in Figure 1A . Cell extracts were processed for western blotting and probed for GBP5 expression before and after induction and reinduction as indicated in Figure 1A . α - Tubulin ( Tubulin ) was used as a loading control . ( G ) Cells constitutively expressing STAT1 ( as in B ) were primed with IFNγ , followed by IFNγ washout . After 48h , naïve and primed cells were induced by IFNγ for different time points ( 5 , 15 , 30 , 60 , 180 min ) . Cell extracts were prepared at indicated timepoints and processed for western blotting . Immunoblot of pSTAT1 - Y701 , and α - Tubulin ( Tubulin ) as a loading control .

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Figure 5(A) Single-cell RNA-seq from HeLa cells for STAT1 Each dot represents STAT1 expression in one cell in naïve (n=91), induction (n=90) and reinduction (n=92). Error bars, SEM.(C) Blot probing for STAT1 before and after IFNγ induction in STAT1 knockout (STAT1KO), STAT1 rescued (STAT1, constitutive) and parental control (WT), to confirm knockout and rescue status. α-Tubulin (Tubulin) was used as a loading control.(D) STAT1 rescue cells and their parental control were subjected to IFNγ induction and reinduction regime as outlined in Figure 1A. Cell extracts were processed for western blotting and probed for GBP5 expression before and after induction and reinduction as indicated in Figure 1A. α-Tubulin (Tubulin) was used as a loading control.(G) Cells constitutively expressing STAT1 (as in B) were primed with IFNγ, followed by IFNγ washout. After 48h, naïve and primed cells were induced by IFNγ for different time points (5, 15, 30, 60, 180 min). Cell extracts were prepared at indicated timepoints and processed for western blotting. Immunoblot of pSTAT1-Y701, and α-Tubulin (Tubulin) as a loading control.


Figure 6 ( A ) HeLa cells were subjected to IFNγ induction and reinduction regime as outlined in Figure 1A with 2 days and 7 days recovery time ( primed state ) after IFNγ washout . Cell extracts were prepared at indicated timepoints , processed for western blotting and probed for STAT1 , pSTAT1 - Y701 and pSTAT1 - S727 . α - Tubulin ( Tubulin ) as a loading control . ( C ) ( H ) STAT1KO : : STAT1 - S727E and STAT1KO : : STAT1 - S727D expressing cells or their parental controls ( WT ) were subjected to IFNγ induction and reinduction regime as outlined in Figure 1A , RNA was isolated and GBP5 mRNA level was determined by RT - qPCR and normalized to ACTB mRNA level . Statistical significance was determined using Ordinary one - way ANOVA . Data are shown as mean ( error bars , SD ; n = 3 biological replicates ) . ( E ) Characterization of dTAG - induced destruction and recovery . STAT1 - EGFP - dTAG expressing cells were induced with IFNγ for 24h , then treated with dTAG13 ( 100 nM ) for DMSO vehicle control ( I ) or STAT1 depletion ( II ) for 24h . To determine recovery of STAT1 expression , cells were washed three times with medium and harvested 48h after IFNγ washout ( 120 hour timepoint , lane 4 ) . In Parallel , ( III ) cells were treated with dTAG13 ( 100 nM ) for 48h during 24h induction with IFNγ . Immunoblot of STAT1 - GFP - dTAG at indicated treatments confirm STAT1 depletion and recovery , respectively . α - Tubulin ( Tubulin ) was used as a loading control . ( G ) RNA was isolated and GBP5 mRNA level as indicated in ( F ) was determined by RT - qPCR , normalized to ACTB mRNA level . Statistical significance was determined using two - way ANOVA . Data are shown as mean ( error bars , SD ; n = 3 biological replicates )

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Figure 6(A) HeLa cells were subjected to IFNγ induction and reinduction regime as outlined in Figure 1A with 2 days and 7 days recovery time (primed state) after IFNγ washout. Cell extracts were prepared at indicated timepoints, processed for western blotting and probed for STAT1, pSTAT1-Y701 and pSTAT1-S727. α-Tubulin (Tubulin) as a loading control.(C) (H) STAT1KO::STAT1-S727E and STAT1KO::STAT1-S727D expressing cells or their parental controls (WT) were subjected to IFNγ induction and reinduction regime as outlined in Figure 1A, RNA was isolated and GBP5 mRNA level was determined by RT-qPCR and normalized to ACTB mRNA level. Statistical significance was determined using Ordinary one-way ANOVA. Data are shown as mean (error bars, SD; n=3 biological replicates).(E) Characterization of dTAG-induced destruction and recovery. STAT1-EGFP-dTAG expressing cells were induced with IFNγ for 24h, then treated with dTAG13 (100 nM) for DMSO vehicle control (I) or STAT1 depletion (II) for 24h. To determine recovery of STAT1 expression, cells were washed three times with medium and harvested 48h after IFNγ washout (120 hour timepoint, lane 4). In Parallel, (III) cells were treated with dTAG13 (100 nM) for 48h during 24h induction with IFNγ. Immunoblot of STAT1-GFP-dTAG at indicated treatments confirm STAT1 depletion and recovery, respectively. α-Tubulin (Tubulin) was used as a loading control.(G) RNA was isolated and GBP5 mRNA level as indicated in (F) was determined by RT-qPCR, normalized to ACTB mRNA level. Statistical significance was determined using two-way ANOVA. Data are shown as mean (error bars, SD; n=3 biological replicates)


Figure 1A Western blot analysis STAT expression and phosphorylation . Bone marrow derived macrophages ( BMDMs ) of wild type ( WT ) , Stat1Y701F / + ( WT / YF ) or Stat1 - / - ( S1 ) mice were treated with 250 IU / mL of IFNβ or 5 ng / mL of IFNγ for 0 . 5 , 6 , 12 and 24 h . Whole cell extracts were collected and tested in western blot for levels of phosphorylation of STAT1 ( Y701 ) and STAT2 ( Y689 ) and total level of STAT1 and STAT2 . The blots are representative of more than 3 independent experiments . B Effect of STAT1Y701Fheterozygosity on the expression of type I IFN - induced genes ( ISG ) . BMDMs of wild type ( WT ) , Stat1Y701F / + ( WT / YF ) or Stat1 - / - ( S1 ) mice were treated with 250 IU / mL of IFNβ for 4 and 48 h . Gene expression was measured by Q - PCR and normalized to Gapdh and to the expression levels in untreated wild type cells . The bars represent mean values with the standard deviations ( SD ) of three independent experiments . C Effect of STAT1Y701Fheterozygosity on the expression of IFNγ - induced genes . BMDMs of wild type ( WT ) , Stat1Y701F / + ( WT / YF ) or Stat1 - / - ( S1 ) mice were treated with 5 ng / mL of IFNγ for 4 and 48 h . Gene expression was measured by Q - PCR and normalized to Gapdh and to the expression levels in untreated wild type cells . The bars represent mean values with the standard deviations ( SD ) of three independent experiments .

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Figure 1A Western blot analysis STAT expression and phosphorylation. Bone marrow derived macrophages (BMDMs) of wild type (WT), Stat1Y701F/+ (WT/YF) or Stat1-/- (S1) mice were treated with 250 IU/mL of IFNβ or 5 ng/mL of IFNγ for 0.5, 6, 12 and 24 h. Whole cell extracts were collected and tested in western blot for levels of phosphorylation of STAT1 (Y701) and STAT2 (Y689) and total level of STAT1 and STAT2. The blots are representative of more than 3 independent experiments.B Effect of STAT1Y701Fheterozygosity on the expression of type I IFN-induced genes (ISG). BMDMs of wild type (WT), Stat1Y701F/+ (WT/YF) or Stat1-/- (S1) mice were treated with 250 IU/mL of IFNβ for 4 and 48 h. Gene expression was measured by Q-PCR and normalized to Gapdh and to the expression levels in untreated wild type cells. The bars represent mean values with the standard deviations (SD) of three independent experiments.C Effect of STAT1Y701Fheterozygosity on the expression of IFNγ-induced genes. BMDMs of wild type (WT), Stat1Y701F/+ (WT/YF) or Stat1-/- (S1) mice were treated with 5 ng/mL of IFNγ for 4 and 48 h. Gene expression was measured by Q-PCR and normalized to Gapdh and to the expression levels in untreated wild type cells. The bars represent mean values with the standard deviations (SD) of three independent experiments.


Figure 2A Effect of STAT1Y701F homozygosity on STAT1 expression in mouse cells and organs . Spleens , livers or bone marrow derived macrophages ( BMDMs ) were isolated from wild type ( WT ) , Stat1Y701F ( YF ) and Stat1 - / - ( S1 ) mice . Whole cell extracts were collected and tested for levels of total Stat1 in western blot . The blots are representative of more than 3 independent experiments . B Effect of STAT1Y701F homozygosity on STAT1 phosphorylation at Y701 . BMDMs were isolated from wild type ( WT ) , Stat1Y701F ( YF ) and Stat1 - / - ( S1 ) mice and stimulated for 30 min with 250 IU / mL of IFNβ or 5 ng / mL of IFNγ . Whole cell extracts were collected and tested for levels of STAT1 phosphorylation on Y701 in western blot . The blots are representative of more than 3 independent experiments . C Effect of STAT1Y701Fhomozygosity on the expression of IFNβ - induced genes . BMDMs of wild type ( WT ) , Stat1Y701F ( YF ) and Stat1 - / - ( S1 ) mice were treated with 5 ng / mL of IFNγ for 4 or 48 h . Gene expression was measured by Q - PCR and normalized to Gapdh and to the expression levels in untreated wild type cells . Bars represent a mean value of 3 independent experiments . Error bars represent standard error of the mean ( SEM ) ; asterisks denote the level of statistical significance ( ns , p > 0 . 05 ) ; the p - values were calculated using paired ratio t - test . D Effect of STAT1Y701Fhomozygosity on the expression of type I IFN - induced genes ( ISG ) . BMDMs were isolated from wild type ( WT ) , Stat1Y701F , Stat1 - / - , Stat2 - / - and IRF9 - / - mice treated with 250 IU / mL of IFNβ for 4 , 8 , 12 , 24 or 48 h . Gene expression was measured by Q - PCR and normalized to Gapdh and to the expression levels in untreated wild type cells . Bars represent a mean value of 3 independent experiments . Error bars represent standard error of the mean ( SEM ) ; asterisks denote the level of statistical significance ( ns , p > 0 . 05 ; * , p ≤ 0 . 05 ; * * , p ≤ 0 . 01 ) ; the p - values were calculated using paired ratio t - test . E STAT1 and STAT2 phosphorylation in Stat1 - / - , Stat1Y701F , Stat2 - / - and Irf9 - / - macrophages . BMDMs were isolated from wild type ( WT ) , Stat1Y701F ( YF ) , Stat1 - / - ( S1 ) , Stat2 - / - ( S2 ) and IRF9 - / - ( IRF9 ) mice and treated with 250 IU / mL of IFNβ for 30 min or 6 , 12 or 24 h . The whole cell extracts were collected and tested in western blot for levels of phosphorylation of STAT1 on Y701 and of STAT2 on Y689 . The same cell extracts were tested for total levels of STAT1 and STAT2 . The blots are representative of more than 3 independent experiments .

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

Figure 2A Effect of STAT1Y701F homozygosity on STAT1 expression in mouse cells and organs. Spleens, livers or bone marrow derived macrophages (BMDMs) were isolated from wild type (WT), Stat1Y701F (YF) and Stat1-/- (S1) mice. Whole cell extracts were collected and tested for levels of total Stat1 in western blot. The blots are representative of more than 3 independent experiments.B Effect of STAT1Y701F homozygosity on STAT1 phosphorylation at Y701. BMDMs were isolated from wild type (WT), Stat1Y701F (YF) and Stat1-/- (S1) mice and stimulated for 30 min with 250 IU/mL of IFNβ or 5 ng/mL of IFNγ. Whole cell extracts were collected and tested for levels of STAT1 phosphorylation on Y701 in western blot. The blots are representative of more than 3 independent experiments.C Effect of STAT1Y701Fhomozygosity on the expression of IFNβ-induced genes. BMDMs of wild type (WT), Stat1Y701F (YF) and Stat1-/- (S1) mice were treated with 5 ng/mL of IFNγ for 4 or 48 h. Gene expression was measured by Q-PCR and normalized to Gapdh and to the expression levels in untreated wild type cells. Bars represent a mean value of 3 independent experiments. Error bars represent standard error of the mean (SEM); asterisks denote the level of statistical significance (ns, p>0.05); the p-values were calculated using paired ratio t-test.D Effect of STAT1Y701Fhomozygosity on the expression of type I IFN-induced genes (ISG). BMDMs were isolated from wild type (WT), Stat1Y701F, Stat1-/-, Stat2-/- and IRF9-/- mice treated with 250 IU/mL of IFNβ for 4, 8, 12, 24 or 48 h. Gene expression was measured by Q-PCR and normalized to Gapdh and to the expression levels in untreated wild type cells. Bars represent a mean value of 3 independent experiments. Error bars represent standard error of the mean (SEM); asterisks denote the level of statistical significance (ns, p>0.05; *, p≤ 0.05; **, p≤ 0.01); the p-values were calculated using paired ratio t-test.E STAT1 and STAT2 phosphorylation in Stat1-/-, Stat1Y701F, Stat2-/- and Irf9-/- macrophages. BMDMs were isolated from wild type (WT), Stat1Y701F (YF), Stat1-/- (S1), Stat2-/- (S2) and IRF9-/- (IRF9) mice and treated with 250 IU/mL of IFNβ for 30 min or 6, 12 or 24 h. The whole cell extracts were collected and tested in western blot for levels of phosphorylation of STAT1 on Y701 and of STAT2 on Y689. The same cell extracts were tested for total levels of STAT1 and STAT2. The blots are representative of more than 3 independent experiments.


Figure 3A IFNβ - stimulated binding of STAT2 to ISRE sequences of Mx2 and IRF7 promoters . Bone marrow derived macrophages ( BMDMs ) of wild type ( WT ) , Stat1Y701F ( YF ) , Stat1 - / - ( S1 ) , Stat2 - / - ( S2 ) and IRF9 - / - ( IRF9 ) mice were treated with 250 IU / mL of IFNβ for 2 or 24 h . Cells were crosslinked , sonicated and immunoprecipitated with STAT2 - specific antibody . The amount of precipitated DNA was measured by Q - PCR . Bars represent a mean value of 3 independent experiments . Error bars represent standard error of the mean ( SEM ) ; asterisks denote the level of statistical significance ( * * , p ≤ 0 . 01 ) ; the p - values were calculated using paired ratio t - test . B Impact of STAT2 deficiency on IFNβ - stimulated STAT1 association with the Mx2 ISRE . BMDMs of wild type ( WT ) , Stat1 - / - ( S1 ) and Stat2 - / - ( S2 ) mice were treated with 250 IU / mL of IFNβ for 2 or 24 h . Cells were crosslinked , sonicated and immunoprecipitated with STAT1 - specific antibody . The amount of precipitated DNA was measured by Q - PCR . Bars represent mean values of three independent experiments ; error bars represent standard error of mean ( SEM ) . C Simultaneous association of STAT1 and STAT2 with the Mx2 ISRE analyzed by ChIP - reChIP . BMDMs of wild type ( WT ) mice were treated with 250 IU / mL of IFNβ for 2 or 24 h . Cells were crosslinked , sonicated and immunoprecipitated with either STAT1 - specific antibody and re - immunoprecipitated with STAT2 - specific antibody or vice versa . The amount of precipitated DNA was measured by Q - PCR . Bars represent mean values of three independent experiments ; error bars represent standard deviation ( SD ) . D Impact of STAT1Y701F on delayed , STAT2 - mediated expression of IFN - induced genes . Stat1 - / - fibroblasts were transfected with plasmids driving expression of the indicated proteins . 24 h after transfection , 250 IU / ml of IFNβ was added to the transfected cells and ISG expression was determined by Q - PCR after 48h of cytokine treatment . Gene expression was measured by Q - PCR and normalized to Gapdh . Bars represent a mean value of 3 independent experiments . Error bars represent standard error of the mean ( SEM ) and asterisks denote the level of statistical significance ( * , p ≤ 0 . 05 ) ; the p - values were calculated using paired ratio t - test .

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Figure 3A IFNβ-stimulated binding of STAT2 to ISRE sequences of Mx2 and IRF7 promoters. Bone marrow derived macrophages (BMDMs) of wild type (WT), Stat1Y701F (YF), Stat1-/- (S1), Stat2-/- (S2) and IRF9-/- (IRF9) mice were treated with 250 IU/mL of IFNβ for 2 or 24 h. Cells were crosslinked, sonicated and immunoprecipitated with STAT2-specific antibody. The amount of precipitated DNA was measured by Q-PCR. Bars represent a mean value of 3 independent experiments. Error bars represent standard error of the mean (SEM); asterisks denote the level of statistical significance (**, p≤ 0.01); the p-values were calculated using paired ratio t-test.B Impact of STAT2 deficiency on IFNβ -stimulated STAT1 association with the Mx2 ISRE. BMDMs of wild type (WT), Stat1-/- (S1) and Stat2-/- (S2) mice were treated with 250 IU/mL of IFNβ for 2 or 24 h. Cells were crosslinked, sonicated and immunoprecipitated with STAT1-specific antibody. The amount of precipitated DNA was measured by Q-PCR. Bars represent mean values of three independent experiments; error bars represent standard error of mean (SEM).C Simultaneous association of STAT1 and STAT2 with the Mx2 ISRE analyzed by ChIP-reChIP. BMDMs of wild type (WT) mice were treated with 250 IU/mL of IFNβ for 2 or 24 h. Cells were crosslinked, sonicated and immunoprecipitated with either STAT1-specific antibody and re-immunoprecipitated with STAT2-specific antibody or vice versa. The amount of precipitated DNA was measured by Q-PCR. Bars represent mean values of three independent experiments; error bars represent standard deviation (SD).D Impact of STAT1Y701F on delayed, STAT2-mediated expression of IFN-induced genes. Stat1-/- fibroblasts were transfected with plasmids driving expression of the indicated proteins. 24 h after transfection, 250 IU/ml of IFNβ was added to the transfected cells and ISG expression was determined by Q-PCR after 48h of cytokine treatment. Gene expression was measured by Q-PCR and normalized to Gapdh. Bars represent a mean value of 3 independent experiments. Error bars represent standard error of the mean (SEM) and asterisks denote the level of statistical significance (*, p≤ 0.05); the p-values were calculated using paired ratio t-test.


Figure 4A Analysis of STAT2 nuclear translocation by immunofluorescence . Bone marrow derived macrophages ( BMDMs ) of wild type ( WT ) , Stat1Y701F ( YF ) , Stat1 - / - ( S1 ) , Stat2 - / - ( S2 ) and IRF9 - / - ( IRF9 ) mice were seeded on cover slips and stimulated with 250 IU / mL of IFNβ for 30 min or 24 h . The cells were fixed and stained for STAT2 specific antibody followed by Alexafluor ® 488 conjugated secondary antibody ( green ) . Nuclei were stained with DAPI ( blue ) . Studies are representative of more than three independent experiments . The scale bars represent 10 µm . B Quantitative evaluation of STAT2nuclear translocation . The intensity of STAT2 - dependent immunofluorescence over DNA staining ( DAPI ) was quantified using ImageJ software in 20 cells from two independent experiments . Bars represent a mean with standard deviation ( SD ) and asterisks denote the level of statistical significance ( * * * , p ≤ 0 . 001 ) ; p - value was calculated using unpaired t - test .

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Figure 4A Analysis of STAT2 nuclear translocation by immunofluorescence. Bone marrow derived macrophages (BMDMs) of wild type (WT), Stat1Y701F (YF), Stat1-/- (S1), Stat2-/- (S2) and IRF9-/- (IRF9) mice were seeded on cover slips and stimulated with 250 IU/mL of IFNβ for 30 min or 24 h. The cells were fixed and stained for STAT2 specific antibody followed by Alexafluor® 488 conjugated secondary antibody (green). Nuclei were stained with DAPI (blue). Studies are representative of more than three independent experiments. The scale bars represent 10 µm.B Quantitative evaluation of STAT2nuclear translocation. The intensity of STAT2-dependent immunofluorescence over DNA staining (DAPI) was quantified using ImageJ software in 20 cells from two independent experiments. Bars represent a mean with standard deviation (SD) and asterisks denote the level of statistical significance (***, p≤ 0.001); p-value was calculated using unpaired t-test.


Figure 5A Legionella pneumophila growth in unstimulated macrophages . Bone marrow derived macrophages ( BMDMs ) of wild type ( WT ) , Stat1Y701F , Stat1 - / - mice were seeded on in the 24 - well plates and infected with L . pneumophila ( JR32 Fla - , MOI 0 . 25 ) . The numbers of colony forming units ( CFUs ) were determined 24 h , 48 h and 72 h after infection on charcoal yeast extract plates ( CYE ) . The 0 time point was collected 1 . 5 h after the infection . B Legionella pneumophila growth in IFNβ - treated macrophages . Bone marrow derived macrophages ( BMDMs ) of wild type ( WT ) , Stat1Y701F , Stat1 - / - mice were seeded in 24 - well plates , treated with 500 U / mL of IFNβ and then infected with L . pneumophila ( JR32 Fla - , MOI 0 . 25 ) . The numbers of colony forming units ( CFUs ) were determined 24 h , 48 h and 72 h after infection on charcoal yeast extract plates ( CYE ) . The 0 time point was collected 1 . 5 h after the infection .

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Figure 5A Legionella pneumophila growth in unstimulated macrophages. Bone marrow derived macrophages (BMDMs) of wild type (WT), Stat1Y701F, Stat1-/- mice were seeded on in the 24-well plates and infected with L. pneumophila (JR32 Fla-, MOI 0.25). The numbers of colony forming units (CFUs) were determined 24 h, 48 h and 72 h after infection on charcoal yeast extract plates (CYE). The 0 time point was collected 1.5 h after the infection.B Legionella pneumophila growth in IFNβ-treated macrophages. Bone marrow derived macrophages (BMDMs) of wild type (WT), Stat1Y701F, Stat1-/- mice were seeded in 24-well plates, treated with 500 U/mL of IFNβ and then infected with L. pneumophila (JR32 Fla-, MOI 0.25). The numbers of colony forming units (CFUs) were determined 24 h, 48 h and 72 h after infection on charcoal yeast extract plates (CYE). The 0 time point was collected 1.5 h after the infection.


Figure 6A Western blot analysis of STAT1 tyrosine phosphorylation . Bone marrow derived macrophages ( BMDMs ) of wild type ( WT ) , Stat1Y701F ( YF ) and Stat1 - / - ( S1 ) mice were infected with L . monocytogenes ( LO28 , MOI 10 ) for 5 or 6 h . Whole cell extracts were collected and tested in western blot for levels of total STAT1 and phosphorylation of STAT1 on tyrosine ( Y701 ) . The blots are representative of more than 3 independent experiments . B Impact of Stat1Y701F mutation , or of deletion of ISGF3 subunits on the expression of the IFNβ gene . BMDMs of wild type ( WT ) , Stat1Y701F , Stat1 - / - , Stat2 - / - and IRF9 - / - mice were infected with L . monocytogenes ( LO28 , MOI 10 ) for 4 , 8 , 12 , 24 or 48 h . Levels of Ifnβ gene expression were determined by Q - PCR . Bars represent mean values of three independent experiments . Error bars represent standard error of mean ( SEM ) .

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Figure 6A Western blot analysis of STAT1 tyrosine phosphorylation. Bone marrow derived macrophages (BMDMs) of wild type (WT), Stat1Y701F (YF) and Stat1-/- (S1) mice were infected with L. monocytogenes (LO28, MOI 10) for 5 or 6 h. Whole cell extracts were collected and tested in western blot for levels of total STAT1 and phosphorylation of STAT1 on tyrosine (Y701). The blots are representative of more than 3 independent experiments.B Impact of Stat1Y701F mutation, or of deletion of ISGF3 subunits on the expression of the IFNβ gene. BMDMs of wild type (WT), Stat1Y701F, Stat1-/-, Stat2-/- and IRF9-/- mice were infected with L. monocytogenes (LO28, MOI 10) for 4, 8, 12, 24 or 48 h. Levels of Ifnβ gene expression were determined by Q-PCR. Bars represent mean values of three independent experiments. Error bars represent standard error of mean (SEM).


Figure 7A Impact of Stat1 deficiency or of STAT1Y701F mutation on the growth of Listeria monocytogenes in macrophages . Bone marrow derived macrophages ( BMDMs ) of wild type ( WT ) , Stat1Y701F and Stat1 - / - mice were infected with L . monocytogenes ( LO28 , MOI 10 ) . Colony forming unit ( CFU ) numbers were determined 1 , 2 , 4 , 6 or 8 h after infection by plating on brain - heart - infusion ( BHI ) agar plates . The graph represents biological triplicates and the data are represented as mean values . Error bars represent standard deviation ( SD ) and asterisks denote statistically significant differences ( ns , p > 0 . 05 ; * * , p ≤ 0 . 01 ; * * * , p ≤ 0 . 001 ) ; p - values were calculated using unpaired t - test . B Survival of mice infected with L . monocytogenes . 10 wild type ( WT ) , Stat1Y701F and Stat1 - / - mice per group were infected by intraperitoneal injection of 1x 102 viable L . monocytogenes . Survival was monitored over 10 days . The study is representative of more than 3 independent experiments . C Organ pathogen burdens of mice infected with L . monocytogenes . Wild type ( WT ) , Stat1Y701F ( YF ) and Stat1 - / - ( S1 ) mice were infected by intraperitoneal injection of 1x 102 viable L . monocytogenes . Number of colony forming units ( CFU ) in organs was determined at 48 h , 72 h or at the terminal stage of infection by plating homogenates on brain - heart - infusion ( BHI ) agar plates or Oxford agar plates ( for lungs ) . Dots represent pooled data of 3 independent experiments . Lines represent the median and asterisks denote statistically significant differences ( ns , p > 0 . 05 ; * , p ≤ 0 . 05 ; * * , p ≤ 0 . 01 ; * * * , p ≤ 0 . 001 ; * * * * , p ≤ 0 . 0001 ) ; p - values were calculated using Mann - Whitney test .

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Figure 7A Impact of Stat1 deficiency or of STAT1Y701F mutation on the growth of Listeria monocytogenes in macrophages. Bone marrow derived macrophages (BMDMs) of wild type (WT), Stat1Y701F and Stat1-/- mice were infected with L. monocytogenes (LO28, MOI 10). Colony forming unit (CFU) numbers were determined 1, 2, 4, 6 or 8 h after infection by plating on brain-heart-infusion (BHI) agar plates. The graph represents biological triplicates and the data are represented as mean values. Error bars represent standard deviation (SD) and asterisks denote statistically significant differences (ns, p>0.05; **, p≤0.01; ***, p≤ 0.001); p-values were calculated using unpaired t-test.B Survival of mice infected with L. monocytogenes. 10 wild type (WT), Stat1Y701F and Stat1-/- mice per group were infected by intraperitoneal injection of 1x 102 viable L. monocytogenes. Survival was monitored over 10 days. The study is representative of more than 3 independent experiments.C Organ pathogen burdens of mice infected with L. monocytogenes. Wild type (WT), Stat1Y701F (YF) and Stat1-/- (S1) mice were infected by intraperitoneal injection of 1x 102 viable L. monocytogenes. Number of colony forming units (CFU) in organs was determined at 48 h, 72 h or at the terminal stage of infection by plating homogenates on brain-heart-infusion (BHI) agar plates or Oxford agar plates (for lungs). Dots represent pooled data of 3 independent experiments. Lines represent the median and asterisks denote statistically significant differences (ns, p>0.05; *, p≤ 0.05; **, p≤ 0.01; ***, p≤ 0.001; ****, p≤ 0.0001); p-values were calculated using Mann-Whitney test.


Figure 8A Immunohistochemical analysis of infection and inflammatory infiltrates . Wild type ( WT ) , Stat1Y701F and Stat1 - / - mice were infected by intraperitoneal injection of 1x102 viable L . monocytogenes for 48 h . Liver sections were examined by immunohistochemistry with L . monocytogenes or Ly6C / Ly6G specific antibody . The scale bars on 20x magnification images represent 100 µm and 50 µm on the 63x magnification images . B Quantitative evaluation of inflammatory infiltrates . Infiltrates representing the entire surface of sections from five animals per genotype were counted and categorized according to their size . C Liver pathology in infected mice . Wild type ( WT ) , Stat1Y701F ( YF ) and Stat1 - / - ( S1 ) mice were infected by intraperitoneal injection of 1x105 viable L . monocytogenes for 72 h . Serum was collected and tested for ALT activity . Lines represent the median and asterisks denote statistically significant differences ( * , p ≤ 0 . 05 ; * * * , p ≤ 0 . 001 ) ; p - values were calculated using unpaired t - test .

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Figure 8A Immunohistochemical analysis of infection and inflammatory infiltrates. Wild type (WT), Stat1Y701F and Stat1-/- mice were infected by intraperitoneal injection of 1x102 viable L. monocytogenes for 48 h. Liver sections were examined by immunohistochemistry with L. monocytogenes or Ly6C/Ly6G specific antibody. The scale bars on 20x magnification images represent 100 µm and 50 µm on the 63x magnification images.B Quantitative evaluation of inflammatory infiltrates. Infiltrates representing the entire surface of sections from five animals per genotype were counted and categorized according to their size.C Liver pathology in infected mice. Wild type (WT), Stat1Y701F (YF) and Stat1-/- (S1) mice were infected by intraperitoneal injection of 1x105 viable L. monocytogenes for 72 h. Serum was collected and tested for ALT activity. Lines represent the median and asterisks denote statistically significant differences (*, p≤ 0.05; ***, p≤ 0.001); p-values were calculated using unpaired t-test.


Figure 1Western blot analysis ( B ) of DCLK1 in aortas of LFD and HFD - fed ApoE - / - mice . β - actin was used as the loading control ( n = 10 biological replicates ) . ( E ) Representative immunofluorescence staining of CD31 ( green ) and DCLK1 ( red ) in aortic roots ( scale bar = 25 μm ) . ( F ) Representative immunofluorescence staining of α - SMA ( green ) and DCLK1 ( red ) in aortic roots ( scale bar = 25 μm ) . ( G ) Representative immunofluorescence staining of F4 / 80 ( green ) and DCLK1 ( red ) in aortic roots ( scale bar = 25 μm ) . White arrows indicate co - location of F4 / 80 and DCLK1 . ( H - I ) Time - course of DCLK1 induction in response to oxLDL in mouse primary peritoneal macrophages ( MPMs ) . MPMs were exposed to oxLDL ( 50 μg / mL ) for indicated time . Western blot analysis ( H ) and densitometric quantification ( I ) of DCLK1 were shown . GAPDH was used as the loading control ( n = 3 biological replicates ) . Data information : Data were shown as mean ± SEM ; * , P < 0 . 05 ; ns , not significant , two - tailed unpaired Student ' s t - test .

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Figure 1Western blot analysis (B) of DCLK1 in aortas of LFD and HFD-fed ApoE-/- mice. β-actin was used as the loading control (n=10 biological replicates).(E) Representative immunofluorescence staining of CD31 (green) and DCLK1 (red) in aortic roots (scale bar=25 μm). (F) Representative immunofluorescence staining of α-SMA (green) and DCLK1 (red) in aortic roots (scale bar=25 μm). (G) Representative immunofluorescence staining of F4/80 (green) and DCLK1 (red) in aortic roots (scale bar=25 μm). White arrows indicate co-location of F4/80 and DCLK1.(H-I) Time-course of DCLK1 induction in response to oxLDL in mouse primary peritoneal macrophages (MPMs). MPMs were exposed to oxLDL (50 μg/mL) for indicated time. Western blot analysis (H) and densitometric quantification (I) of DCLK1 were shown. GAPDH was used as the loading control (n=3 biological replicates). Data information: Data were shown as mean ± SEM; *, P < 0.05; ns, not significant, two-tailed unpaired Student's t-test.


Figure 2Representative en face Oil Red O staining ( A ) of Oil Red O - positive plaque lesion area in aortas . Plaque area was defined as percentage of total surface area of the aorta ( n = 10 biological replicates ) . Representative images ( C ) of plaque lesion area in aortic arches ( n = 10 biological replicates ) . ( E - F ) Representative images of Oil Red O staining ( E ) and quantification ( F ) of atherosclerotic lesion in aortic roots ( scale bar = 250 μm , n = 10 biological replicates ) . Plaque area was quantified by the proportion of plaque area to aortic root area . Data information : Data were shown as mean ± SEM ; * , P < 0 . 05 ; ns , not significant , two - tailed unpaired Student ' s t - test . ( G - H ) Representative images of Masson ' s Trichrome staining ( G ) and quantification ( H ) for collagen deposition in aortic roots ( scale bar = 25 μm , n = 10 biological replicates ) . Data information : Data were shown as mean ± SEM ; * , P < 0 . 05 ; ns , not significant , two - tailed unpaired Student ' s t - test .

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Figure 2Representative en face Oil Red O staining (A) of Oil Red O-positive plaque lesion area in aortas. Plaque area was defined as percentage of total surface area of the aorta (n=10 biological replicates).Representative images (C) of plaque lesion area in aortic arches (n=10 biological replicates).(E-F) Representative images of Oil Red O staining (E) and quantification (F) of atherosclerotic lesion in aortic roots (scale bar=250 μm, n=10 biological replicates). Plaque area was quantified by the proportion of plaque area to aortic root area. Data information: Data were shown as mean ± SEM; *, P < 0.05; ns, not significant, two-tailed unpaired Student's t-test.(G-H) Representative images of Masson's Trichrome staining (G) and quantification (H) for collagen deposition in aortic roots (scale bar=25 μm, n=10 biological replicates). Data information: Data were shown as mean ± SEM; *, P < 0.05; ns, not significant, two-tailed unpaired Student's t-test.


Figure 3Representative immunofluorescence staining images ( A ) of F4 / 80 ( green ) in aortic roots . Tissues were counterstained with DAPI ( blue ) . Scale bar = 250 μm , n = 10 biological replicates . Representative immunohistochemistry staining images of Ly6G ( C in aortic roots ( scale bar = 25 μm , n = 10 biological replicates ) . Representative immunohistochemistry staining images of Ly6C ( E in aortic roots ( scale bar = 25 μm , n = 10 biological replicates ) . Scatter diagram ( G ) and quantification of monocyte in plasma measured by an automated blood cell analyzer ( n = 10 biological replicates ) . SSC , side scatter ; SFL , side fluorescence . Data information : Data were shown as mean ± SEM ; * , P < 0 . 05 ; ns , not significant , two - tailed unpaired Student ' s t - test . ( J - K ) Protein ( J ) and mRNA ( K ) levels of inflammatory cytokines TNF - α and IL - 6 in serum and aortas . The values of mRNA levels were normalized to Rn18s ( n = 10 biological replicates ) . Data information : Data were shown as mean ± SEM ; * , P < 0 . 05 ; ns , not significant , two - tailed unpaired Student ' s t - test . ( L ) mRNA levels of proinflammatory chemokines and adhesion molecules in aortas ( n = 10 biological replicates ) . The values were normalized to Rn18s . Data information : Data were shown as mean ± SEM ; * , P < 0 . 05 ; ns , not significant , two - tailed unpaired Student ' s t - test .

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Figure 3Representative immunofluorescence staining images (A) of F4/80 (green) in aortic roots. Tissues were counterstained with DAPI (blue). Scale bar=250 μm, n=10 biological replicates.Representative immunohistochemistry staining images of Ly6G (C in aortic roots (scale bar=25 μm, n=10 biological replicates).Representative immunohistochemistry staining images of Ly6C (E in aortic roots (scale bar=25 μm, n=10 biological replicates).Scatter diagram (G) and quantification of monocyte in plasma measured by an automated blood cell analyzer (n=10 biological replicates). SSC, side scatter; SFL, side fluorescence. Data information: Data were shown as mean ± SEM; *, P < 0.05; ns, not significant, two-tailed unpaired Student's t-test.(J-K) Protein (J) and mRNA (K) levels of inflammatory cytokines TNF-α and IL-6 in serum and aortas. The values of mRNA levels were normalized to Rn18s (n=10 biological replicates). Data information: Data were shown as mean ± SEM; *, P < 0.05; ns, not significant, two-tailed unpaired Student's t-test.(L) mRNA levels of proinflammatory chemokines and adhesion molecules in aortas (n=10 biological replicates). The values were normalized to Rn18s. Data information: Data were shown as mean ± SEM; *, P < 0.05; ns, not significant, two-tailed unpaired Student's t-test.