Fig 2: ATF5 promotes intestinal barrier homeostasis via regulation of leptin signaling(A) Representative image of Atf5flox/flox and Atf5?IEC mice (n = 4). Scale bar indicates 1.5 cm.(B and C) Bodyweight (B) and feeding behavior (C) of Atf5flox/flox and Atf5?IEC mice fed a standard diet. Day 0 represents start of experiment at 6 weeks of age. Data represent mean ± standard error of the mean (n = 10; *p < 0.05, **p < 0.01 using the Student’s t test).(D) Serum leptin levels in 6-week-old Atf5flox/flox and Atf5?IEC mice. Data represent mean ± standard error of the mean (n = 5; ****p < 0.0001 using the Student’s t test).(E and F) Bodyweight (E) and feeding behavior (F) of Atf5flox/flox and Atf5?IEC mice intraperitoneally injected with leptin and fed a standard diet. Day 0 represents start of experiment at 6 weeks of age. Data represent mean ± standard error of the mean (n = 8; ns, non-significant using the Student’s t test).(G) Serum FITC-dextran levels in Atf5flox/flox and Atf5?IEC mice intraperitoneally injected with leptin. Data represent mean ± standard error of the mean (n = 5; ns, non-significant, *p < 0.05, ***p < .001 using the Student’s t test).(H and I) Immunoblot analysis and quantification of DAO (H) and I-FABP (I) serum protein levels in Atf5flox/flox and Atf5?IEC mice intraperitoneally injected with leptin. Coomassie stained SDS-PAGE gels are shown as loading controls. Data represent mean ± standard error of the mean (n = 4; **p < 0.01, ***p < 0.001, ****p < 0.0001 using the Student’s t test).(J) Survival of Atf5flox/flox, Atf5?IEC, and leptin-treated Atf5?IEC mice challenged with Salmonella (n = 5). See Table S1 for all statistics pertaining to survival analysis.

Fig 3: ATF5 prevents enteric pathogen dissemination by promoting intestinal barrier function(A) Serum FITC-dextran levels in Atf5flox/flox and Atf5?IEC mice. Data represent mean ± standard error of the mean (n = 5; **p < 0.01, ***p < 0.001, ****p < 0.0001 using the Student’s t test).(B) Immunohistochemistry of Atf5flox/flox and Atf5?IEC intestinal samples using anti-E-cadherin antibody and DAPI co-stain (n = 3). Representative images shown (boxed region denotes enlarged area). Scale bars indicate 100 µm (wide images) or 10 µm (enlarged images).(C) Immunoblot analysis and quantification of E-cadherin protein levels in Atf5flox/flox and Atf5?IEC mice. Actin was used as a loading control. Data represent mean ± standard error of the mean (n = 5; ****p < 0.0001 using the Student’s t test).(D) Immunohistochemistry of Atf5flox/flox and Atf5?IEC intestinal samples using anti-ZO-1 antibody and DAPI co-stain (n = 3). Representative images shown (boxed region denotes enlarged area). Scale bars indicate 100 µm (wide images) or 10 µm (enlarged images).(E) Immunoblot analysis and quantification of ZO-1 protein levels in Atf5flox/flox and Atf5?IEC mice. Actin was used as a loading control. Data represent mean ± standard error of the mean (n = 5; *p < 0.05 using the Student’s t test).(F–H) Immunoblot analysis and quantification of I-FABP (F), DAO (G), and ZO-1 (H) serum levels in Atf5flox/flox and Atf5?IEC intestinal samples. Coomassie stained SDS-PAGE gels are shown as loading controls (n = 5; ****p < 0.0001 using the Student’s t test).(I and J). Bodyweight (I) and DAI scores (J) of Atf5flox/flox and Atf5?IEC mice exposed to 4% DSS. Data represent mean ± standard error of the mean (n = 5; *p < 0.05, ****p < 0.0001 using the Student’s t test).(K) Representative image and quantification of colon lengths in Atf5flox/flox and Atf5?IEC mice exposed to DSS (n = 5). Data represent mean ± standard error of the mean (n = 5; ****p < 0.0001 using the Student’s t test). Scale bar, 1 cm.(L and M) Representative histological analysis and pathology score table of colon tissue sections from Atf5flox/flox and Atf5?IEC mice exposed to DSS (arrow and asterisk represent epithelial hyperplasia and extensive ulceration and inflammation, respectively; n = 3). Scale bars, 200 µm.

Fig 4: ATF5 supports intestinal barrier function by preventing hyperglycemia and aberrant glycolytic flux(A and B) Blood glucose (A) and insulin (B) levels in Atf5flox/flox and Atf5?IEC mice. Data represent mean ± standard error of the mean (n = 5; **p < 0.01, ***p < 0.001 using the Student’s t test).(C) Serum FITC-dextran levels in Atf5flox/flox and Atf5?IEC mice intraperitoneally injected with 2-DG. Data represent mean ± standard error of the mean (n = 5; ns, non-significant using the Student’s t test).(D and E) Immunoblot analysis and quantification of DAO (D) and I-FABP (E) serum protein levels in Atf5flox/flox and Atf5?IEC mice, with or without intraperitoneal 2-DG injection. Coomassie stained SDS-PAGE gels are shown as loading controls. Data represent mean ± standard error of the mean (n = 4; **p < 0.01, ****p < 0.0001 using the Student’s t test).(F and G) Clinical scores in Atf5flox/flox and Atf5?IEC mice, with or without intraperitoneal 2-DG injection, challenged with Salmonella (F) or C. rodentium (G). Data represent mean ± standard error of the mean (n = 5; ns, non-significant using the Student’s t test).(H and I) Survival of Atf5flox/flox and Atf5?IEC mice, with or without intraperitoneal 2-DG injection, challenged with Salmonella (H) or C. rodentium (I) (n = 5). See Table S1 for all statistics pertaining to survival analysis.(J–M) CFU counts of liver and spleen samples from Salmonella- (J and K) or C. rodentium-infected (L and M) Atf5flox/flox and Atf5?IEC mice, with or without intraperitoneal 2-DG injection. Data represent mean ± standard error of the mean (n = 5; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 using the Student’s t test).