Journal: Cell reports

Article Title: The mitochondrial UPR regulator ATF5 promotes intestinal barrier function via control of the satiety response

doi: 10.1016/j.celrep.2022.111789

Figure Lengend Snippet: (A) Immunoblot analysis and quantification of ATF5 protein levels in the intestine, liver, and spleens of Atf5 flox/flox and Atf5 ΔIEC mice. Actin was used as a loading control. Data represent mean ± standard error of the mean (n = 5; ns, non-significant, *p < 0.05 using the Student’s t test). (B) Immunoblot analysis and quantification of UPR mt -related proteins in the presence or absence of Salmonella infection in Atf5 flox/flox and Atf5 ΔIEC mice. Actin was used as a loading control. Data represent mean ± standard error of the mean (n = 8; ***p < 0.001,****p < 0.0001 using the Student’s t test). (C–E) Oxygen consumption rate (OCR) (C), ATP production (D), and oxidative damage (E) from small intestine samples of Atf5 flox/flox and Atf5 IEC mice in the presence or absence of Salmonella infection. Data represent mean ± standard error of the mean (n = 5; ns, non-significant, *p < 0.05,**p < 0.001, ***p < 0.001,****p < 0.0001 using the Student’s t test). (F) Mitochondrial membrane potential quantification using TMRE from small intestine samples of Atf5 flox/flox and Atf5 ΔIEC mice in the presence or absence of Salmonella infection. Data represent mean ± standard error of the mean (n = 5 ; **** p < 0.0001 using the Student’s t test). (G and H) Changes in bodyweight (G) and feeding (H) of Atf5 flox/flox and Atf5 ΔIEC mice during challenge with Salmonella . Data represent mean ± standard error of the mean (n = 5 ; * p < 0.05 , ** p < 0.001 using the Student’s t test). (I–K) Colony-forming units (CFU) of intestine (I), liver (J) and spleen (K) samples from Salmonella infected Atf5 flox/flox and Atf5 ΔIEC mice. (n = 5; ns, non-significant, ****p < 0.0001 using the Student’s t test). (L) Survival of Atf5 flox/flox and Atf5 ΔIEC mice during challenge with Salmonella (n = 5). See for all statistics pertaining to survival analysis. (M and N) Representative histological analysis (M) and pathology score table (N) of small intestine tissue sections from Atf5 flox/flox and Atf5 ΔIEC mice during challenge with Salmonella (closed arrowhead represents reduced villi height, open arrowhead represents increased spacing between intestinal crypts, arrow and asterisk represent neutrophilic inflammation and villous fusion, respectively; n = 3). Scale bars, 200 μm.

Article Snippet: Atf5 flox/flox , Cyagen Inc. , This manuscript.

Techniques: Western Blot, Control, Infection, Membrane

Journal: Cell reports

Article Title: The mitochondrial UPR regulator ATF5 promotes intestinal barrier function via control of the satiety response

doi: 10.1016/j.celrep.2022.111789

Figure Lengend Snippet: (A) Serum FITC-dextran levels in Atf5 flox/flox and Atf5 ΔIEC 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 Atf5 flox/flox and Atf5 ΔIEC 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 Atf5 flox/flox and Atf5 ΔIEC 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 Atf5 flox/flox and Atf5 ΔIEC 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 Atf5 flox/flox and Atf5 ΔIEC 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 Atf5 flox/flox and Atf5 ΔIEC 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 Atf5 flox/flox and Atf5 ΔIEC 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 Atf5 flox/flox and Atf5 ΔIEC 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 Atf5 flox/flox and Atf5 ΔIEC mice exposed to DSS (arrow and asterisk represent epithelial hyperplasia and extensive ulceration and inflammation, respectively; n = 3). Scale bars, 200 μm.

Article Snippet: Atf5 flox/flox , Cyagen Inc. , This manuscript.

Techniques: Immunohistochemistry, Staining, Western Blot, Control, SDS Page

Journal: Cell reports

Article Title: The mitochondrial UPR regulator ATF5 promotes intestinal barrier function via control of the satiety response

doi: 10.1016/j.celrep.2022.111789

Figure Lengend Snippet: (A) Immunoblot analysis and quantification of UPR mt -related proteins in the presence or absence of 1.5g/L doxycycline in Atf5 flox/flox and Atf5 ΔIEC mice. Actin was used as a loading control. Data represent mean ± standard error of the mean (n = 4; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 using the Student’s t test). (B) Serum FITC-dextran levels in Atf5 flox/flox and Atf5 ΔIEC mice pre-treated with 1.5 g/L doxycycline and subsequently exposed to 4% DSS or mock control. Data represent mean ± standard error of the mean (n = 5; ns, non-significant, *p < 0.05, ****p < 0.0001 using the Student’s t test). (C and D) Bodyweight of Atf5 flox/flox (C) and Atf5 ΔIEC (D) mice pre-treated with 1.5 g/L doxycycline and subsequently exposed to 4% DSS. Data represent mean ± standard error of the mean (n = 5; ns, non-significant, ****p < 0.0001 by the Student’s t test). (E and F) DAI scores of Atf5 flox/flox (E) and Atf5 ΔIEC (F) mice pre-treated with 1.5 g/L doxycycline and subsequently exposed to 4% DSS or mock control. Data represent mean ± standard error of the mean (n = 5; ns, non-significant , *** p < 0.001 by the Student’s t test). (G and H) Survival of Atf5 flox/flox (G) and Atf5 ΔIEC (H) mice pre-treated with 1.5 g/L doxycycline and subsequently exposed to 4% DSS or mock control (n = 5). See for all statistics pertaining to survival analysis. (I) Representative image and quantification of colon lengths in Atf5 flox/flox and Atf5 ΔIEC mice exposed to 4% DSS, in the presence or absence of 1.5 g/L doxycycline pre-treatment. Data represent mean ± standard error of the mean (n = 5; ns, non-significant, *p < 0.05, ***p < 0.001, ****p < 0.0001 using the Student’s t test). Scale bar, 1 cm. (J and K) Representative histological analysis and pathology score table of small intestine tissue sections from Atf5 flox/flox and Atf5 ΔIEC mice pre-treated with 1.5 g/L doxycycline, followed by exposure to 4% DSS (arrow and asterisk represent epithelial hyperplasia and extensive ulceration and inflammation, respectively; n = 3). Scale bars, 200 μm.

Article Snippet: Atf5 flox/flox , Cyagen Inc. , This manuscript.

Techniques: Western Blot, Control

Journal: Cell reports

Article Title: The mitochondrial UPR regulator ATF5 promotes intestinal barrier function via control of the satiety response

doi: 10.1016/j.celrep.2022.111789

Figure Lengend Snippet: (A) Representative image of Atf5 flox/flox and Atf5 ΔIEC mice ( n = 4 ). Scale bar indicates 1.5 cm. (B and C) Bodyweight (B) and feeding behavior (C) of Atf5 flox/flox and Atf5 ΔIEC 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 Atf5 flox/flox and Atf5 ΔIEC 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 Atf5 flox/flox and Atf5 ΔIEC 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 Atf5 flox/flox and Atf5 ΔIEC 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 Atf5 flox/flox and Atf5 ΔIEC 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 Atf5 flox/flox , Atf5 ΔIEC , and leptin-treated Atf5 ΔIEC mice challenged with Salmonella (n = 5). See for all statistics pertaining to survival analysis.

Article Snippet: Atf5 flox/flox , Cyagen Inc. , This manuscript.

Techniques: Injection, Western Blot, Staining, SDS Page

Journal: Cell reports

Article Title: The mitochondrial UPR regulator ATF5 promotes intestinal barrier function via control of the satiety response

doi: 10.1016/j.celrep.2022.111789

Figure Lengend Snippet: (A) Heatmap of differentially expressed genes in intestinal tissues of Atf5 ΔIEC mice compared with Atf5 flox/flox controls, including 269 up and 588 downregulated genes (n = 3). (B) Top 20 URMs predicted based on an ingenuity pathway analysis of genes differentially expressed in intestinal tissues, showing the inferred activation of URMs in Atf5 ΔIEC mice relative to Atf5 flox/flox controls. Z-scores for URM activation are based on observed patterns of gene expression for genes downstream of respective URMs, where the magnitude of the Z score represents evidence for differential activation in Atf5 ΔIEC mice relative Atf5 flox/flox controls, and the sign of the Z score indicates the direction of activation (positive) or repression (negative) in Atf5 ΔIEC mice relative to Atf5 flox/flox controls (n = 3). (C) Top 15 enriched KEGG pathways from differentially expressed genes in Atf5 ΔIEC mice compared with Atf5 flox/flox controls, with distributions of fold change for differentially expressed genes within each KEGG pathway. (D) Cck transcript levels measured by qRT-PCR in Atf5 flox/flox and Atf5 ΔIEC mice. Data represent mean ± standard error of the mean (n = 5; ****p < 0.0001 using the Student’s t test). (E) Serum CCK levels in Atf5 flox/flox and Atf5 ΔIEC mice. Data represent mean ± standard error of the mean (n = 5; ***p < 0.001 using the Student’s t test). (F) Serum leptin levels in Atf5 flox/flox and Atf5 ΔIEC mice, with or without intraperitoneal CCK injection. Data represent mean ± standard error of the mean (n = 5; ns, non-significant, *p < 0.05, ***p < 0.001 using the Student’s t test). (G and H) Bodyweight (G) and feeding behavior (H) of Atf5 flox/flox and Atf5 ΔIEC mice intraperitoneally injected with CCK. Day 0 represents start of experiment at 6 weeks of age. Data represent mean ± standard error of the mean (n = 5; ns, non-significant using the Student’s t test). (I) Serum FITC-dextran levels in Atf5 flox/flox and Atf5 ΔIEC mice, with or without intraperitoneal CCK injection. Data represent mean ± standard error of the mean (n = 5; ns, non-significant , * p < 0.05, **p < 0.01 using the Student’s t test). (J and K) Immunoblot analysis and quantification of DAO (J) and I-FABP (K) serum protein levels in Atf5 flox/flox and Atf5 ΔIEC mice, with or without intraperitoneal CCK injection. Coomassie-stained SDS-PAGE gels are shown as loading controls. Data represent mean ± standard error of the mean (n = 4; *p < 0.05, **p < 0.01, ***p < 0.001 using the Student’s t test). (L) Survival of Atf5 flox/flox and Atf5 ΔIEC mice, with or without intraperitoneal CCK injection, challenged with Salmonella ( n = 5 ). See for all statistics pertaining to survival analysis.

Article Snippet: Atf5 flox/flox , Cyagen Inc. , This manuscript.

Techniques: Activation Assay, Gene Expression, Quantitative RT-PCR, Injection, Western Blot, Staining, SDS Page

Journal: Cell reports

Article Title: The mitochondrial UPR regulator ATF5 promotes intestinal barrier function via control of the satiety response

doi: 10.1016/j.celrep.2022.111789

Figure Lengend Snippet: (A and B) Blood glucose (A) and insulin (B) levels in Atf5 flox/flox and Atf5 ΔIEC 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 Atf5 flox/flox and Atf5 ΔIEC 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 Atf5 flox/flox and Atf5 ΔIEC 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 Atf5 flox/flox and Atf5 ΔIEC 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 Atf5 flox/flox and Atf5 ΔIEC mice, with or without intraperitoneal 2-DG injection, challenged with Salmonella (H) or C . rodentium (I) (n = 5). See 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) Atf5 flox/flox and Atf5 ΔIEC 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).

Article Snippet: Atf5 flox/flox , Cyagen Inc. , This manuscript.

Techniques: Injection, Western Blot, Staining, SDS Page, Infection

Journal: Cell reports

Article Title: The mitochondrial UPR regulator ATF5 promotes intestinal barrier function via control of the satiety response

doi: 10.1016/j.celrep.2022.111789

Figure Lengend Snippet:

Article Snippet: Atf5 flox/flox , Cyagen Inc. , This manuscript.

Techniques: Recombinant, Virus, Isolation, Enzyme-linked Immunosorbent Assay, RNA Sequencing, Software