Volume 153, Issue 5, Pages 1338-1350.e3 (November 2017) Activation of Nuclear Factor−κB by Tumor Necrosis Factor in Intestinal Epithelial Cells and Mouse Intestinal Epithelia Reduces Expression of the Chloride Transporter SLC26A3  Anoop Kumar, Ishita Chatterjee, Tarunmeet Gujral, Anas Alakkam, Hayley Coffing, Arivarasu N. Anbazhagan, Alip Borthakur, Seema Saksena, Ravinder K. Gill, Waddah A. Alrefai, Pradeep K. Dudeja  Gastroenterology  Volume 153, Issue 5, Pages 1338-1350.e3 (November 2017) DOI: 10.1053/j.gastro.2017.08.024 Copyright © 2017 AGA Institute Terms and Conditions

Figure 1 TNF decreases DRA expression in HT-29 cells. HT-29 cells grown on Transwell inserts for 10–12 days were treated with different concentrations of TNF (in culture medium supplemented with 0.2% bovine serum albumin) (10–50 ng/mL) for 24 hours (A). Protein lysates were prepared, run on 7.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis, transblotted, and Western blotting was performed utilizing anti-DRA antibody. Representative blot of 3 different experiments. Densitometric analysis shows relative expression of DRA normalized to GAPDH. ∗∗P < .001 vs control (Con). (B) Total RNA was extracted and quantitative real-time reverse transcription PCR was performed utilizing SYBR green fluorescent dye. DRA mRNA levels were normalized to respective GAPDH mRNA (internal control) levels. Results are expressed as fold-changes in mRNA levels in treated cells compared with control. Values are means ± SEM from ≥3 different experiments performed in triplicate. ∗∗P < .001 vs control (C) TNF decreased apical Cl−/HCO3− exchange activity in T-84 cells. Cells were treated with TNF (50 ng/mL) for 24 hours from basolateral sides. Cl−/HCO3− exchange activity was measured as DIDS (4,4′-Diisothiocyano-2,2′-stilbenedisulfonic acid)-sensitive 125I− uptake for 5 minutes. Results are expressed as percent of control and represent means ± SEM of 6 separate experiments performed in triplicate. ∗∗P < .001 vs control. Gastroenterology 2017 153, 1338-1350.e3DOI: (10.1053/j.gastro.2017.08.024) Copyright © 2017 AGA Institute Terms and Conditions

Figure 2 TNF decreases DRA expression in 3D culture of Caco-2 cells. Caco-2 cells grown in 3D culture system were treated with TNF (50 ng/mL, 24 hours). (A) Protein lysates were prepared, run on 7.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis, transblotted, and Western blotting was performed utilizing anti-DRA antibody. Representative blot of 3 different experiments is presented. Densitometric analysis shows relative expression of DRA normalized to GAPDH. ∗∗P < .001 vs control (Con). (B) Untreated or TNF (50 ng/mL, 24 hours) treated Caco-2 cells in 3D culture system were stained for DRA (green), phalloidin (red), and 4′,6-diamidino-2-phenylindole (DAPI) (blue) and visualized by confocal microscopy. Images were obtained with a Zeiss LSM 710 confocal microscope. (C) Total RNA was extracted and quantitative real-time reverse transcription PCR was performed utilizing SYBR green fluorescent dye. Data represent the relative expression of DRA normalized to the respective GAPDH mRNA (internal control) levels. Results are expressed as fold-change in mRNA levels in treated cells compared with control. Data represent mean ± SEM of 4 separate experiments. ∗∗P < .001 compared with control. Gastroenterology 2017 153, 1338-1350.e3DOI: (10.1053/j.gastro.2017.08.024) Copyright © 2017 AGA Institute Terms and Conditions

Figure 3 TNF decreases mouse DRA but not mouse PAT1 mRNA expression in crypt-derived mouse ileal enteroids. Crypt-derived mouse ileal enteroids grown on Matrigel were treated for 6 or 24 hours with TNF (50 ng/mL). Total RNA was extracted and quantitative real-time reverse transcription PCR was performed utilizing SYBR green fluorescent dye. Data represent the relative expression of DRA (A) or PAT1 (B) normalized to the respective GAPDH mRNA (internal control) levels. Results are expressed as fold-change in mRNA levels in treated cells compared with control (Con). Values are means ± SEM from ≥3 different experiments performed in triplicate. ∗∗P < .001 vs control (Con). Gastroenterology 2017 153, 1338-1350.e3DOI: (10.1053/j.gastro.2017.08.024) Copyright © 2017 AGA Institute Terms and Conditions

Figure 4 TNF decreases DRA promoter activity via NF-κB pathway. HT-29 cells were transiently transfected with human DRA promoter construct (p-1183/+114) along with the mammalian expression vector for β-galactosidase (pCMV-β-gal). Twenty-four hours post-transfection, HT-29 cells were treated with different doses of TNF ranging from 5 to 100 ng/mL for 24 hours (A) and with and without caffeic acid phenethyl ester (CAPE) (B). Promoter activity was measured by luciferase assays. Values were normalized to β-galactosidase activity to correct for transfection efficiency. (C) HT-29 cells grown on Transwell inserts for 10–12 days were treated for 24 hours with TNF (50 ng/mL) and with or without, NF-κB inhibitor, CAPE (25 μM) (D) Crypt-derived mouse Ileal enteroids grown on Matrigel were treated for 24 hours with TNF (50 ng/mL) with and without CAPE. Total RNA was extracted and quantitative real-time reverse transcription PCR was performed utilizing SYBR green fluorescent dye. Data represent the relative expression of DRA normalized to the respective GAPDH or B2M mRNA (internal control) levels. Results are expressed as fold-change in mRNA levels in treated cells compared with control. (E) HT-29 cells were transiently transfected with NF-κB promoter luciferase reporter construct along with the mammalian expression vector for β-galactosidase (pCMV-β-gal). Twenty-four hours post-transfection, HT-29 cells were treated with 20 or 50 ng/mL TNF for 24 hours. Promoter activity was measured by luciferase assays. Values were normalized to β-galactosidase activity to correct for transfection efficiency, (F) HT-29 cells grown on Transwell inserts for 10–12 days were treated for 24 hours with 20 or 50 ng/mL TNF. Nuclear lysates were prepared, run on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, transblotted, and Western blotting was performed utilizing anti-p65 or p50 subunit antibody. Representative blot of 3 different experiments is shown. Results represent mean ± SEM of 3–4 separate experiments performed in triplicate and expressed as percent of control comparing transfected cells treated with TNF to untreated cells (control). ∗∗P < .001 compared with control; ∗P < .05 compared with control. ##P < .001 compared with TNF; #P < .05 compared with TNF. Gastroenterology 2017 153, 1338-1350.e3DOI: (10.1053/j.gastro.2017.08.024) Copyright © 2017 AGA Institute Terms and Conditions

Figure 5 siRNA knockdown of IκBα decreases DRA expression: Caco-2 cells were transfected with scrambled or IκB/α-specific siRNA for 48 hours. siRNA-treated knockdown of IκB/α was confirmed by mRNA (A) and immunoblotting (B) utilizing primers specific for IκB/α and anti-IκB/α antibody. (C) Total RNA was extracted and quantitative real-time RT-PCR for DRA was performed utilizing SYBR green fluorescent dye. Data represent the relative expression of DRA normalized to the respective GAPDH mRNA (internal control) levels. Results are expressed as fold-change in mRNA levels in treated cells compared with control. Values represent means ± SEM of 3 separate experiments. ∗∗P < .001 compared with control. (D) Protein lysates were prepared, run on 7.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis, transblotted, and Western blotting was performed utilizing anti-DRA antibody. Representative blot of 3 different experiments is shown. Densitometric analysis shows relative expression of DRA normalized to GAPDH. ∗∗P < .001 vs control. Gastroenterology 2017 153, 1338-1350.e3DOI: (10.1053/j.gastro.2017.08.024) Copyright © 2017 AGA Institute Terms and Conditions

Figure 6 Ectopic expression of NF-κB subunit (p65 and p50) inhibits DRA expression (A). Caco-2 cells were transiently transfected with DRA promoter construct with and without p65 or p50 subunit expression vector or together along with the mammalian expression vector for β-galactosidase (pCMV-β-gal). Twenty-four hours post-transfection, promoter activity was measured by luciferase assay. Values were normalized to β-galactosidase activity. (B) Caco-2 cells were transiently transfected with and without p65 or p50 subunit expression vector or both together. Total RNA was extracted and quantitative real-time reverse transcription PCR was performed utilizing SYBR green fluorescent dye. Data represent the relative expression of DRA normalized to the respective GAPDH mRNA (internal control) levels. (C) Caco-2 cells were transiently transfected with and without p65 or p50 subunit expression vector or both. Protein lysates were prepared, run on 7.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis, transblotted, and Western blotting was performed utilizing anti-DRA antibody. Representative blot of 3 different experiments is shown. Densitometric analysis shows relative expression of DRA normalized to GAPDH. (D) Identification of the NF-κB responsive region in DRA promoter. Caco-2 cells transiently transfected with and without p65 subunit expression vector were fixed in formaldehyde, chromatin was sonicated, and immunoprecipitated with specific antibody against p65. After reverse cross-linking and DNA extraction, immunoprecipitated DNA was used as the template for real-time quantitative PCR utilizing primers spanning nucleotides −1183/+114 of DRA. Enrichment of the p65 was observed at −375/−84 bp and −935/−629 bp regions of DRA promoter. Values are means ± SEM from 3 or more different experiments. ∗P < .05; ∗∗P < .001 vs control. Gastroenterology 2017 153, 1338-1350.e3DOI: (10.1053/j.gastro.2017.08.024) Copyright © 2017 AGA Institute Terms and Conditions

Figure 7 In vivo effects of TNF on DRA expression in C57BL/6 mice. TNF (5 μg/mouse in 200 μL PBS) or vehicle alone was injected intraperitoneally into C57BL/6 mice. After 3 or 6 hours, mice were sacrificed, intestines were removed, and mucosa scraped from ileal and colonic regions for RNA isolation. Mouse DRA mRNA was measured by real-time PCR using gene-specific primers. Relative DRA mRNA expression in ileal (A) and colonic mucosa (B) of mice treated with vehicle or TNF. Values are means ± SEM from 5 mice. ∗∗P < .001; ∗P < .05 vs control. TNF decreases DRA immunostaining in ileum (C) and colon (D). Green: DRA; red: villin; blue: nuclei. Gastroenterology 2017 153, 1338-1350.e3DOI: (10.1053/j.gastro.2017.08.024) Copyright © 2017 AGA Institute Terms and Conditions

Supplementary Figure 1 HT-29 cells were transiently transfected with DRA promoter construct (p-1183/+114) along with the mammalian expression vector for β-galactosidase (pCMV-β-gal). 6 hours or 12 hours post-transfection, HT-29 cells were treated with different doses of TNF ranging from 5 to 100 ng/mL for 6 hours (A) or 12 hours (B). Promoter activity was measured by luciferase assays. Values were normalized to β-galactosidase activity to correct for transfection efficiency. Results represent mean ± SEM of 3 separate experiments performed in triplicate and expressed as percent of control comparing transfected cells treated with TNF to untreated cells (control). ∗∗P < .001 compared with control. Gastroenterology 2017 153, 1338-1350.e3DOI: (10.1053/j.gastro.2017.08.024) Copyright © 2017 AGA Institute Terms and Conditions

Supplementary Figure 2 Caco-2 cells were transfected with scrambled or IκB/α-specific siRNA for 48 hours. Scrambled or IκB/α-treated Caco-2 cells were stained for p65 (green), 4′,6-diamidino-2-phenylindole (DAPI) (blue), and visualized by confocal microscopy. Images were obtained with a Zeiss LSM 710 confocal microscope. Gastroenterology 2017 153, 1338-1350.e3DOI: (10.1053/j.gastro.2017.08.024) Copyright © 2017 AGA Institute Terms and Conditions

Supplementary Figure 3 Caco-2 cells were transiently transfected with DRA promoter construct with and without p65 or p50 subunit expression vector. Twenty-four hours post-transfection. Nuclear lysates were prepared, run on 7.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis, transblotted, and Western blotting was performed utilizing anti-p65 or p50 subunit antibody. Representative blot of 3 different experiments is shown. Gastroenterology 2017 153, 1338-1350.e3DOI: (10.1053/j.gastro.2017.08.024) Copyright © 2017 AGA Institute Terms and Conditions

Supplementary Figure 4 Caco-2 cells were transiently transfected with DRA promoter construct with (A) different doses of p65 (0.2–1 μg/well) or (B) p50 (0.2–1 μg/well) β-galactosidase (pCMV-β-gal). Twenty-four hours post-transfection, promoter activity was measured by luciferase assays. Values were normalized to β-galactosidase activity. (C) T-84 cells were transiently transfected with DRA promoter construct with and without p65 or p50 subunit expression vector or together along with the mammalian expression vector for β-galactosidase (pCMV-β-gal). Twenty-four hours post-transfection, promoter activity was measured by luciferase assays. Values were normalized to β-galactosidase activity. Results represent mean ± SEM of 3 separate experiments performed in triplicate and expressed as percent of control comparing p65 or vehicle transfected (control). ∗∗P < .001 compared with control. Gastroenterology 2017 153, 1338-1350.e3DOI: (10.1053/j.gastro.2017.08.024) Copyright © 2017 AGA Institute Terms and Conditions

Supplementary Figure 5 Caco-2 cells grown in 3D culture system were treated with TNF (50 ng/mL, 24 hours). Total RNA was extracted and quantitative real-time reverse transcription PCR was performed utilizing SYBR green fluorescent dye. Data represent the relative expression of NHE2 (A) and ZO-1 (B) normalized to the respective GAPDH mRNA (internal control) levels. Results are expressed as fold-change in mRNA levels in treated cells compared with control. Data represent mean ± SEM of 3 separate experiments. ∗∗P < .001 compared with control. Gastroenterology 2017 153, 1338-1350.e3DOI: (10.1053/j.gastro.2017.08.024) Copyright © 2017 AGA Institute Terms and Conditions