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. Author manuscript; available in PMC: 2018 Nov 1.
Published in final edited form as: Gastroenterology. 2017 Aug 18;153(5):1338–1350.e3. doi: 10.1053/j.gastro.2017.08.024

Figure 4. TNF Decreases DRA promoter activity via NF-κ B pathway.

Figure 4

HT-29 cells were transiently transfected with human DRA promoter construct (p-1183/+114) along with the mammalian expression vector for β-galactosidase (pCMV-βgal). 24h post-transfection, HT-29 cells were treated with different doses of TNF ranging from 5–100 ng/ml for 24h (A) and with and without 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 24h 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 h with TNF (50 ng/ml) with and without CAPE. Total RNA was extracted and quantitative real-time RT-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 NFκB promoter luciferase reporter construct along with the mammalian expression vector for β-galactosidase (pCMV-β-gal). 24h post-transfection, HT-29 cells were treated with 20 or 50 ng/ml of TNF for 24h. 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 24h with 20 or 50 ng/ml TNF. Nuclear lysates were prepared, run on 10% SDS-PAGE, 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 % of control comparing transfected cells treated with TNF to untreated cells (control). **p<0.001 compared with control, *p<0.05, compared with control. ##p<0.001 compared with TNF, #p<0.05, compared with TNF.