Fig. 3

β-catenin regulates RelA gene expression in colon cancer cells. a Representative images depicting immunohistochemical (IHC) staining of β-catenin, TCF4 and RelA conducted in tissue sections derived from human normal colon and colon carcinoma samples. Images were captured at 200X magnification. b Scatter plots representing the mean H-scores of β-catenin, TCF4 and RelA in normal (n = 22) and colon carcinoma tissue (n = 45) samples, respectively. c Determination of Spearman’s rank correlation coefficient (rs) between the mean H-scores of β-catenin and RelA, analyzed from both normal and colon carcinoma tissues in combination. d Multiple colon cancer cell lines, grown in 60 mm cell culture dishes were transfected with 4 μg of pGZ-β-catenin or EV. Cell lysates were prepared, 36 h post transfection and probed for the indicated proteins by IB. ‘Exo’ and ‘Endo’ depicts exogenous and endogenous β-catenin, respectively. e Colon cancer cell lines, grown in 60 mm cell culture dishes were transfected with 4 μg of scr or β-catenin shRNA (represented as sh β-cat) plasmids. f HCT 116 cells were seeded in 60 mm plates and were transfected with scr or sh β-cat plasmids in a dose-dependent manner as indicated. For both (e) and (f) cell lysates were prepared, 48 h post transfection and probed for the indicated proteins by IB. Densitometry values below the blots in panels (d), (e) and (f) are relative to the loading control (Actin). g Colon cancer cell lines as indicated were seeded in 35 mm plates and transfected with 2 μg of pGZ- β-catenin or EV. Total RNA was extracted 36 h post transfection, followed by analysis of RelA mRNA by qRT–PCR. h Colon cancer cell lines as indicated were seeded in 35 mm plates and transfected with 2 μg of scr or sh β-cat. Total RNA was extracted, 48 h post transfection followed by analysis of RelA mRNA by qRT–PCR. For both (g) and (h) Cyclin D1 and c-Myc mRNA expression served as positive controls for β-catenin transfection. 18S rRNA was used for normalization. i HCT 116 (left) and SW-480 (right) cells were seeded in 35 mm plates and transfected with β-catenin siRNA (si β-cat,1 μl) or scr siRNA (control, 1 μl) along with pGL3-RelA-prom (1 μg) and Renilla luciferase plasmid (50 ng). The luciferase activity was measured, 48 h post transfection. j HCT 116 (left) and SW-480 (right) cells were seeded in 35 mm plates and co-transfected with either EV or pcDNA3.1-myc-his-Wnt3a (1 μg) along with pGL3-RelA-prom (1 μg) and Renilla luciferase construct (50 ng). k HCT 116 (left panel) and SW-480 (right panel) cells seeded in 35 mm plates were transfected with either pcDNA3.1-myc-his-Wnt3a or pcDNA3.1-myc-his (EV,1 μg) along with pGL3-WT-RelA-prom or its deletion constructs (1 μg) and Renilla luciferase plasmid (50 ng). For both (j) and (k) the luciferase activity was measured, 36 h post transfection. For (i), (j) and (k) cells co-transfected with (1 μg) of EV/control, pGL3-WT-RelA-prom and Renilla luciferase plasmid (50 ng) served as control. Renilla luciferase activity was used for normalization and data is represented as fold activity with respect to control cells. Error bars in all the indicated sub-figures represent mean (+) s.d from three independent biological repeats. Indicated P-values were calculated using Student’s t-test and P < 0.0001 is represented as ****