Fig. 2

p68 transcriptionally regulates RelA gene expression through its occupancy at the TBE sites of the RelA promoter. a Multiple colon cancer cell lines as indicated were seeded in 35 mm plates and transfected with 2 μg of pGZ-p68 or EV. Total RNA was extracted 36 h post transfection followed by analysis of RelA mRNA by qRT–PCR. b Multiple colon cancer cell lines as indicated were seeded in 35 mm plates and transfected with 2 μg of scr (control), or sh p68–1 or sh p68–2. Total RNA was extracted 48 h post transfection, followed by analysis of RelA mRNA by qRT–PCR. For both (a) and (b) Cyclin D1 (CCND1) mRNA expression served as positive control. 18S rRNA was used for normalization. c HCT 116 (left) and SW-480 (right) cells were seeded in 35 mm plates and co-transfected with pGL3-RelA-prom (1 μg), Renilla luciferase plasmid (50 ng) and either EV or pIRES-p68 (1 μg). The luciferase activity was measured, 36 h post transfection. d HCT 116 (left) and SW-480 (right) cells were seeded in 35 mm plates and transfected with either shRNA1 (1 μg) or shRNA2 (1 μg) of p68 or scr (control- EV) (1 μg) along with pGL3-RelA-prom (1 μg) and Renilla luciferase plasmid (50 ng). The luciferase activity was measured, 48 h post transfection. For both (c) and (d) cells co-transfected with 1 μg of EV, pGL3-RelA-prom and Renilla luciferase plasmid (50 ng) served as control. e Schematic representation of the RelA promoter showing multiple TCF-binding elements (TBE) sites. f HCT 116 cells were grown in 100 mm cell culture dishes followed by Chromatin immunoprecipitation (ChIP) assay using the indicated antibodies. Amplification of Cyclin D1 promoter region containing TBE served as positive control for both p68 and β-catenin. Actin promoter served as positive control for Pol II. PCR amplification of the immunoprecipitated DNAs was performed using primers designed from the RelA promoter region – TBE [1–3]-(349 bp), flanking TBE1, TBE2 and TBE3; TBE4-(296 bp), flanking TBE4; TBE5-(140 bp), flanking TBE5 and TBE [6–7]-(213 bp), flanking TBE6 & TBE7. g HCT 116 cells were grown in 100 mm plates and transfected with 8 μl scr (control) or siRNA against p68 (si p68) followed by ChIP assay using the indicated antibodies; 48 h post transfection. Quantification of the immunoprecipitated DNA was done by qRT–PCR. Results were determined first as percentage of input and then presented as fold enrichment relative to IgG. For both (f) and (g) ChIP using antibody against RNA Polymerase II (Pol II) and IgG served as positive and negative control, respectively. DNA extract (10% without ChIP) was used as input. NS denotes ‘not significant’. h HCT 116 (left) and SW-480 (right) cells seeded in 35 mm plates were transfected with either pIRES-p68 or pIRES-EV (1 μg) along with pGL3-WT-RelA-prom or its deletion constructs (1 μg) and Renilla luciferase plasmid (50 ng). Cells co-transfected with (1 μg) of EV (pIRES), pGL3-WT-RelA-prom and Renilla luciferase plasmid (50 ng) served as control. The luciferase activity was measured, 36 h post transfection. For (c), (d) and (h) Renilla luciferase activity was used for normalization and data is represented as fold activity with respect to control. 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 ****