Figure 6

Activity of the mouse Pit1 promoter is upregulated by p65/ NFκB. (A) Schematic representation of mPit1p cloned upstream of the luciferase cDNA in a pGL3-LUC plasmid (mPiT1p-LUC). In silico analysis identified six putative binding sites for the p65 subunit of NF-κB (upper panel) and two putative binding sites of AP-1 (lower panel). (B) Analysis of mPit1p activity by luciferase assay following p65 transfection. HEK293 cells were cotransfected with mPit1-LUC (or pGL3-LUC) and sport6 control plasmid or plasmid encoding p65, p105, or EKLF (positive control). Luciferase activity was normalized to that of the pGL3-LUC, which is devoid of any regulator region upstream of the luciferase cDNA. Data are means ± S.E.M. of at least three experiments. Mann-Whitney Rank Sum test was performed. ^#p < 0.05; ^##p < 0.01 vs pGL3-LUC + sport6 condition. (C) Analysis of mPit1p activity by luciferase assay following AP-1 transfection. HEK293 cells were cotransfected with m Pit1-LUC (or pGL3-LUC) and sport6 control plasmid or plasmid encoding the c-Jun or c-Fos. Luciferase activity was normalized to that of the pGL3-LUC. Data are means ± S.E.M. of at least three experiments. Mann-Whitney Rank Sum test was performed. ^#p < 0.05; ^##p < 0.01 vs pGL3-LUC + sport6 condition. (D) RT-qPCR analysis of Pit1 mRNA expression in WT MEFs treated with 20 μM BAY11-7082 or vehicle for 30 minutes prior to stimulation with 100 ng/ml. Data are means ± S.E.M. of at least three experiments. Student’s unpaired t-test or an Unpaired t-test with Welch correction for groups with unequal variance was performed ^#indicates comparison with the untreated condition: ^##p < 0.01; *indicates comparison with LPS only treated cells; **p < 0.01. (E) Representative western blot of PiT1 in WT MEFs treated with 20 μM BAY11-7082 or vehicle for 30 minutes prior to stimulation with 100 ng/ml LPS for 4 h. Full-length blots are presented in Supplementary Fig. 9. (F) q-PCR analysis of p65 ChIP experiments. DNA immunoprecipitated with p65 from MEFs treated (black bar) or not (white bar) with 100 ng/ml LPS for 90 min was analyzed by quantitative PCR using primers located along the mouse Pit1 promoter. Negative controls (grey bars) were performed using DNA incubated with beads but without anti-p65 antibody. Cxcl2 was used as positive control. Means ± S.E.M. of three experiments are presented. Student’s unpaired t-test or an Unpaired t-test with Welch correction for groups with unequal variance was performed *indicates significant difference from untreated control cells at p < 0.05.