Figure 4

Effect of NFκB in resistin-induced SDF-1 mRNA expression and promoter activity. (A) SDF-1 mRNA and (B) SDF-1-1010-Luc activity were determined in TSGH 9201 cells pretreated with vehicle (DMSO), NF-κB inhibitor Pyrrolidinedithiocarbamate Ammonium (PDTC), or peptides (SN50), transfected with control siRNA (si-CL), si-p65, or si-p50), and then stimulated with 25 ng/mL resistin for 4 h. SDF-1 mRNA levels were determined through real-time PCR in TSGH cells and normalized to 18S rRNA. (C) SDF-1-1010-Luc activity were determined in TSGH cells with or without resistin for 2 h. The SDF-1 promoter p1010-Luc plasmid and several deletion promoter constructs. Right panel: TSGH9201 cells were cotransfected with 5′-deletion constructs and stimulated with 25 ng/mL resistin for 2 h. SDF-1 promoter activity was measured using a luciferase assay normalized to β-galactosidase activity and was shown to be relative to that of TSGH cells transfected with p1010-Luc (set to 100%). All bar graphs represent folds of CL cells. *P < 0.05 versus CL (Vehicle control, DMSO and p1010-Luc). (D) TSGH 9201 cells were transfected with control siRNA (si-CL), or a specific siRNA of si-p65 and si-p50 and then the cells were treated with 25 ng/mL resistin for 6 h. Total cell lysates were extracted, and the proteins of p65, p50 and β-actin were immunodetected.