Fig. 5.

FoxO6 induced inflammation through PAR2 in HepG2 cells and PAR2-KO mice. (A) Cells transfected with the FoxO6 vector (100 MOI), pre-incubated with PAR2-siRNA (25 nM) for 24 h were subjected to qRT-PCR analysis using actin as a control. FoxO6, PAR2, IL-1β, TF, and AP-1 mRNA levels were assessed. The results were normalized with respect to actin levels. ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 vs. Normal; ^∗p < 0.05, ^∗∗p < 0.01 vs. FoxO6 treated group. (B) HepG2 cells were transiently transfected pre-incubated with PAR2-siRNA (25 nM) for 24 h with or without FoxO6 (100 MOI). Cells were analyzed by Western blotting using FoxO6, IL-1β, PAR2, p-ERK1/2, ERK1/2, AP-1 (c-Jun), PEPCK, p-JNK1/2, JNK1/2, p-IRS (S307), p-IRS (T632), IRS, p-Akt, Akt, and β-actin antibodies. Bars in densitometry data represent means ± SE, and significance was determined using an unpaired t-test: ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 vs. Normal; ^∗∗p < 0.01, ^∗∗∗p < 0.001 vs. FoxO6 treated cells. (C) FoxO6, p-c-Jun, c-Jun, PAR2, p-IRS (S307), p-IRS (T632), IRS, p-Akt, Akt, p-JNK1/2, and JNK1/2 levels were assessed using nuclear and cytosolic proteins isolated from the liver samples of PAR2-KO mice. TFIIB was the loading control of the nuclear fraction. β-actin was the loading control of the cytosolic fractions. Results are representative of three independent experiments. Bars in densitometry data represent means (n = 4) ± SE, and significance was determined using an unpaired t-test: ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 vs. wt.