Figure 4
Induction of AhR signaling and COX-2 expression by VRCZ. (A) HaCaT cells were exposed to 30 μM VRCZ or vehicle (DMSO; 0.07%) for 3 hours. Cells were then fixed and stained with the polyclonal IgG rabbit anti-human AhR Ab followed by incubation with FITC-labeled anti-rabbit IgG secondary antibody. VRCZ induced the translocation of AhR to nucleus. Scale bar: 50 µm. Representative date (n = 3). (B) HaCaT KCs were exposed to 100 μM VRCZ or vehicle for 2 hours. mRNA levels of AhR target genes (CYP1A1, 1A2 and 1B1) were analyzed using real time RT-PCR. Bars indicate mean ± SEM (n = 3). Student’s t test. (C,D) HaCaT cells were preincubated with AhR antagonist CH223191 (10 µM) or GNF351 (500 nM) for 30 minutes and incubated with various concentrations of VRCZ at 10 or 100 μM for 2 hours. The expression levels of AhR-target gene CYP1A1 and COX-2 were monitored. The results are expressed as folds of induction after VRCZ treatments. Bars indicate mean ± SEM (n = 3). Student’s t test. (E–G) HaCaT cells transfected with siRNA-control or siRNA-AhRs were exposed to VRCZ or vehicle for 2 hours. The mRNA expressions of AhR, CYP1A1 and COX-2 were analyzed by real-time RT-PCR. Bars indicate mean ± SEM (E, F: n = 3; G: n = 6). Student’s t test. (H,I) Relative CYP1A1 and COX-2 mRNA expression augmented by VRCZ and its metabolites (VNO and P-VNO) were examined in HaCaT cells after 2 hours incubation with each substance. Date are normalized to GAPDH and expressed as fold increase from DMSO-treated controls. Bars indicate mean ± SEM (n = 3). Student’s t test.
