Figure 3

AHR regulated FLG expression via OVOL1 in NHEKs. (a–d) Data are expressed as mean±S.E.M.; n=3 for each group; *P<0.05. NHEKs were transfected with control siRNA or AHR siRNA and then treated with FICZ (100 nM) or Glyteer (0.001%) for 24 h. Expression of OVOL1 and FLG in the NHEKs was analyzed by qRT-PCR. (e–k) NHEKs transfected with control siRNA, AHR siRNA, or OVOL1 siRNA were treated with DMSO or FICZ (100 nM) for 24 h and then stained with an anti-FLG antibody (primary antibody) and an Alexa Fluor 546-conjugated anti-mouse IgG antibody (secondary). The nuclei were counterstained with DAPI (blue). (e) Control siRNA-transfected NHEKs treated with DMSO, (f) control siRNA-transfected NHEKs treated with FICZ, (g) AHR siRNA-transfected NHEKs treated with DMSO, (h) AHR siRNA-transfected NHEKs treated with FICZ, (i) OVOL1 siRNA-transfected NHEKs treated with DMSO, and (j) OVOL1 siRNA-transfected NHEKs treated with FICZ. (k) Isotype negative control. Confocal laser scanning images revealed increased FLG expression (red) in control siRNA-transfected NHEKs treated with Glyteer (f) as compared with control siRNA-transfected NHEKs treated with DMSO (e); this upregulation was abrogated in AHR siRNA- or OVOL1 siRNA-transfected NHEKs treated with FICZ (h and j). The data are representative of experiments repeated three times with similar results. The scale bar is 25 μm. (l) 3D-cultured NHEKs were treated with CH-223191 (10 μM), FICZ (1 μM), or CH-223191 (10 μM) plus FICZ (1 μM), or CH-223191 (10 μM), Glyteer (0.01%), or CH-223191 (10 μM) plus Glyteer (0.01%) for 48 h. Total cell lysates were prepared and subjected to western blot analysis with an anti-FLG antibody and an anti-OVOL1 antibody. The data are representative of experiments repeated three times with similar results