Figure 4

Nuclear translocation of OVOL1 was likely to be inhibited in AD skin, leading to the reduced FLG expression in AD skin. Normal skin (a) and AD skin (d) were stained with hematoxylin and eosin. The scale bar is 100 μm. Expression of FLG and OVOL1 in the epidermis of the same skin lesion was analyzed by IHC staining for FLG (red) or OVOL1 (red). The expression of FLG was observed in normal skin (b) and was low in AD skin (e). The expression of OVOL1 was observed mainly in the nuclei of keratinocytes in normal skin (c); however, nuclear OVOL1 expression was lower in AD skin (f). For semiquantitative analysis of IHC staining, microscopic visual fields of the samples from each group were randomly chosen and examined. In a high-power field (× 400 magnification), the nuclear-OVOL1-stained cells of the epidermis were counted, as were all the cells with hematoxylin staining. Nuclear OVOL1 expression was lower in AD skin (AD) compared with normal skin (NS) (g). NHEKs treated with DMSO (h), FICZ (100 nM) (i), IL-4 (10 ng/ml) (j), or FICZ plus IL-4 (k) for 24 h were stained with an anti-OVOL1 antibody (primary antibody) and an Alexa Fluor 488-conjugated anti-rabbit IgG antibody (secondary). The nuclei were counterstained with DAPI (blue). Confocal laser scanning images revealed that OVOL1 expression was noticeable mainly in the cytoplasm in a steady state (h) and that the AHR activation by FICZ induced nuclear translocation of OVOL1 (i). In contrast, IL-4 did not induce nuclear translocation of OVOL1, and the latter was retained in the cytoplasm (j). IL-4-mediated blockade of the nuclear translocation of OVOL1 was overridden by treatment with FICZ (k). (l) Isotype negative control. The scale bar is 25 μm. The data are representative of experiments repeated three times with similar results. (m) NHEKs were treated with FICZ (100 nM) in the absence or presence of IL-4 (10 ng/ml) for 18 h. Cellular nuclear protein was extracted using a biochemical subcellular fractionation technique. The OVOL1 levels in the nuclear protein fraction of NHEKs were evaluated by western blotting. The activation of AHR by FICZ increased the nuclear OVOL1 expression; in contrast, IL-4 did not change nuclear expression of OVOL1. The IL-4-mediated blockade of the OVOL1 nuclear translocation was partially reversed by treatment with FICZ. The data are representative of experiments repeated three times with similar results