Fig 1: Silencing of Pmel17 in MCs by lentivirus-based shRNA fails to affect the expression of FHL2 and E-cad and the cytoskeletal remodeling of KCs. (a) MCs were transfected with shRNA #3 and then were irradiated with 30 mJ/cm2 UVB. Western blotting was performed to confirm the protein levels of Pmel17. The histogram shows the densitometric quantification of data with means ± SD from 3 independent experiments. ∗P < 0.05; ∗∗P < 0.01. (b) KCs were treated with CM from MCs transfected with or without Pmel17 shRNA (MC-CM/shPmel17-2) or irradiated with or without 30 mJ/cm2 UVB. Western blotting was performed to detect the protein levels of FHL2 and E-cad; representative blots are shown on the left. The histograms show the densitometric quantification of data with means ± SD from 3 independent experiments. ∗P < 0.05; ∗∗P < 0.01. (c) Representative images of immunostaining for F-actin (green, upper panels) and E-cad (green, lower panels). DAPI (blue) was used to visualize cell nuclei. Scale bars = 20 μm. Histograms (on the right) show the length-to-width ratio and the fluorescence intensity of 20 cells. Data represent means ± SD of three independent experiments. ns: no significant difference.
Fig 2: Expression profiles of FHL2/sPmel17 and E-cad/Dct in matched skin from healthy subjects and from depigmented and from repigmented vitiligo. Skin specimens were biopsied from normal skin, depigmented skin, and repigmented skin. (a) Representative images of immunofluorescence costaining of FHL2 (green) and sPmel17 (red) in normal skin (upper panels), in depigmented lesional skin (middle panels), and in repigmented lesional skin (lower panels). Scale bar = 20 μm. (b) Representative images of immunofluorescence costaining of E-cadherin (green) and DCT (red) in biopsies collected from normal skin, depigmented skin, and repigmented skin, high-magnification images of the boxed areas in merged image panel. Scale bar = 50 μm. Histograms show the FHL2, sPmel17, and E-cadherin relative fluorescence intensity of 10 fields. Data represent means ± SD of three independent experiments. ∗P < 0.05; ∗∗P < 0.01.
Fig 3: Expression profiles of FHL2 and E-cad in KCs treated with the CM from UVB-exposed or UVB-unexposed MCs. Primary human epidermal KCs were isolated from juvenile foreskin tissues, then treated with the CM from MCs, which had been exposed or unexposed to 30 mJ/cm2 UVB. (a) qRT-PCR was performed to measure mRNA levels of FHL2 and CDH1 (encoding E-cad) in KCs treated with the CM from UVB-exposed (MC-CM/UVB) or UVB-unexposed (MC-CM/Sham) MCs. The results were normalized to the housekeeping gene GAPDH. The data represent means ± SD from 3 independent experiments. ∗P < 0.05; ∗∗P < 0.01. (b) Protein levels of FHL2 and E-cad were analyzed by western blotting. Representative blots are shown on the left. The histograms show the densitometric quantification of data with means ± SD from 3 independent experiments. ∗P < 0.05; ∗∗P < 0.01. (c) Representative images of immunostaining for F-actin (labeled by FITC-phalloidin; upper panel) and E-cad (lower panel). Nuclei were counterstained with DAPI (blue). Histograms (on the right) show the length-to-width ratio and the fluorescence intensity of 20 cells. Data represent means ± SD of three independent experiments. ∗∗P < 0.01. Scale bars = 20 μm.
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