Figure 4.

Ultraviolet radiation (UVR) activates the Ca²⁺/protein kinase C (PKC)/microphthalmia‐associated transcription factor (MITF) signalling pathway through opsin 5 (OPN5) and upregulates expression of tyrosinase (TYR), tyrosinase‐related protein 1 (TRP1) and TRP2. (a, b) Images of representative human epidermal melanocytes (HEMs) and PIG1 human melanocyte cells loaded with the Ca²⁺ indicator Fluo‐3 and irradiated with UVR. Scale bar = 20 µm. HEMs and PIG1 calcium fluxes were quantified by flow cytometry. Data are shown as mean ± SEM of three independent experiments. Statistical significance was determined by one‐way anova followed by Tukey’s test. **P < 0·01 ***P < 0·001. (c, d) Cells were transfected with (c) short interfering (si)RNA against OPN5, or (d) lentivirus overexpressing OPN5 (H‐OPN5), then irradiated with or without UVR and lysed after 48 h. Lysates were analysed by Western blotting using the indicated antibodies. (e, f) Cells were irradiated with and without UVR, and mitogen‐activated protein kinase (MAPK) and PKC protein and phosphorylated (p‐) protein levels were determined by Western blot analysis. (g) Cells were transfected with short interfering RNA (RNAi) against OPN5 irradiated with UVR and lysed after 48 h. Lysates were analysed by Western blotting using the indicated antibodies. (h, i) After inhibiting the expression of MITF with ML329, cells were irradiated with or without UVR, and lysed 48 h later to observe PKC protein expression. Beta‐actin was used as a loading control. CAMKII, calmodulin‐dependent protein kinase II.