1. Melanocytes Sense Blue Light and Regulate Pigmentation through Opsin-3
Claire Regazzetti, Laura Sormani, Delphine Debayle, Françoise Bernerd, Meri K. Tulic, Gian Marco De Donatis, Bérengère Chignon- Sicard, Stéphane Rocchi, Thierry Passeron 
Journal of Investigative Dermatology 
Volume 138, Issue 1, Pages (January 2018)
DOI: /j.jid
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2. Figure 1
Blue light stimulates melanogenesis. NHMs (skin type IV) were stimulated with 50J/cm2 of 415-nm light. Cells were lysed (a) immediately after stimulation or (b, c) 3 days later. (a, b) The lysate was analyzed by Western blot with indicated antibodies, and (c) the relative protein level was quantified (n = 5). NHMs (skin type V) from two different donors were stimulated one time with 50J/cm2 of 415-nm light. (d) After 10 days, cells were observed with bright field microscopy. Scale bar = 100 μm. (e) Skin abdominoplasty was stimulated with 5 to 90 J/cm2 of 415-nm light, biopsied, and placed in ex vivo culture for 5 days. (f) Biopsy samples were lysed, and proteins were analyzed by Western blot with indicated antibodies. ∗∗P < DCT, dopachrome tautomerase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NHM, normal human melanocyte.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2017 The Authors Terms and Conditions

3. Figure 2
OPN3 senses blue light in melanocytes and mediates melanogenesis. Relative expression level of opsins in NHMs (skin type IV) analyzed by quantitative PCR. (a) OPN levels were normalized with actin level (n = 6). NHMs (skin type IV) were transfected with siRNA directed against OPN3 or control. Two days later, cells were stimulated with 50 J/cm2 of 415-nm light and lysed (c) immediately or (d) 3 days later. The lysate was analyzed by Western blot with indicated antibodies. The efficiency of siRNA was determined by (b) quantitative PCR. Ctl, control; ct, control; DCT, dopachrome tautomerase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; si, small interfering; TYR, tyrosinase.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2017 The Authors Terms and Conditions

4. Figure 3
Melanogenesis induced by blue light and OPN3 stimulation is calcium dependent. NHM (skin type IV) were stimulated with 50J/cm2 of 415nm light. Immediately after the stimulation, calcium flux was quantified with Fluo4 (a) and protein lysate was analyzed by western blot (b–c). NHM transfected with siRNA directed against OPN3 or control were stimulated with 50J/cm2 of 415nm light, calcium flux was quantified with Fluo4 (d) and protein lysate was analyzed by western blot (e) NHM treated with BAPTA (3μM) or KN-93 (5μM) were stimulated with 50J/cm2 of 415nm light and protein lysate was analyzed after stimulation (f). ∗∗P < ct, control; Ctrl, control; ERK, extracellular signal-regulated kinase; NHM, normal human melanocyte; p, phosphorylated.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2017 The Authors Terms and Conditions

5. Figure 4
Blue light induces formation of tyr/p complex leading to a sustained tyrosinase activity in dark phototype melanocytes. NHM (skin type IV) were stimulated with 50 mJ/cm2 of 415-nm light. (a) Multimeric tyrosinase/TYRP protein was observed on tyrosinase Western blot. NHMs were transfected with siRNA directed against OPN3 or control. (b) Two days later, cells were stimulated with 50 J/cm2 of 415-nm light and lysed immediately. NHMs from phototypes I and II or V and VI (two lots/conditions) were stimulated with 50 mJ/cm2 of 415-nm light. (d) Two weeks later, tyrosinase activity was visualized by immunofluorescence (scale bar = 100 μm), and (c) the lysate after stimulation was analyzed by Western blot. Ct, control; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NHM, normal human melanocyte; si, small interfering.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2017 The Authors Terms and Conditions