Apigenin promotes pigmentation through the c-KIT-CRTCs/CREB signaling pathway. (A,B) HEMCs were pretreated with or without 10 μM ISCK03 (c-KIT inhibitor) for 1 h before apigenin was added for an additional 1 h. Phosphorylated CREB (p-CREB) and nuclear levels of CRTC1 were measured by Western blotting. Histone H3 served as a reference for nuclear proteins, and β-actin served as a reference for cytosolic proteins. (C,D) HEMCs were pretreated with or without 10 μM ISCK03 for 1 h before apigenin was added for 48 h. Melanin content and the expression levels of Tyrosinase, MITF, Cdc42, and Rab27a were measured as described previously. (E,F) HEMCs were transfected with si-NC or si-c-KIT for 24 h, followed by apigenin (10 μM) treatment for 48 h. Melanin content and the expression levels of Tyrosinase, MITF, Cdc42, and Rab27a were measured as described previously. (G) Binding of c-KIT with apigenin was analyzed by microscale thermophoresis (MST). The binding curve represents data points from 3 measurements. The calculated Kd is 2.6 ± 0.14 µM. Data are expressed as means ± SEM (n = 3). *p < 0.05 vs. non-treated cells, #p < 0.05 vs. apigenin-treated cells. c-KIT, cellular-KIT; CREB, cAMP response element-binding protein; CRTC, CREB-regulated co-activator; HEMCs, human epidermal melanocytes; MITF, Melanocytes inducing transcription factor; Cdc42, cell division cycle 42; Rab27a, Ras-related protein Rab-27a.
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