Fig 1: Confirmation of the quaternary ceRNA network regulating TYR. (A) After transfection with siRNAs of ENST00000606533 in MCs for 48 h, the transfection efficiency of si-ENST00000606533 mix (mixing the three siRNAs) was most significant, and the expression of circ_0091223, miR-1291, and TYR were detected by transfection with si-ENST00000606533 mix. (B) After transfection with siRNAs of circ_0091223 for 48 h, the transfection efficiency of si-circ_0091223 2 was most significant, and the expression of ENST00000606533, miR-1291, and TYR were detected by transfection with si-circ_0091223 2. (C) After transfection with miR-1291 mimics for 48 h, the transfection efficiency, the expression of ENST00000606533, circ_0091223, and TYR were detected. (D) After treated with a-MSH, the MCs were transfected with si-ENST00000606533 mix, or si-circ_0091223 2, or miR-1291 mimics, and the expression of ENST00000606533, circ_0091223, miR-1291, and TYR were detected. (E) After co-transfection with siRNAs of ENST00000606533 and miRNA_1291 inhibitor for 48 h, the expression of ENST00000606533, circ_0091223, miR-1291, and TYR were detected. (F) After co-transfection with siRNA of circ_0091223 and miR-1291 inhibitor for 48 h, the expression of ENST00000606533, circ_0091223, miR-1291, and TYR were detected. (G) After treated with a-MSH and/or si-ENST00000606533 mix, or si-circ_0091223 2, or miR-1291 mimics, the protein level of TYR were detected. (H) After treated with a-MSH and/or si-ENST00000606533 mix, or si-circ_0091223 2, or miR-1291 mimics, the Masson-Fontana melanin staining were detected.
Fig 2: Red fluorescence was labelled for Sox9 as a marker for HFSCs. Tyr-positive cells labelled by green fluorescence were located in the hair bulbs and epidermis. HFSCs and melanocytes were decreased as shown by the merged images in the UVA group. There was an increase of Tyr-positive cells in the epidermis of the UVA group.
Fig 3: a-MSH-treatment induced ENST00000606533 and circ_0091223 expression but inhibits miR-1291 expression. (A–C) ENST00000606533, ENST00000532071, circ_0091223, circ_0031728, miR-1291, and miR-4530 levels were detected by qRT-PCR in MCs after 150 nM a-MSH treatment. (D) Luciferase reporter assay was used to detect the binding of miR-1291 to circ_0091223 and ENST00000606533. (E, F) Schematic model for wild type (WT) or mutant (mut) transcripts of ENST00000606533 or circ_0091223 luciferase reporters. (G) The binding site of miR-1291 with TYR predicted by TargetScan software.
Fig 4: Relative protein expression by Western blot. (A and B) Compared with the control group, relative protein expression of Wnt10b was increased in both the UVB and the UVA groups. (A and C) The expression of β-catenin was also higher in both the UVB and the UVA groups as compared to the control group. (A and D) LEF1 expression was higher in the UVB and UVA groups than in the control group. (A and E) Western blot showed that UV irradiation up-regulated the expression of Tyr. (*P<0.05, **P<0.01).
Fig 5: Effects of CRE and CSE on mRNA expression of genes involved in melanogenesis. Total RNA from B16F10 cells treated with CRE and CSE extracts were collected at 48 h, and levels of (A) MITF (B) TYR (C) TRP-1 (D) TRP-2 mRNA were determined by qPCR, using GAPDH as an internal control. ##P < .01, ###P < .001: statistically significant vs control group and *P < .05, *P < .01, ***P < .001 vs α -MSH group.
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