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(A) Immunofluorescence images of PIG1 cells transfected <t>with</t> <t>MC1R-mEmerald</t> (green) and mock-treated, or treated with UV, 100 nM α-MSH, or both. Cells were treated with α-MSH for 36 h in the absence of serum after UV exposure. Cells were stained with the Arl13b antibody (red). Scale bar, 5 μm. (B) MC1R ciliary enrichment analysis of panel A as previously reported . Ciliary and cell body intensity of MC1R was measured using ImageJ. Enrichment at the cilium is expressed as: (integrated density at the cilium)/(integrated density in the cell body). Enrichment > 1 indicates higher localization of mEmerald tagged MC1R at the primary cilium than at the cell body. Every replicate was represented as a superplot. ( n = 30 ciliated cells from 3 different replicates) (C, D) Immunofluorescence images (C) of A375 MC1R-KO cells transfected with WT, R151C, or R160W MC1R-mEmerald (green) and mock-treated or treated with 100 nM α-MSH for 36 h in the absence of serum after UV exposure. Cells were stained with the Arl13b antibody (red). The percentage of ciliated cells with ciliary MC1R localization was quantified in panel D ( n = 3 independent experiments). Scale bar, 5 μm. (E) Immunofluorescence images of MC1R-mEmerald transfected primary human melanocytes treated with UV/α-MSH (100 nM). Cells were treated with α-MSH for 36 h in the presence of serum after UV exposure. Cells were stained with the Arl13b (red) and TYRP1 (magenta) antibodies. Nuclei were stained with DAPI (blue). Representative images for non-ciliated cells, ciliated cells without ciliary MC1R (MC1R − cilium), and ciliated cells with ciliary MC1R (MC1R + cilium) were selected from the same slides. Scale bar, 10 μm. (F) Quantification of the number of TYRP1 puncta ( n = 20 cells) as described in E. (G) Tyrosinase activity of primary human melanocytes that were mock treated, treated with 100 nM α-MSH, 100 nM α-MSH/30 μm ciliobrevin A (Cilio A), or 100 nM α-MSH/2 mM CH for 36 h in the absence of serum after UV exposure ( n = 3 independent experiments). (H) Quantitative RT-PCR analysis of melanogenesis-related genes in primary human melanocytes treated as described in G ( n = 3 independent experiments). TYR, tyrosinase; DCT, dopachrome tautomerase; <t>MITF,</t> melanocyte inducing transcription factor; TYRP1, tyrosinase related protein 1; PMEL, premelanosome protein. (I) Immunoblot analysis of melanogenesis-related proteins in primary human melanocytes treated as described in G. GAPDH served as a control. (J) Tyrosinase activity of A375 MC1R-KO cells rescued with different forms of MC1R ( n = 5 independent experiments). Cilio-MC1R was constructed by fusing the ciliary protein Arl13b with WT, R151C, or R160W MC1R. Cells were treated with or without 100 nM α-MSH for 36 h in the absence of serum and UV. Data are presented as mean ± SD. Statistical significance was determined by one-way ANOVA; * p < 0.05, ** p < 0.01, *** p < 0.001; ns, not significant. See also . The underlying data for this figure can be found in . The uncropped blots are included in . α-MSH, α-melanocyte-stimulating hormone; CH, chloral hydrate; MC1R, melanocortin 1 receptor; UV, ultraviolet.
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(A) Immunofluorescence images of PIG1 cells transfected with MC1R-mEmerald (green) and mock-treated, or treated with UV, 100 nM α-MSH, or both. Cells were treated with α-MSH for 36 h in the absence of serum after UV exposure. Cells were stained with the Arl13b antibody (red). Scale bar, 5 μm. (B) MC1R ciliary enrichment analysis of panel A as previously reported . Ciliary and cell body intensity of MC1R was measured using ImageJ. Enrichment at the cilium is expressed as: (integrated density at the cilium)/(integrated density in the cell body). Enrichment > 1 indicates higher localization of mEmerald tagged MC1R at the primary cilium than at the cell body. Every replicate was represented as a superplot. ( n = 30 ciliated cells from 3 different replicates) (C, D) Immunofluorescence images (C) of A375 MC1R-KO cells transfected with WT, R151C, or R160W MC1R-mEmerald (green) and mock-treated or treated with 100 nM α-MSH for 36 h in the absence of serum after UV exposure. Cells were stained with the Arl13b antibody (red). The percentage of ciliated cells with ciliary MC1R localization was quantified in panel D ( n = 3 independent experiments). Scale bar, 5 μm. (E) Immunofluorescence images of MC1R-mEmerald transfected primary human melanocytes treated with UV/α-MSH (100 nM). Cells were treated with α-MSH for 36 h in the presence of serum after UV exposure. Cells were stained with the Arl13b (red) and TYRP1 (magenta) antibodies. Nuclei were stained with DAPI (blue). Representative images for non-ciliated cells, ciliated cells without ciliary MC1R (MC1R − cilium), and ciliated cells with ciliary MC1R (MC1R + cilium) were selected from the same slides. Scale bar, 10 μm. (F) Quantification of the number of TYRP1 puncta ( n = 20 cells) as described in E. (G) Tyrosinase activity of primary human melanocytes that were mock treated, treated with 100 nM α-MSH, 100 nM α-MSH/30 μm ciliobrevin A (Cilio A), or 100 nM α-MSH/2 mM CH for 36 h in the absence of serum after UV exposure ( n = 3 independent experiments). (H) Quantitative RT-PCR analysis of melanogenesis-related genes in primary human melanocytes treated as described in G ( n = 3 independent experiments). TYR, tyrosinase; DCT, dopachrome tautomerase; MITF, melanocyte inducing transcription factor; TYRP1, tyrosinase related protein 1; PMEL, premelanosome protein. (I) Immunoblot analysis of melanogenesis-related proteins in primary human melanocytes treated as described in G. GAPDH served as a control. (J) Tyrosinase activity of A375 MC1R-KO cells rescued with different forms of MC1R ( n = 5 independent experiments). Cilio-MC1R was constructed by fusing the ciliary protein Arl13b with WT, R151C, or R160W MC1R. Cells were treated with or without 100 nM α-MSH for 36 h in the absence of serum and UV. Data are presented as mean ± SD. Statistical significance was determined by one-way ANOVA; * p < 0.05, ** p < 0.01, *** p < 0.001; ns, not significant. See also . The underlying data for this figure can be found in . The uncropped blots are included in . α-MSH, α-melanocyte-stimulating hormone; CH, chloral hydrate; MC1R, melanocortin 1 receptor; UV, ultraviolet.

Journal: PLOS Biology

Article Title: Melanocortin 1 receptor mediates melanin production by interacting with the BBSome in primary cilia

doi: 10.1371/journal.pbio.3002940

Figure Lengend Snippet: (A) Immunofluorescence images of PIG1 cells transfected with MC1R-mEmerald (green) and mock-treated, or treated with UV, 100 nM α-MSH, or both. Cells were treated with α-MSH for 36 h in the absence of serum after UV exposure. Cells were stained with the Arl13b antibody (red). Scale bar, 5 μm. (B) MC1R ciliary enrichment analysis of panel A as previously reported . Ciliary and cell body intensity of MC1R was measured using ImageJ. Enrichment at the cilium is expressed as: (integrated density at the cilium)/(integrated density in the cell body). Enrichment > 1 indicates higher localization of mEmerald tagged MC1R at the primary cilium than at the cell body. Every replicate was represented as a superplot. ( n = 30 ciliated cells from 3 different replicates) (C, D) Immunofluorescence images (C) of A375 MC1R-KO cells transfected with WT, R151C, or R160W MC1R-mEmerald (green) and mock-treated or treated with 100 nM α-MSH for 36 h in the absence of serum after UV exposure. Cells were stained with the Arl13b antibody (red). The percentage of ciliated cells with ciliary MC1R localization was quantified in panel D ( n = 3 independent experiments). Scale bar, 5 μm. (E) Immunofluorescence images of MC1R-mEmerald transfected primary human melanocytes treated with UV/α-MSH (100 nM). Cells were treated with α-MSH for 36 h in the presence of serum after UV exposure. Cells were stained with the Arl13b (red) and TYRP1 (magenta) antibodies. Nuclei were stained with DAPI (blue). Representative images for non-ciliated cells, ciliated cells without ciliary MC1R (MC1R − cilium), and ciliated cells with ciliary MC1R (MC1R + cilium) were selected from the same slides. Scale bar, 10 μm. (F) Quantification of the number of TYRP1 puncta ( n = 20 cells) as described in E. (G) Tyrosinase activity of primary human melanocytes that were mock treated, treated with 100 nM α-MSH, 100 nM α-MSH/30 μm ciliobrevin A (Cilio A), or 100 nM α-MSH/2 mM CH for 36 h in the absence of serum after UV exposure ( n = 3 independent experiments). (H) Quantitative RT-PCR analysis of melanogenesis-related genes in primary human melanocytes treated as described in G ( n = 3 independent experiments). TYR, tyrosinase; DCT, dopachrome tautomerase; MITF, melanocyte inducing transcription factor; TYRP1, tyrosinase related protein 1; PMEL, premelanosome protein. (I) Immunoblot analysis of melanogenesis-related proteins in primary human melanocytes treated as described in G. GAPDH served as a control. (J) Tyrosinase activity of A375 MC1R-KO cells rescued with different forms of MC1R ( n = 5 independent experiments). Cilio-MC1R was constructed by fusing the ciliary protein Arl13b with WT, R151C, or R160W MC1R. Cells were treated with or without 100 nM α-MSH for 36 h in the absence of serum and UV. Data are presented as mean ± SD. Statistical significance was determined by one-way ANOVA; * p < 0.05, ** p < 0.01, *** p < 0.001; ns, not significant. See also . The underlying data for this figure can be found in . The uncropped blots are included in . α-MSH, α-melanocyte-stimulating hormone; CH, chloral hydrate; MC1R, melanocortin 1 receptor; UV, ultraviolet.

Article Snippet: Primary antibodies used in this study included the following: β-actin (Abways, AB0035; 1:1,000), ace-tubulin (Sigma-Aldrich, T7451; 1:1,000), ATP1A1 (Proteintech, 14418-1-AP; 1:1,000), Arl13b (Proteintech, 17711-1-AP; 1:2,000), BBS1 (Proteintech, 21118-1-AP; 1:1,000), BBS2 (Proteintech, 11188-2-AP; 1:1,000), BBS4 (Proteintech, 12766-1-AP; 1:1,000), BBS9 (Proteintech, 14460-1-AP; 1:1,000), Flag (Sigma-Aldrich, F1804; 1:2,000; Proteintech, 20543-1-AP; 1:1,000), DCT (Santa Cruz Biotechnology, sc-74439; 1:1,000), GFP (Abways, AB0045; 1:1,000; Abways, AB0005; 1:1,000), HA (Sigma-Aldrich, H9658; 1:5,000), MC1R (Signalway Antibody, 36969; 1:500), MITF (Proteintech, 13092-1-AP; 1:1,000; Santa Cruz Biotechnology, sc-515925; 1:1,000), TYR (Santa Cruz Biotechnology, sc-20035; 1:1,000), TYRP1 (Santa Cruz Biotechnology, sc-166857; 1:1,000).

Techniques: Immunofluorescence, Transfection, Staining, Activity Assay, Quantitative RT-PCR, Western Blot, Control, Construct

(A, B) Detection of ciliary cAMP levels by cilia-targeted cADDis (green) and the reference ciliary marker 5HT6-mCherry (red) in A375 MC1R-KO cells rescued with MC1R or cilio-MC1R (constructed by fusing the ciliary protein Arl13b with MC1R). Cells were pre-serum-starved for 24 h and cultured in the absence UV. Panel A shows time-lapse images of cADDis (green) and cilia (red) after the addition of 1 μm α-MSH (denoted by arrow). Then, 100 μm forskolin was added at 6 min (denoted by arrow). Scale bar, 2.5 μm. In panel B, the fluorescence intensities were normalized to the time point of −1 min ( n = 5 ciliated cells from 5 independent experiments). The normalized fluorescence intensity is expressed as: (the fluorescence intensity of mCherry / the fluorescence intensity of cADDis) / (the fluorescence intensity of mCherry at −1 min / the fluorescence intensity of cADDis at −1 min). (C) Time course of total cellular cAMP levels of A375 MC1R-KO cells rescued with MC1R or cilio-MC1R upon stimulation with 1 μm α-MSH. Cells were pre-serum-starved for 24 h and cultured in the absence UV. (D) Volcano plots of differentially expressed genes in A375 MC1R-KO cells rescued with WT MC1R or cilio-MC1R. Cells were cultured in the absence of serum and UV, and treated with 100 nM α-MSH for 12 h before mRNA extraction. Genes with fold change >2 and adjusted p -value <0.05 were considered as differentially expressed. Melanogenesis-related genes were highlighted and marked on the spot. (E) Quantitative RT-PCR analysis of the indicated genes of A375 MC1R-KO cells transfected with the control vector, MC1R, or cilio-MC1R. Cell were treated with 100 nM α-MSH for 12 h before mRNA extraction ( n = 3 independent experiments). Cells were cultured in the absence of serum and UV. (F) Immunoblot analysis of the indicated proteins in A375 MC1R-KO cells rescued with the control vector, MC1R, or cilio-MC1R. Cells were treated with 100 nM α-MSH for 24 h in the absence of serum and UV before lysis. GAPDH served as a control. (G, H) Immunoprecipitation and immunoblotting showing the interaction between Sox-3×Flag and MITF-HA. HEK293T cells were transfected with the indicated plasmids and immunoprecipitated with antibodies against Flag (G) and HA (H). (I) Dual-luciferase reporter assay of the pGL3-basic or DCT promoter transfected with the indicated plasmids. The relative luciferase activity was calculated as the luciferase activity normalized with the control vector ( n = 3 independent experiments). (J) Tyrosinase activity of A375 MC1R-KO cells rescued with the control vector, MC1R, or cilio-MC1R. Cells were transfected with control or Sox9 shRNAs and treated with 100 nM α-MSH for 24 h in the absence of serum and UV before detection ( n = 3 independent experiments). (K) Immunoblot analysis of the indicated proteins in A375 MC1R-KO cells rescued with the control vector, MC1R, or cilio-MC1R. Cells were transfected with control or Sox9 shRNAs and treated with α-MSH for 24 h in the absence of serum and UV before lysis. GAPDH served as a control. Data are presented as mean ± SD. Statistical significance was determined by unpaired two-tailed Student’s t test (B and C) or one-way ANOVA (E, I, and J); * p < 0.05, ** p < 0.01, *** p < 0.001; ns, not significant. See also . The underlying data for this figure can be found in . The uncropped blots are included in . α-MSH, α-melanocyte-stimulating hormone; MC1R, melanocortin 1 receptor; UV, ultraviolet.

Journal: PLOS Biology

Article Title: Melanocortin 1 receptor mediates melanin production by interacting with the BBSome in primary cilia

doi: 10.1371/journal.pbio.3002940

Figure Lengend Snippet: (A, B) Detection of ciliary cAMP levels by cilia-targeted cADDis (green) and the reference ciliary marker 5HT6-mCherry (red) in A375 MC1R-KO cells rescued with MC1R or cilio-MC1R (constructed by fusing the ciliary protein Arl13b with MC1R). Cells were pre-serum-starved for 24 h and cultured in the absence UV. Panel A shows time-lapse images of cADDis (green) and cilia (red) after the addition of 1 μm α-MSH (denoted by arrow). Then, 100 μm forskolin was added at 6 min (denoted by arrow). Scale bar, 2.5 μm. In panel B, the fluorescence intensities were normalized to the time point of −1 min ( n = 5 ciliated cells from 5 independent experiments). The normalized fluorescence intensity is expressed as: (the fluorescence intensity of mCherry / the fluorescence intensity of cADDis) / (the fluorescence intensity of mCherry at −1 min / the fluorescence intensity of cADDis at −1 min). (C) Time course of total cellular cAMP levels of A375 MC1R-KO cells rescued with MC1R or cilio-MC1R upon stimulation with 1 μm α-MSH. Cells were pre-serum-starved for 24 h and cultured in the absence UV. (D) Volcano plots of differentially expressed genes in A375 MC1R-KO cells rescued with WT MC1R or cilio-MC1R. Cells were cultured in the absence of serum and UV, and treated with 100 nM α-MSH for 12 h before mRNA extraction. Genes with fold change >2 and adjusted p -value <0.05 were considered as differentially expressed. Melanogenesis-related genes were highlighted and marked on the spot. (E) Quantitative RT-PCR analysis of the indicated genes of A375 MC1R-KO cells transfected with the control vector, MC1R, or cilio-MC1R. Cell were treated with 100 nM α-MSH for 12 h before mRNA extraction ( n = 3 independent experiments). Cells were cultured in the absence of serum and UV. (F) Immunoblot analysis of the indicated proteins in A375 MC1R-KO cells rescued with the control vector, MC1R, or cilio-MC1R. Cells were treated with 100 nM α-MSH for 24 h in the absence of serum and UV before lysis. GAPDH served as a control. (G, H) Immunoprecipitation and immunoblotting showing the interaction between Sox-3×Flag and MITF-HA. HEK293T cells were transfected with the indicated plasmids and immunoprecipitated with antibodies against Flag (G) and HA (H). (I) Dual-luciferase reporter assay of the pGL3-basic or DCT promoter transfected with the indicated plasmids. The relative luciferase activity was calculated as the luciferase activity normalized with the control vector ( n = 3 independent experiments). (J) Tyrosinase activity of A375 MC1R-KO cells rescued with the control vector, MC1R, or cilio-MC1R. Cells were transfected with control or Sox9 shRNAs and treated with 100 nM α-MSH for 24 h in the absence of serum and UV before detection ( n = 3 independent experiments). (K) Immunoblot analysis of the indicated proteins in A375 MC1R-KO cells rescued with the control vector, MC1R, or cilio-MC1R. Cells were transfected with control or Sox9 shRNAs and treated with α-MSH for 24 h in the absence of serum and UV before lysis. GAPDH served as a control. Data are presented as mean ± SD. Statistical significance was determined by unpaired two-tailed Student’s t test (B and C) or one-way ANOVA (E, I, and J); * p < 0.05, ** p < 0.01, *** p < 0.001; ns, not significant. See also . The underlying data for this figure can be found in . The uncropped blots are included in . α-MSH, α-melanocyte-stimulating hormone; MC1R, melanocortin 1 receptor; UV, ultraviolet.

Article Snippet: Primary antibodies used in this study included the following: β-actin (Abways, AB0035; 1:1,000), ace-tubulin (Sigma-Aldrich, T7451; 1:1,000), ATP1A1 (Proteintech, 14418-1-AP; 1:1,000), Arl13b (Proteintech, 17711-1-AP; 1:2,000), BBS1 (Proteintech, 21118-1-AP; 1:1,000), BBS2 (Proteintech, 11188-2-AP; 1:1,000), BBS4 (Proteintech, 12766-1-AP; 1:1,000), BBS9 (Proteintech, 14460-1-AP; 1:1,000), Flag (Sigma-Aldrich, F1804; 1:2,000; Proteintech, 20543-1-AP; 1:1,000), DCT (Santa Cruz Biotechnology, sc-74439; 1:1,000), GFP (Abways, AB0045; 1:1,000; Abways, AB0005; 1:1,000), HA (Sigma-Aldrich, H9658; 1:5,000), MC1R (Signalway Antibody, 36969; 1:500), MITF (Proteintech, 13092-1-AP; 1:1,000; Santa Cruz Biotechnology, sc-515925; 1:1,000), TYR (Santa Cruz Biotechnology, sc-20035; 1:1,000), TYRP1 (Santa Cruz Biotechnology, sc-166857; 1:1,000).

Techniques: Marker, Construct, Cell Culture, Fluorescence, Extraction, Quantitative RT-PCR, Transfection, Control, Plasmid Preparation, Western Blot, Lysis, Immunoprecipitation, Luciferase, Reporter Assay, Activity Assay, Two Tailed Test