mouse mc1r  (Thermo Fisher)


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    Structured Review

    Thermo Fisher mouse mc1r
    (CKPV) 2 induces macrophage M1 to M2 polarization. Cytokine release profile (IL-1β, IL-6 and IL-10) and arginase activity after indicated treatment/s in primary cultured macrophages transfected with or without <t>MC1R</t> RNAi. LPS:5 ng/ml, IFN-γ:10 ng/ml, α-MSH: 10 µM, and (CKPV) 2 (0.1, 1 and 5 µM) (B–E). The supernatant of the above macrophages were collected and added to L929 cells, after 20 hours, cell viability was measured by MTT assay, the inhibitory rate was calculated by 100%-cell viability OD of treatment group/cell viability OD of untreated control group (A). * p
    Mouse Mc1r, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    mouse mc1r - by Bioz Stars, 2022-10
    93/100 stars

    Images

    1) Product Images from "The Synthetic Melanocortin (CKPV)2 Exerts Anti-Fungal and Anti-Inflammatory Effects against Candida albicans Vaginitis via Inducing Macrophage M2 Polarization"

    Article Title: The Synthetic Melanocortin (CKPV)2 Exerts Anti-Fungal and Anti-Inflammatory Effects against Candida albicans Vaginitis via Inducing Macrophage M2 Polarization

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0056004

    (CKPV) 2 induces macrophage M1 to M2 polarization. Cytokine release profile (IL-1β, IL-6 and IL-10) and arginase activity after indicated treatment/s in primary cultured macrophages transfected with or without MC1R RNAi. LPS:5 ng/ml, IFN-γ:10 ng/ml, α-MSH: 10 µM, and (CKPV) 2 (0.1, 1 and 5 µM) (B–E). The supernatant of the above macrophages were collected and added to L929 cells, after 20 hours, cell viability was measured by MTT assay, the inhibitory rate was calculated by 100%-cell viability OD of treatment group/cell viability OD of untreated control group (A). * p
    Figure Legend Snippet: (CKPV) 2 induces macrophage M1 to M2 polarization. Cytokine release profile (IL-1β, IL-6 and IL-10) and arginase activity after indicated treatment/s in primary cultured macrophages transfected with or without MC1R RNAi. LPS:5 ng/ml, IFN-γ:10 ng/ml, α-MSH: 10 µM, and (CKPV) 2 (0.1, 1 and 5 µM) (B–E). The supernatant of the above macrophages were collected and added to L929 cells, after 20 hours, cell viability was measured by MTT assay, the inhibitory rate was calculated by 100%-cell viability OD of treatment group/cell viability OD of untreated control group (A). * p

    Techniques Used: Activity Assay, Cell Culture, Transfection, MTT Assay

    (CKPV) 2 promotes cAMP production via MC1R. (A) Upper panel: the effects of MC1R siRNA (S1, S2 and S3, see sequence on Tab. 1) on MC1R mRNA level in primary cultured macrophages. Lower panel: MC1R siRNA knockdown almost blocked (CKPV) 2 -induced cAMP production in macrophages. (B) Upper panel, RT-PCR results confirms (CKPV) 2 mRNA expression in MC1R cDNA-transfected COS-7 cells (COS-7/MC1R) or nonsense-cDNA-transfected control cells (COS-7). Lower panel: (CKPV) 2 -induced cAMP production in negative control-cDNA-transfected (NC) or MC1R cDNA-transfected COS-7 cells. * p
    Figure Legend Snippet: (CKPV) 2 promotes cAMP production via MC1R. (A) Upper panel: the effects of MC1R siRNA (S1, S2 and S3, see sequence on Tab. 1) on MC1R mRNA level in primary cultured macrophages. Lower panel: MC1R siRNA knockdown almost blocked (CKPV) 2 -induced cAMP production in macrophages. (B) Upper panel, RT-PCR results confirms (CKPV) 2 mRNA expression in MC1R cDNA-transfected COS-7 cells (COS-7/MC1R) or nonsense-cDNA-transfected control cells (COS-7). Lower panel: (CKPV) 2 -induced cAMP production in negative control-cDNA-transfected (NC) or MC1R cDNA-transfected COS-7 cells. * p

    Techniques Used: Sequencing, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Expressing, Transfection, Negative Control

    2) Product Images from "Palmitoylation-dependent activation of MC1R prevents melanomagenesis"

    Article Title: Palmitoylation-dependent activation of MC1R prevents melanomagenesis

    Journal: Nature

    doi: 10.1038/nature23887

    Palmitoylation of MC1R in melanocytes a-b , MC1R RHC-variant HPMs exposed to α-MSH and UVB irradiated were treated with BSA-conjugated fatty acids. b , cells were exposed to palmitic acid +/− 2-BP. cAMP were calculated by three independent experiments shown as mean ± SD. c-f , HPMs ( c ), HPMs expressing WT, mutant or variant Flag-MC1R ( d-f ) were incubated with α-MSH and processed for ABE analysis. g-k , HPMs ( g ), HPMs expressing WT, mutant or variant Flag-MC1R ( h-k ) were treated with α-MSH, irradiated with UVB, and harvested for ABE analysis. Western blots shown were representative of three independent experiments. **p
    Figure Legend Snippet: Palmitoylation of MC1R in melanocytes a-b , MC1R RHC-variant HPMs exposed to α-MSH and UVB irradiated were treated with BSA-conjugated fatty acids. b , cells were exposed to palmitic acid +/− 2-BP. cAMP were calculated by three independent experiments shown as mean ± SD. c-f , HPMs ( c ), HPMs expressing WT, mutant or variant Flag-MC1R ( d-f ) were incubated with α-MSH and processed for ABE analysis. g-k , HPMs ( g ), HPMs expressing WT, mutant or variant Flag-MC1R ( h-k ) were treated with α-MSH, irradiated with UVB, and harvested for ABE analysis. Western blots shown were representative of three independent experiments. **p

    Techniques Used: Variant Assay, Irradiation, Expressing, Mutagenesis, Incubation, Western Blot

    Activating MC1R palmitoylation rescues the defect of MC1R RHC variants a-b , MC1R-depleted hTERT/p53DD/CDK4(R24C)/BRAF V600E melanocytes expressing Flag-MC1R ( a ) or cells further infected with shZDHHC13 and/or WT HA-ZDHHC13 virus ( b ) were pre-incubated with α-MSH, UVB irradiated and assayed for clonogenic survival. Results were calculated as mean ± SD from three independent experiments. c-e , Growth curves ( c ), dissected tumors ( d ) and tumor weight ( e ) for the xenograft experiments with indicated cells inoculated subcutaneously into each flank of nude mice (n=8). Visible tumors were measured at the indicated days. Error bars represent ±SEM. *p
    Figure Legend Snippet: Activating MC1R palmitoylation rescues the defect of MC1R RHC variants a-b , MC1R-depleted hTERT/p53DD/CDK4(R24C)/BRAF V600E melanocytes expressing Flag-MC1R ( a ) or cells further infected with shZDHHC13 and/or WT HA-ZDHHC13 virus ( b ) were pre-incubated with α-MSH, UVB irradiated and assayed for clonogenic survival. Results were calculated as mean ± SD from three independent experiments. c-e , Growth curves ( c ), dissected tumors ( d ) and tumor weight ( e ) for the xenograft experiments with indicated cells inoculated subcutaneously into each flank of nude mice (n=8). Visible tumors were measured at the indicated days. Error bars represent ±SEM. *p

    Techniques Used: Expressing, Infection, Incubation, Irradiation, Mouse Assay

    Palmitoylation of MC1R in melanocytes (a) Endogenous MC1R protein is palmitoylated. B16 cells were incubated with 1 μM α-MSH for 3.5 h. Cells were then harvested for IP by specific anti-MC1R antibody, and then for ABE and IB analysis. (b) Exogenous MC1R protein is palmitoylated. B16 cells were infected with retrovirus encoding Flag-MC1R WT and incubated with 1 μM α-MSH for 3.5 h. Cells were then harvested for IP, ABE and IB analysis. (c) MC1R is palmitoylated at C315. B16 cells with stable depletion of endogenous MC1R by shRNA were infected with the indicated Flag-MC1R encoding retroviral constructs. Cells were treated with 1 μM α-MSH for 3.5 h and then harvested for IP, ABE and IB analysis. (d) MC1R RHC variants are defective in palmitoylation. B16 cells with stable depletion of endogenous MC1R by shRNA were infected with the indicated Flag-MC1R encoding retroviral constructs. Cells were treated with 1 μM α-MSH for 3.5 h and then harvested for IP, ABE and IB analysis. (e-f) R151C does not create a palmitoylation site. B16 cells (e) or HPMs (f) with stable depletion of endogenous MC1R by shRNA were infected with the indicated Flag-MC1R encoding retroviral constructs. Cells were treated with 1 μM α-MSH for 3.5 h and then harvested for IP, ABE and IB analysis. (g) UVB enhances MC1R palmitoylation. B16 cells were incubated with 1 μM α-MSH for 30 min followed by 100 J/m 2 UVB irradiation. Cells were harvested for IP by specific anti-MC1R antibody, then for ABE and IB analysis 3 h after UVB exposure. (h) UVB irradiation enhances MC1R palmitoylation. B16 cells were infected with retroviral constructs encoding Flag-MC1R WT and pre-treated with 1 μM α-MSH for 30 min followed by 100 J/m 2 UVB irradiation. Cells were harvested for IP, ABE and IB analysis 3 h after UVB exposure. (i) MC1R C315S mutant does not respond to UVB irradiation. B16 with stable depletion of MC1R by shRNA were infected with the indicated Flag-MC1R encoding retroviral constructs. Cells were pre-treated with 1 μM α-MSH for 30 min followed by 100 J/m 2 UVB irradiation. Cells were harvested for IP, ABE and IB analysis 3 h after UVB exposure. (j-m) MC1R RHC variants are defective in palmitoylation after UVB. B16 or HPMs with stable depletion of MC1R by shRNA were infected with the indicated Flag-MC1R encoding retroviral constructs. Cells were pre-treated with 1 μM α-MSH for 30 min followed by 100 J/m 2 UVB irradiation. Cells were harvested for IP, ABE and IB analysis 3 h after UVB exposure.
    Figure Legend Snippet: Palmitoylation of MC1R in melanocytes (a) Endogenous MC1R protein is palmitoylated. B16 cells were incubated with 1 μM α-MSH for 3.5 h. Cells were then harvested for IP by specific anti-MC1R antibody, and then for ABE and IB analysis. (b) Exogenous MC1R protein is palmitoylated. B16 cells were infected with retrovirus encoding Flag-MC1R WT and incubated with 1 μM α-MSH for 3.5 h. Cells were then harvested for IP, ABE and IB analysis. (c) MC1R is palmitoylated at C315. B16 cells with stable depletion of endogenous MC1R by shRNA were infected with the indicated Flag-MC1R encoding retroviral constructs. Cells were treated with 1 μM α-MSH for 3.5 h and then harvested for IP, ABE and IB analysis. (d) MC1R RHC variants are defective in palmitoylation. B16 cells with stable depletion of endogenous MC1R by shRNA were infected with the indicated Flag-MC1R encoding retroviral constructs. Cells were treated with 1 μM α-MSH for 3.5 h and then harvested for IP, ABE and IB analysis. (e-f) R151C does not create a palmitoylation site. B16 cells (e) or HPMs (f) with stable depletion of endogenous MC1R by shRNA were infected with the indicated Flag-MC1R encoding retroviral constructs. Cells were treated with 1 μM α-MSH for 3.5 h and then harvested for IP, ABE and IB analysis. (g) UVB enhances MC1R palmitoylation. B16 cells were incubated with 1 μM α-MSH for 30 min followed by 100 J/m 2 UVB irradiation. Cells were harvested for IP by specific anti-MC1R antibody, then for ABE and IB analysis 3 h after UVB exposure. (h) UVB irradiation enhances MC1R palmitoylation. B16 cells were infected with retroviral constructs encoding Flag-MC1R WT and pre-treated with 1 μM α-MSH for 30 min followed by 100 J/m 2 UVB irradiation. Cells were harvested for IP, ABE and IB analysis 3 h after UVB exposure. (i) MC1R C315S mutant does not respond to UVB irradiation. B16 with stable depletion of MC1R by shRNA were infected with the indicated Flag-MC1R encoding retroviral constructs. Cells were pre-treated with 1 μM α-MSH for 30 min followed by 100 J/m 2 UVB irradiation. Cells were harvested for IP, ABE and IB analysis 3 h after UVB exposure. (j-m) MC1R RHC variants are defective in palmitoylation after UVB. B16 or HPMs with stable depletion of MC1R by shRNA were infected with the indicated Flag-MC1R encoding retroviral constructs. Cells were pre-treated with 1 μM α-MSH for 30 min followed by 100 J/m 2 UVB irradiation. Cells were harvested for IP, ABE and IB analysis 3 h after UVB exposure.

    Techniques Used: Incubation, Infection, shRNA, Construct, Irradiation, Mutagenesis

    Palm-B activates MC1R palmitoylation and rescues the defect of MC1R RHC variants (a) HPMs were infected with retrovirus encoding Flag-MC1R WT and incubated with 1 μM α-MSH for 3.5 h. The medium was replaced with fresh medium with vehicle or 1 μM Palm-B and cells were treated with indicated time. Cells were harvested for IP, ABE and IB analysis. (b) B16 and HPMs with stable depletion of MC1R by shRNA were infected with the indicated Flag-MC1R encoding retroviral constructs. Cells were pre-treated with 1 μM α-MSH and 1 μM Palm-B for 30 min followed by 100 J/m 2 UVB irradiation. Cells were harvested for IP, ABE and IB analysis 3 h after UVB exposure. (c) B16 and HPMs with stable depletion of MC1R by shRNA were infected with the indicated Flag-MC1R encoding retroviral constructs. Cells were pre-treated with 1 μM α-MSH and 1 μM Palm-B for 30 min followed by 100 J/m 2 UVB irradiation. After 3 h, cells were harvested for cAMP immunoassay. These data were compiled from three independent experiments. Data are represented as mean ± SD. (d-e) B16 and HPMs with stable depletion of MC1R by shRNA were infected with the indicated Flag-MC1R encoding retroviral constructs. Cells were pre-treated with 1 μM α-MSH and 1 μM Palm-B for 30 min followed by 100 J/m 2 UVB irradiation. After 3 h, total RNA were collected for reverse transcription and cDNA were then used for qRT-PCR by specific primers targeting mouse and/or human MITF or TYR. Three independent experiments were quantified. Data are represented as mean ± SD. (f) HPMs with stable depletion of MC1R by shRNA were infected with the indicated Flag-MC1R encoding retro-viral constructs. Cells were pre-treated with 1 μM α-MSH and 1 μM Palm-B for 30 min followed by 100 J/m 2 UVB irradiation. Genomic DNA were extracted at the different time points indicated and photoproducts were detected by ELISA. Cyclobutane pyrimidine dimer (CPD) or 6–4 pyrimidine photoproduct (6–4PP) antibodies were used. Three independent experiments were measured and calculated as mean ± SD. (g) C57BL/6 mice or C57BL/6-MC1R e / e -MC1R R151C -tg mice were given a 10 mg/kg Palm-B or vehicle injection intraperitoneally 3 h before UVB irradiation (500 J/m 2 ). 3 h after UVB, whole skins were collected and the lysates were subjected for IP, ABE and IB analysis. (h-i) C57BL/6 mice or C57BL/6-MC1R e / e -MC1R R151C -tg mouse were injected intraperitoneally with 10 mg/kg Palm-B 3 h before UVB irradiation (500 J/ m 2 ). Melanocytes were isolated by flow cytometry, then DNA were extracted and subjected to ELISA 3 h after UVB irradiation. Results were calculated as mean ± SD from three independent experiments. (j) B16 with stable depletion of MC1R by shRNA were infected with the indicated Flag-MC1R encoding retroviral constructs. Cells were pre-treated with 1 μM α-MSH and 1 μM Palm-B for 30 min followed by 25 J/m 2 UVB irradiation. Cells were subjected to SA-β-gal staining assay 7 days after UVR. The quantification of the percentage of SA-β-gal positive cells and representative pictures are shown. Data shown correspond to one representative experiment out of three independent experiments. Data are represented as mean ± SD. (k) The cells generated as indicated were seeded (10,000 cells per well) in 0.5% low-melting-point agarose in DMEM with 10% FBS, layered onto 0.8% agarose in DMEM+10% FBS. The plates were cultured for 30 days where upon the colonies > 50 μm were counted under a light microscope. Colony numbers were plotted as mean ± SD from three independent experiments.
    Figure Legend Snippet: Palm-B activates MC1R palmitoylation and rescues the defect of MC1R RHC variants (a) HPMs were infected with retrovirus encoding Flag-MC1R WT and incubated with 1 μM α-MSH for 3.5 h. The medium was replaced with fresh medium with vehicle or 1 μM Palm-B and cells were treated with indicated time. Cells were harvested for IP, ABE and IB analysis. (b) B16 and HPMs with stable depletion of MC1R by shRNA were infected with the indicated Flag-MC1R encoding retroviral constructs. Cells were pre-treated with 1 μM α-MSH and 1 μM Palm-B for 30 min followed by 100 J/m 2 UVB irradiation. Cells were harvested for IP, ABE and IB analysis 3 h after UVB exposure. (c) B16 and HPMs with stable depletion of MC1R by shRNA were infected with the indicated Flag-MC1R encoding retroviral constructs. Cells were pre-treated with 1 μM α-MSH and 1 μM Palm-B for 30 min followed by 100 J/m 2 UVB irradiation. After 3 h, cells were harvested for cAMP immunoassay. These data were compiled from three independent experiments. Data are represented as mean ± SD. (d-e) B16 and HPMs with stable depletion of MC1R by shRNA were infected with the indicated Flag-MC1R encoding retroviral constructs. Cells were pre-treated with 1 μM α-MSH and 1 μM Palm-B for 30 min followed by 100 J/m 2 UVB irradiation. After 3 h, total RNA were collected for reverse transcription and cDNA were then used for qRT-PCR by specific primers targeting mouse and/or human MITF or TYR. Three independent experiments were quantified. Data are represented as mean ± SD. (f) HPMs with stable depletion of MC1R by shRNA were infected with the indicated Flag-MC1R encoding retro-viral constructs. Cells were pre-treated with 1 μM α-MSH and 1 μM Palm-B for 30 min followed by 100 J/m 2 UVB irradiation. Genomic DNA were extracted at the different time points indicated and photoproducts were detected by ELISA. Cyclobutane pyrimidine dimer (CPD) or 6–4 pyrimidine photoproduct (6–4PP) antibodies were used. Three independent experiments were measured and calculated as mean ± SD. (g) C57BL/6 mice or C57BL/6-MC1R e / e -MC1R R151C -tg mice were given a 10 mg/kg Palm-B or vehicle injection intraperitoneally 3 h before UVB irradiation (500 J/m 2 ). 3 h after UVB, whole skins were collected and the lysates were subjected for IP, ABE and IB analysis. (h-i) C57BL/6 mice or C57BL/6-MC1R e / e -MC1R R151C -tg mouse were injected intraperitoneally with 10 mg/kg Palm-B 3 h before UVB irradiation (500 J/ m 2 ). Melanocytes were isolated by flow cytometry, then DNA were extracted and subjected to ELISA 3 h after UVB irradiation. Results were calculated as mean ± SD from three independent experiments. (j) B16 with stable depletion of MC1R by shRNA were infected with the indicated Flag-MC1R encoding retroviral constructs. Cells were pre-treated with 1 μM α-MSH and 1 μM Palm-B for 30 min followed by 25 J/m 2 UVB irradiation. Cells were subjected to SA-β-gal staining assay 7 days after UVR. The quantification of the percentage of SA-β-gal positive cells and representative pictures are shown. Data shown correspond to one representative experiment out of three independent experiments. Data are represented as mean ± SD. (k) The cells generated as indicated were seeded (10,000 cells per well) in 0.5% low-melting-point agarose in DMEM with 10% FBS, layered onto 0.8% agarose in DMEM+10% FBS. The plates were cultured for 30 days where upon the colonies > 50 μm were counted under a light microscope. Colony numbers were plotted as mean ± SD from three independent experiments.

    Techniques Used: Infection, Incubation, shRNA, Construct, Irradiation, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Mouse Assay, Injection, Isolation, Flow Cytometry, Staining, Generated, Cell Culture, Light Microscopy

    ZDHHC13 is a major PAT of MC1R (a) HEK293 cells were co-transfected with constructs encoding Flag-MC1R and HA-ZDHHCs in 6-well plate. Cells lysates were harvested for IP by anti-Flag antibody, and then for ABE and IB analysis. IB for total MC1R and HA-ZDHHC proteins was shown. (b) B16 cells were infected with Flag-MC1R and indicated ZDHHC13 WT or C456S mutant encoding retroviral constructs. Cells were treated with 1 μM α-MSH for 3.5 h and then harvested for IP, ABE and IB analysis. (c) B16 cells with deletion of ZDHHC13 by selective shRNAs were pre-treated with 1 μM α-MSH for 30 min followed by 100 J/m 2 UVB irradiation and harvested for IP, ABE and IB analysis 3 h after UVB exposure. (d) B16 cells with stable depletion of MC1R by shRNA were infected with the indicated Flag-MC1R encoding retroviral constructs, then cells were infected with shZDHHC13 and/or WT HA-ZDHHC13 expressing virus. Finally, cells were pre-treated with 1 μM α-MSH for 30 min followed by 100 J/m 2 UVB irradiation and harvested for IP, ABE and IB analysis 3 h after UVB exposure. (e-f) B16 cells or HPMs with stable depletion of MC1R by shRNA were infected with the Flag-MC1R R151C or R151C+C315S double mutant encoding retroviral constructs, then cells were infected with WT HA-ZDHHC13 expressing virus. Finally, cells were pre-treated with 1 μM α-MSH for 3.5 h and harvested for IP, ABE and IB analysis. (g) HPMs were incubated with 1 μM α-MSH or vehicle for 30 min followed by 100 J/m 2 UVB irradiation. Cells were harvested for IP by specific anti-MC1R antibody, then for ABE and IB analysis 3 h after UVB exposure. (h) HPMs were incubated with 1 μM α-MSH or vehicle for 30 min followed by 100 J/m 2 UVB irradiation. Cells were harvested for IP by specific anti-MC1R antibody, then for IB analysis 3 h after UVB exposure. (i) A schematic showing the conserved SQ motif of ZDHHC13. (j) HPMs were infected with WT ZDHHC13 or ZDHHC13 S8A mutant encoding retroviral constructs, then cells were irradiated with 100 J/m 2 UVB irradiation and harvested for IP and IB analysis 3 h after UVB exposure. (k) WT Flag-ATR or the kinase-dead (KD) Flag-ATR mutant transfected HEK293 cells were irradiated before Flag beads immunoprecipitation. Then immunoprecipitated WT ZDHHC13 or S8A mutant were incubated with immunoprecipitated WT ATR or the KD ATR mutant in kinase buffer. After reaction, proteins were collected for IB analysis. (l) HPMs were infected with WT ZDHHC13 or ZDHHC13 S8A mutant and Flag-MC1R encoding retroviral constructs, then cells were irradiated with 100 J/m 2 UVB irradiation and harvested for IP and IB analysis 3 h after UVB exposure. (m) HPMs cells with stable depletion of ZDHHC13 by shRNA were infected with the indicated ZDHHC13 and Flag-MC1R encoding retroviral constructs. Then cells were pre-treated with 1 μM α-MSH for 30 min followed by 100 J/m 2 UVB irradiation and harvested for IP, ABE and IB analysis 3 h after UVB exposure.
    Figure Legend Snippet: ZDHHC13 is a major PAT of MC1R (a) HEK293 cells were co-transfected with constructs encoding Flag-MC1R and HA-ZDHHCs in 6-well plate. Cells lysates were harvested for IP by anti-Flag antibody, and then for ABE and IB analysis. IB for total MC1R and HA-ZDHHC proteins was shown. (b) B16 cells were infected with Flag-MC1R and indicated ZDHHC13 WT or C456S mutant encoding retroviral constructs. Cells were treated with 1 μM α-MSH for 3.5 h and then harvested for IP, ABE and IB analysis. (c) B16 cells with deletion of ZDHHC13 by selective shRNAs were pre-treated with 1 μM α-MSH for 30 min followed by 100 J/m 2 UVB irradiation and harvested for IP, ABE and IB analysis 3 h after UVB exposure. (d) B16 cells with stable depletion of MC1R by shRNA were infected with the indicated Flag-MC1R encoding retroviral constructs, then cells were infected with shZDHHC13 and/or WT HA-ZDHHC13 expressing virus. Finally, cells were pre-treated with 1 μM α-MSH for 30 min followed by 100 J/m 2 UVB irradiation and harvested for IP, ABE and IB analysis 3 h after UVB exposure. (e-f) B16 cells or HPMs with stable depletion of MC1R by shRNA were infected with the Flag-MC1R R151C or R151C+C315S double mutant encoding retroviral constructs, then cells were infected with WT HA-ZDHHC13 expressing virus. Finally, cells were pre-treated with 1 μM α-MSH for 3.5 h and harvested for IP, ABE and IB analysis. (g) HPMs were incubated with 1 μM α-MSH or vehicle for 30 min followed by 100 J/m 2 UVB irradiation. Cells were harvested for IP by specific anti-MC1R antibody, then for ABE and IB analysis 3 h after UVB exposure. (h) HPMs were incubated with 1 μM α-MSH or vehicle for 30 min followed by 100 J/m 2 UVB irradiation. Cells were harvested for IP by specific anti-MC1R antibody, then for IB analysis 3 h after UVB exposure. (i) A schematic showing the conserved SQ motif of ZDHHC13. (j) HPMs were infected with WT ZDHHC13 or ZDHHC13 S8A mutant encoding retroviral constructs, then cells were irradiated with 100 J/m 2 UVB irradiation and harvested for IP and IB analysis 3 h after UVB exposure. (k) WT Flag-ATR or the kinase-dead (KD) Flag-ATR mutant transfected HEK293 cells were irradiated before Flag beads immunoprecipitation. Then immunoprecipitated WT ZDHHC13 or S8A mutant were incubated with immunoprecipitated WT ATR or the KD ATR mutant in kinase buffer. After reaction, proteins were collected for IB analysis. (l) HPMs were infected with WT ZDHHC13 or ZDHHC13 S8A mutant and Flag-MC1R encoding retroviral constructs, then cells were irradiated with 100 J/m 2 UVB irradiation and harvested for IP and IB analysis 3 h after UVB exposure. (m) HPMs cells with stable depletion of ZDHHC13 by shRNA were infected with the indicated ZDHHC13 and Flag-MC1R encoding retroviral constructs. Then cells were pre-treated with 1 μM α-MSH for 30 min followed by 100 J/m 2 UVB irradiation and harvested for IP, ABE and IB analysis 3 h after UVB exposure.

    Techniques Used: Transfection, Construct, Infection, Mutagenesis, Irradiation, shRNA, Expressing, Incubation, Immunoprecipitation

    Palmitoylation is essential for MC1R function (a) B16 and HPMs with stable depletion of MC1R by shRNA were infected with the indicated Flag-MC1R encoding retroviral constructs. After 3 h, cells were pre-treated with 1 μM α-MSH for 30 min followed by 100 J/m 2 UVB irradiation. Cells were harvested for a cAMP immunoassay. These data were compiled from three independent experiments. Data are represented as mean ± SD. (b-c) B16 and HPMs with stable depletion of MC1R by shRNA were infected with the indicated Flag-MC1R encoding retroviral constructs. After 3 h, cells were pre-treated with 1 μM α-MSH for 30 min followed by 100 J/m 2 UVB irradiation. Total RNA were collected for reverse transcription and cDNA were then used for quantitative real time PCR (qRT-PCR) by specific primers targeting mouse/human MITF or TYR. Three independent experiments were quantified. Data are represented as mean ± SD. (d) HPMs with stable deletion of MC1R by shRNA were infected with the indicated Flag-MC1R encoding retro-viral constructs. Cells were pre-treated with 1 μM α-MSH for 30 min followed by 100 J/m 2 UVB irradiation. Genomic DNA were extracted at the different time points as indicated and photoproducts were detected by ELISA. Cyclobutane pyrimidine dimer (CPD) or 6–4 pyrimidine photoproduct (6–4PP) antibodies were used. Three independent experiments were measured and calculated as mean ± SD. (e) B16 and HPMs with stable depletion of MC1R by shRNA were pre-treated with 1 μM α-MSH for 30 min followed by 25 J/m 2 UVB irradiation. Cells were subjected to SA-β-gal staining assay 7 days after UVR. The quantification of the percentage of SA-β-gal positive cells and representative pictures were shown. Data shown correspond to one representative experiment out of three independent experiments. Data are represented as mean ± SD. (f) B16 and HPMs with stable depletion of MC1R by shRNA were infected with the indicated Flag-MC1R encoding retroviral constructs. Cells were pre-treated with 1 μM α-MSH for 30 min followed by 25 J/m 2 UVB irradiation. Cells were subjected to SA-β-gal staining assay 7 days after UVR. The quantification of the percentage of SA-β-gal positive cells and representative pictures were shown. Data shown correspond to one representative experiment out of three independent experiments. Data are represented as mean ± SD. (g) hTERT/p53DD/CDK4(R24C)/BRAF V600E melanocytes with stable depletion of MC1R by shRNA were pre-incubated with 1 μM α-MSH for 30 min before being irradiated with 20 J/m 2 UVB. Cell lysates were collected for IB analysis. (h) Cells generated in (g) were subjected to clonogenic survival assays 15 days after UVR. Crystal violet was used to stain colonies and the colony numbers were counted from three independent experiments. The relative colony numbers were calculated as mean ± SD. (i) Cells generated in (g) were seeded (10,000 cells per well) in 0.5% low-melting-point agarose in DMEM with 10% FBS, layered onto 0.8% agarose in DMEM with 10% FBS. The plates were cultured for 30 days where upon the colonies > 50 μm were counted under a light microscope. The colony numbers were plotted as mean ± SD from three independent experiments. (j) MC1R-depleted hTERT/p53DD/CDK4(R24C)/BRAF V600E melanocytes were further infected with the indicated Flag-MC1R encoding retroviral constructs. Cells were pre-incubated with 1 μM α-MSH for 30min before being irradiated with 20 J/m 2 UVB. Cell lysates were collected for IB analysis. (k) The cells generated as indicated were seeded (10,000 cells per well) in 0.5% low-melting-point agarose in DMEM with 10% FBS, layered onto 0.8% agarose in DMEM+10% FBS. The plates were cultured for 30 days where upon the colonies > 50 μm were counted under a light microscope. The colony numbers were plotted as mean ± SD from three independent experiments.
    Figure Legend Snippet: Palmitoylation is essential for MC1R function (a) B16 and HPMs with stable depletion of MC1R by shRNA were infected with the indicated Flag-MC1R encoding retroviral constructs. After 3 h, cells were pre-treated with 1 μM α-MSH for 30 min followed by 100 J/m 2 UVB irradiation. Cells were harvested for a cAMP immunoassay. These data were compiled from three independent experiments. Data are represented as mean ± SD. (b-c) B16 and HPMs with stable depletion of MC1R by shRNA were infected with the indicated Flag-MC1R encoding retroviral constructs. After 3 h, cells were pre-treated with 1 μM α-MSH for 30 min followed by 100 J/m 2 UVB irradiation. Total RNA were collected for reverse transcription and cDNA were then used for quantitative real time PCR (qRT-PCR) by specific primers targeting mouse/human MITF or TYR. Three independent experiments were quantified. Data are represented as mean ± SD. (d) HPMs with stable deletion of MC1R by shRNA were infected with the indicated Flag-MC1R encoding retro-viral constructs. Cells were pre-treated with 1 μM α-MSH for 30 min followed by 100 J/m 2 UVB irradiation. Genomic DNA were extracted at the different time points as indicated and photoproducts were detected by ELISA. Cyclobutane pyrimidine dimer (CPD) or 6–4 pyrimidine photoproduct (6–4PP) antibodies were used. Three independent experiments were measured and calculated as mean ± SD. (e) B16 and HPMs with stable depletion of MC1R by shRNA were pre-treated with 1 μM α-MSH for 30 min followed by 25 J/m 2 UVB irradiation. Cells were subjected to SA-β-gal staining assay 7 days after UVR. The quantification of the percentage of SA-β-gal positive cells and representative pictures were shown. Data shown correspond to one representative experiment out of three independent experiments. Data are represented as mean ± SD. (f) B16 and HPMs with stable depletion of MC1R by shRNA were infected with the indicated Flag-MC1R encoding retroviral constructs. Cells were pre-treated with 1 μM α-MSH for 30 min followed by 25 J/m 2 UVB irradiation. Cells were subjected to SA-β-gal staining assay 7 days after UVR. The quantification of the percentage of SA-β-gal positive cells and representative pictures were shown. Data shown correspond to one representative experiment out of three independent experiments. Data are represented as mean ± SD. (g) hTERT/p53DD/CDK4(R24C)/BRAF V600E melanocytes with stable depletion of MC1R by shRNA were pre-incubated with 1 μM α-MSH for 30 min before being irradiated with 20 J/m 2 UVB. Cell lysates were collected for IB analysis. (h) Cells generated in (g) were subjected to clonogenic survival assays 15 days after UVR. Crystal violet was used to stain colonies and the colony numbers were counted from three independent experiments. The relative colony numbers were calculated as mean ± SD. (i) Cells generated in (g) were seeded (10,000 cells per well) in 0.5% low-melting-point agarose in DMEM with 10% FBS, layered onto 0.8% agarose in DMEM with 10% FBS. The plates were cultured for 30 days where upon the colonies > 50 μm were counted under a light microscope. The colony numbers were plotted as mean ± SD from three independent experiments. (j) MC1R-depleted hTERT/p53DD/CDK4(R24C)/BRAF V600E melanocytes were further infected with the indicated Flag-MC1R encoding retroviral constructs. Cells were pre-incubated with 1 μM α-MSH for 30min before being irradiated with 20 J/m 2 UVB. Cell lysates were collected for IB analysis. (k) The cells generated as indicated were seeded (10,000 cells per well) in 0.5% low-melting-point agarose in DMEM with 10% FBS, layered onto 0.8% agarose in DMEM+10% FBS. The plates were cultured for 30 days where upon the colonies > 50 μm were counted under a light microscope. The colony numbers were plotted as mean ± SD from three independent experiments.

    Techniques Used: shRNA, Infection, Construct, Irradiation, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Staining, Incubation, Generated, Cell Culture, Light Microscopy

    MC1R is palmitoylated (a) Fatty acids were dissolved in 100% ethanol (250 mM) mixed with 25 mM BSA and then added to serum-free DMEM/medium 254 at a final concertation of 500 μM. MC1R RHC-variant B16 cells were serum starved for 6 h. For the last 30 min cells were incubated with 1 μM α-MSH, followed by 100 J/m 2 UVB treatment. Lastly cells were treated with indicated BSA-conjugated fatty acid medium for 3 h. Results were calculated as mean ± SD from three independent experiments (n=3). (b) A schematic showing the general process of protein palmitoylation. (c) MC1R RHC-variant B16 melanoma cells were treated as indicated in (a). 2-BP (25 μM) was added or not with palmitic acid for 3 h and cAMP content was determined. Results were calculated as mean ± SD from three independent experiments (n=3). (d-e) MC1R RHC-variant or WT B16 melanoma cells (d) and MC1R RHC-variant or WT HPMs (e) were treated as indicated in Figure 1a . 2-BP (25 μM) was added or not with palmitic acid for 3 h and cAMP content was determined. Results were calculated as mean ± SD from three independent experiments (n=3). (f) A schematic showing the general process of the acyl-biotin exchange (ABE) palmitoylation assay. Unmodified cysteines were irreversibly blocked by N-Ethylmaliemide (NEM), and palmitoylated cysteines were then exposed using HAM and sequentially biotinylated. Biotin levels were determined as a measurement for palmitoylation. (g) Flowchart of palmitoylated protein identification. NEM, N-Ethylmaleimide. HAM, hydroxylamine. 1×10 8 MC1R RHC-variant HPMs were treated with palmitic acid as indicated in (a), and then processed as shown in flowchart. Streptavidin IP was performed and protein samples were separated by SDS-PAGE before staining by Coomassie brilliant blue. Palmitoylation-specific bands were excised for Mass Spectrometry for protein identification. (h) The peptide spectral counts of MC1R from Figure 1c . (i) NBA-palm of MC1R. The prediction shows two possible sites of MC1R palmitoylation. (j) A schematic illustration showing the palmitoylation site at MC1R C315 predicted by NBA-PALM and PEP-FOLD3, and a schematic showing the conserved C-terminal domain of MC1R. (k) Membrane topology of MC1R with indicated RHC, non-RHC and palmitoylation site mutants.
    Figure Legend Snippet: MC1R is palmitoylated (a) Fatty acids were dissolved in 100% ethanol (250 mM) mixed with 25 mM BSA and then added to serum-free DMEM/medium 254 at a final concertation of 500 μM. MC1R RHC-variant B16 cells were serum starved for 6 h. For the last 30 min cells were incubated with 1 μM α-MSH, followed by 100 J/m 2 UVB treatment. Lastly cells were treated with indicated BSA-conjugated fatty acid medium for 3 h. Results were calculated as mean ± SD from three independent experiments (n=3). (b) A schematic showing the general process of protein palmitoylation. (c) MC1R RHC-variant B16 melanoma cells were treated as indicated in (a). 2-BP (25 μM) was added or not with palmitic acid for 3 h and cAMP content was determined. Results were calculated as mean ± SD from three independent experiments (n=3). (d-e) MC1R RHC-variant or WT B16 melanoma cells (d) and MC1R RHC-variant or WT HPMs (e) were treated as indicated in Figure 1a . 2-BP (25 μM) was added or not with palmitic acid for 3 h and cAMP content was determined. Results were calculated as mean ± SD from three independent experiments (n=3). (f) A schematic showing the general process of the acyl-biotin exchange (ABE) palmitoylation assay. Unmodified cysteines were irreversibly blocked by N-Ethylmaliemide (NEM), and palmitoylated cysteines were then exposed using HAM and sequentially biotinylated. Biotin levels were determined as a measurement for palmitoylation. (g) Flowchart of palmitoylated protein identification. NEM, N-Ethylmaleimide. HAM, hydroxylamine. 1×10 8 MC1R RHC-variant HPMs were treated with palmitic acid as indicated in (a), and then processed as shown in flowchart. Streptavidin IP was performed and protein samples were separated by SDS-PAGE before staining by Coomassie brilliant blue. Palmitoylation-specific bands were excised for Mass Spectrometry for protein identification. (h) The peptide spectral counts of MC1R from Figure 1c . (i) NBA-palm of MC1R. The prediction shows two possible sites of MC1R palmitoylation. (j) A schematic illustration showing the palmitoylation site at MC1R C315 predicted by NBA-PALM and PEP-FOLD3, and a schematic showing the conserved C-terminal domain of MC1R. (k) Membrane topology of MC1R with indicated RHC, non-RHC and palmitoylation site mutants.

    Techniques Used: Variant Assay, Incubation, SDS Page, Staining, Mass Spectrometry

    MC1R palmitoylation controls melanomagenesis a , C57BL/6 MC1R variant transgenic mice. b , Eumelanin and pheomelanin content of whole skin from C57BL/6 MC1R variant transgenic mice. Data shown represent the mean ± SD of three independent experiments. c , Melanoma-free survival. Tyr-Cre n=15, Tyr-Cre-BRAF V600E - MC1R e/e n=23, Tyr-Cre-BRAF V600E - MC1R +/+ n=23, Tyr-Cre-BRAF V600E - MC1R e/e - MC1R R151C n=26, Tyr-Cre-BRAF V600E - MC1R e/e - MC1R C315S n=20. By Log-rank test, p=0.0001 (e/e +/+), p=0.0179 (e/e R151C), p=0.8943 (e/e C315S), p=0.0232 (+/+ R151C), p=0.0001 (+/+ C315S), p=0.0233 (R151C C315S). d , Indicated melanocytes were treated with α-MSH and Palm-B before UVB irradiation and assayed for clonogenic survival. Results were calculated as mean ± SD from three independent experiments. e-g , Growth curves ( e ), dissected tumors ( f ) and tumor weight ( g ) for subcutaneous xenograft experiments in nude mice (n=10) using indicated cells. Error bars represent ±SEM. h , Melanoma-free survival of Tyr-Cre-BRAF V600E - MC1R +/+ n=20, Tyr-Cre-BRAF V600E - MC1R +/+ + Palm-B n=20, Tyr-Cre-BRAF V600E - MC1R e/e - MC1R R151C n=17, Tyr-Cre-BRAF V600E - MC1R e/e - MC1R R151C + Palm-B n=18. By Log-rank test, p=0.0241 (R151C R151C + Palm-B), p=0.3711 (MC1R +/+ MC1R +/+ + Palm-B). *p
    Figure Legend Snippet: MC1R palmitoylation controls melanomagenesis a , C57BL/6 MC1R variant transgenic mice. b , Eumelanin and pheomelanin content of whole skin from C57BL/6 MC1R variant transgenic mice. Data shown represent the mean ± SD of three independent experiments. c , Melanoma-free survival. Tyr-Cre n=15, Tyr-Cre-BRAF V600E - MC1R e/e n=23, Tyr-Cre-BRAF V600E - MC1R +/+ n=23, Tyr-Cre-BRAF V600E - MC1R e/e - MC1R R151C n=26, Tyr-Cre-BRAF V600E - MC1R e/e - MC1R C315S n=20. By Log-rank test, p=0.0001 (e/e +/+), p=0.0179 (e/e R151C), p=0.8943 (e/e C315S), p=0.0232 (+/+ R151C), p=0.0001 (+/+ C315S), p=0.0233 (R151C C315S). d , Indicated melanocytes were treated with α-MSH and Palm-B before UVB irradiation and assayed for clonogenic survival. Results were calculated as mean ± SD from three independent experiments. e-g , Growth curves ( e ), dissected tumors ( f ) and tumor weight ( g ) for subcutaneous xenograft experiments in nude mice (n=10) using indicated cells. Error bars represent ±SEM. h , Melanoma-free survival of Tyr-Cre-BRAF V600E - MC1R +/+ n=20, Tyr-Cre-BRAF V600E - MC1R +/+ + Palm-B n=20, Tyr-Cre-BRAF V600E - MC1R e/e - MC1R R151C n=17, Tyr-Cre-BRAF V600E - MC1R e/e - MC1R R151C + Palm-B n=18. By Log-rank test, p=0.0241 (R151C R151C + Palm-B), p=0.3711 (MC1R +/+ MC1R +/+ + Palm-B). *p

    Techniques Used: Variant Assay, Transgenic Assay, Mouse Assay, Irradiation

    MC1R variant and mutant transgenic mice (a) Schematic diagrams of MC1R variant constructs. Transgenic mice were designed to express melanocyte-specific MC1R variants or mutants (controlled by the Tyr enhancer/promoter). (b) C57BL/6 MC1R variant or mutant transgenic mice. (c) Human transgene content in transgenic and control mice. Results were calculated as mean ± SD from three independent experiments. (d) Whole skins from C57BL/6 MC1R variant transgenic mice (8-12 weeks) were collected and stained with Dct antibody. Melanocytes were then isolated and quantified by FACS sorting. Results were calculated as mean ± SD from three independent experiments. (e) Frozen sections of skins from C57BL/6 MC1R variant transgenic mice (8-12 weeks) were stained with Dct antibody. The positive staining represents melanocytes. (f) Illustrations for UVB-induced melanoma development in Tyr-Cre-BRAF CA -MC1R variant mice. (g) H E staining of histological sections and immunohistochemistry staining of S100 of representative cutaneous melanomas. Genotypes were showed as indicated.
    Figure Legend Snippet: MC1R variant and mutant transgenic mice (a) Schematic diagrams of MC1R variant constructs. Transgenic mice were designed to express melanocyte-specific MC1R variants or mutants (controlled by the Tyr enhancer/promoter). (b) C57BL/6 MC1R variant or mutant transgenic mice. (c) Human transgene content in transgenic and control mice. Results were calculated as mean ± SD from three independent experiments. (d) Whole skins from C57BL/6 MC1R variant transgenic mice (8-12 weeks) were collected and stained with Dct antibody. Melanocytes were then isolated and quantified by FACS sorting. Results were calculated as mean ± SD from three independent experiments. (e) Frozen sections of skins from C57BL/6 MC1R variant transgenic mice (8-12 weeks) were stained with Dct antibody. The positive staining represents melanocytes. (f) Illustrations for UVB-induced melanoma development in Tyr-Cre-BRAF CA -MC1R variant mice. (g) H E staining of histological sections and immunohistochemistry staining of S100 of representative cutaneous melanomas. Genotypes were showed as indicated.

    Techniques Used: Variant Assay, Mutagenesis, Transgenic Assay, Mouse Assay, Construct, Staining, Isolation, FACS, Immunohistochemistry

    ZDHHC13 is a major MC1R palmitoyl acyltransferase a-b , B16 cells co-expressing Flag-MC1R and HA-ZDHHCs ( a ) and HPMs expressing Flag-MC1R and ZDHHC13 WT or C456S mutant ( b ) were incubated with α-MSH and processed for ABE analysis. c-f , HPMs expressing ZDHHC13 shRNAs ( c ), HPMs expressing Flag-MC1R together with shZDHHC13 and/or WT HA-ZDHHC13 ( d ), HPMs ( e ) and HPMs expressing Flag-MC1R ( f ) were pre-treated with α-MSH, UVB irradiated and harvested for IP, ABE and IB. g-i , HPMs expressing HA-ZDHHC13 ( g ), HPMs expressing shATR and HA-ZDHHC13 ( h-i ) were pre-treated with α-MSH, UVB irradiated and processed for IP and IB analysis. Western blots shown were representative of three independent experiments. For gel source data, see Supplementary Figure 1 .
    Figure Legend Snippet: ZDHHC13 is a major MC1R palmitoyl acyltransferase a-b , B16 cells co-expressing Flag-MC1R and HA-ZDHHCs ( a ) and HPMs expressing Flag-MC1R and ZDHHC13 WT or C456S mutant ( b ) were incubated with α-MSH and processed for ABE analysis. c-f , HPMs expressing ZDHHC13 shRNAs ( c ), HPMs expressing Flag-MC1R together with shZDHHC13 and/or WT HA-ZDHHC13 ( d ), HPMs ( e ) and HPMs expressing Flag-MC1R ( f ) were pre-treated with α-MSH, UVB irradiated and harvested for IP, ABE and IB. g-i , HPMs expressing HA-ZDHHC13 ( g ), HPMs expressing shATR and HA-ZDHHC13 ( h-i ) were pre-treated with α-MSH, UVB irradiated and processed for IP and IB analysis. Western blots shown were representative of three independent experiments. For gel source data, see Supplementary Figure 1 .

    Techniques Used: Expressing, Mutagenesis, Incubation, Irradiation, Western Blot

    Activating MC1R palmitoylation rescues the defect of MC1R RHC variants (a) B16 and HPMs with stable depletion of MC1R by shRNA were infected with the indicated Flag-MC1R encoding retroviral constructs, then cells were infected with shZDHHC13 and/or WT HA-ZDHHC13 virus. Cells were pre-treated with 1 μM α-MSH for 30 min followed by 100 J/m 2 UVB irradiation. After 3 h, cells were harvested for cAMP immunoassay. These data were compiled from three independent experiments. Data are represented as mean ± SD. (b) B16 and HPMs with stable depletion of MC1R by shRNA were infected with the indicated Flag-MC1R encoding retroviral constructs, then cells were infected with shZDHHC13 and/or WT HA-ZDHHC13 virus. Cells were pre-treated with 1 μM α-MSH for 30 min followed by 100 J/m 2 UVB irradiation. After 3 h, total RNA was collected for reverse transcription and cDNA were then used for qRT-PCR by specific primers targeting mouse MITF. Three independent experiments were quantified. Data are represented as mean ± SD. (c) HPMs with stable depletion of MC1R by shRNA were infected with the indicated Flag-MC1R encoding retro-viral constructs, then cells were infected with shZDHHC13 and/or WT HA-ZDHHC13 virus. Cells were pre-treated with 1 μM α-MSH for 30 min followed by 100J/m 2 UVB irradiation. Genomic DNA was extracted at the different time points indicated and photoproducts detected by ELISA using Cyclobutane pyrimidine dimer (CPD) or 6–4 pyrimidine photoproduct (6–4PP) antibodies. Three independent experiments were measured and calculated as mean ± SD. (d) B16 and HPMs with stable depletion of MC1R by shRNA were infected with the indicated Flag-MC1R encoding retroviral constructs, then cells were infected with shZDHHC13 and/or WT HA-ZDHHC13 virus. Cells were pre-treated with 1 μM α-MSH for 30 min followed by 25 J/m 2 UVB irradiation. Cells were subjected to SA-β-gal staining assay 7 days after UVR. The quantification of the percentage of SA-β-gal positive cells and representative pictures were shown. Data shown correspond to one representative experiment out of three independent experiments. Data are represented as mean ± SD. (e) The cells generated as indicated were seeded (10,000 cells per well) in 0.5% low-melting-point agarose in DMEM with 10% FBS, layered onto 0.8% agarose in DMEM+10% FBS. Plates were cultured for 30 days where upon the colonies > 50 μm were counted under a light microscope. Colony numbers were plotted as mean ± SD from three independent experiments.
    Figure Legend Snippet: Activating MC1R palmitoylation rescues the defect of MC1R RHC variants (a) B16 and HPMs with stable depletion of MC1R by shRNA were infected with the indicated Flag-MC1R encoding retroviral constructs, then cells were infected with shZDHHC13 and/or WT HA-ZDHHC13 virus. Cells were pre-treated with 1 μM α-MSH for 30 min followed by 100 J/m 2 UVB irradiation. After 3 h, cells were harvested for cAMP immunoassay. These data were compiled from three independent experiments. Data are represented as mean ± SD. (b) B16 and HPMs with stable depletion of MC1R by shRNA were infected with the indicated Flag-MC1R encoding retroviral constructs, then cells were infected with shZDHHC13 and/or WT HA-ZDHHC13 virus. Cells were pre-treated with 1 μM α-MSH for 30 min followed by 100 J/m 2 UVB irradiation. After 3 h, total RNA was collected for reverse transcription and cDNA were then used for qRT-PCR by specific primers targeting mouse MITF. Three independent experiments were quantified. Data are represented as mean ± SD. (c) HPMs with stable depletion of MC1R by shRNA were infected with the indicated Flag-MC1R encoding retro-viral constructs, then cells were infected with shZDHHC13 and/or WT HA-ZDHHC13 virus. Cells were pre-treated with 1 μM α-MSH for 30 min followed by 100J/m 2 UVB irradiation. Genomic DNA was extracted at the different time points indicated and photoproducts detected by ELISA using Cyclobutane pyrimidine dimer (CPD) or 6–4 pyrimidine photoproduct (6–4PP) antibodies. Three independent experiments were measured and calculated as mean ± SD. (d) B16 and HPMs with stable depletion of MC1R by shRNA were infected with the indicated Flag-MC1R encoding retroviral constructs, then cells were infected with shZDHHC13 and/or WT HA-ZDHHC13 virus. Cells were pre-treated with 1 μM α-MSH for 30 min followed by 25 J/m 2 UVB irradiation. Cells were subjected to SA-β-gal staining assay 7 days after UVR. The quantification of the percentage of SA-β-gal positive cells and representative pictures were shown. Data shown correspond to one representative experiment out of three independent experiments. Data are represented as mean ± SD. (e) The cells generated as indicated were seeded (10,000 cells per well) in 0.5% low-melting-point agarose in DMEM with 10% FBS, layered onto 0.8% agarose in DMEM+10% FBS. Plates were cultured for 30 days where upon the colonies > 50 μm were counted under a light microscope. Colony numbers were plotted as mean ± SD from three independent experiments.

    Techniques Used: shRNA, Infection, Construct, Irradiation, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Staining, Generated, Cell Culture, Light Microscopy

    Palm-B rescues the defect of MC1R R160W variant (a) B16 and HPMs with stable depletion of MC1R by shRNA were infected with the indicated Flag-MC1R encoding retroviral constructs. Cells were pre-treated with 1 μM α-MSH and 1 μM Palm-B for 30 min followed by 100 J/m 2 UVB irradiation. Cells were harvested for IP, ABE and IB analysis at 3 h after UVB exposure. (b) B16 and HPMs with stable depletion of MC1R by shRNA were infected with the indicated Flag-MC1R encoding retroviral constructs. Cells were pre-treated with 1 μM α-MSH and 1 μM Palm-B for 30 min followed by 100 J/m 2 UVB irradiation. After 3 h, cells were harvested for cAMP immunoassay. These data were compiled from three independent experiments. Data are represented as mean ± SD. (c) B16 and HPMs with stable depletion of MC1R by shRNA were infected with the indicated Flag-MC1R encoding retroviral constructs. Cells were pre-treated with 1 μM α-MSH and 1 μM Palm-B for 30 min followed by 100 J/m 2 UVB irradiation. After 3 h, total RNA was collected for reverse transcription and cDNA then used for qRT-PCR by specific primers targeting mouse and/or human MITF or TYR. Three independent experiments were quantified. Data are represented as mean ± SD. (d) HPMs with stable depletion of MC1R by shRNA were infected with the indicated Flag-MC1R encoding retroviral constructs. Cells were pre-treated with 1 μM α-MSH and 1 μM Palm-B for 30 min followed by 100 J/m 2 UVB irradiation. Genomic DNA were extracted at the different time points indicated and photoproducts were detected by ELISA using anti-cyclobutane pyrimidine dimer (CPD) or 6–4 pyrimidine photoproduct (6–4PP) antibodies. Three independent experiments were measured and calculated as mean ± SD. (e) B16 and HPMs with stable depletion of MC1R by shRNA were infected with the indicated Flag-MC1R encoding retroviral constructs. Cells were pre-treated with 1 μM α-MSH and 1 μM Palm-B for 30 min followed by 25 J/m 2 UVB irradiation. Cells were subjected to SA-β-gal staining assay 7 days after UVR. The quantification of the percentage of SA-β-gal positive cells and representative pictures are shown and correspond to one representative experiment out of three independent experiments. Data are represented as mean ± SD. (f) MC1R-depleted hTERT/p53DD/CDK4(R24C)/BRAF V600E melanocytes were further infected with the indicated Flag-MC1R encoding retro-viral constructs. Cells were pre-incubated with 1 μM α-MSH and 1 μM Palm-B for 30min before being irradiated with 20 J/m 2 UVB, and then subjected to clonogenic survival assays 15 days after UVR. Crystal violet was used to stain colonies and the colony numbers were counted from three independent experiments. The relative colony numbers were calculated as mean ± SD. (g) The cells generated in (k) were seeded (10,000 cells per well) in 0.5% low-melting-point agarose in DMEM with 10% FBS, layered onto 0.8% agarose in DMEM+10% FBS. Plates were cultured for 30 days where upon the colonies > 50 μm were counted under a light microscope. The colony numbers were plotted as mean ± SD from three independent experiments.
    Figure Legend Snippet: Palm-B rescues the defect of MC1R R160W variant (a) B16 and HPMs with stable depletion of MC1R by shRNA were infected with the indicated Flag-MC1R encoding retroviral constructs. Cells were pre-treated with 1 μM α-MSH and 1 μM Palm-B for 30 min followed by 100 J/m 2 UVB irradiation. Cells were harvested for IP, ABE and IB analysis at 3 h after UVB exposure. (b) B16 and HPMs with stable depletion of MC1R by shRNA were infected with the indicated Flag-MC1R encoding retroviral constructs. Cells were pre-treated with 1 μM α-MSH and 1 μM Palm-B for 30 min followed by 100 J/m 2 UVB irradiation. After 3 h, cells were harvested for cAMP immunoassay. These data were compiled from three independent experiments. Data are represented as mean ± SD. (c) B16 and HPMs with stable depletion of MC1R by shRNA were infected with the indicated Flag-MC1R encoding retroviral constructs. Cells were pre-treated with 1 μM α-MSH and 1 μM Palm-B for 30 min followed by 100 J/m 2 UVB irradiation. After 3 h, total RNA was collected for reverse transcription and cDNA then used for qRT-PCR by specific primers targeting mouse and/or human MITF or TYR. Three independent experiments were quantified. Data are represented as mean ± SD. (d) HPMs with stable depletion of MC1R by shRNA were infected with the indicated Flag-MC1R encoding retroviral constructs. Cells were pre-treated with 1 μM α-MSH and 1 μM Palm-B for 30 min followed by 100 J/m 2 UVB irradiation. Genomic DNA were extracted at the different time points indicated and photoproducts were detected by ELISA using anti-cyclobutane pyrimidine dimer (CPD) or 6–4 pyrimidine photoproduct (6–4PP) antibodies. Three independent experiments were measured and calculated as mean ± SD. (e) B16 and HPMs with stable depletion of MC1R by shRNA were infected with the indicated Flag-MC1R encoding retroviral constructs. Cells were pre-treated with 1 μM α-MSH and 1 μM Palm-B for 30 min followed by 25 J/m 2 UVB irradiation. Cells were subjected to SA-β-gal staining assay 7 days after UVR. The quantification of the percentage of SA-β-gal positive cells and representative pictures are shown and correspond to one representative experiment out of three independent experiments. Data are represented as mean ± SD. (f) MC1R-depleted hTERT/p53DD/CDK4(R24C)/BRAF V600E melanocytes were further infected with the indicated Flag-MC1R encoding retro-viral constructs. Cells were pre-incubated with 1 μM α-MSH and 1 μM Palm-B for 30min before being irradiated with 20 J/m 2 UVB, and then subjected to clonogenic survival assays 15 days after UVR. Crystal violet was used to stain colonies and the colony numbers were counted from three independent experiments. The relative colony numbers were calculated as mean ± SD. (g) The cells generated in (k) were seeded (10,000 cells per well) in 0.5% low-melting-point agarose in DMEM with 10% FBS, layered onto 0.8% agarose in DMEM+10% FBS. Plates were cultured for 30 days where upon the colonies > 50 μm were counted under a light microscope. The colony numbers were plotted as mean ± SD from three independent experiments.

    Techniques Used: Variant Assay, shRNA, Infection, Construct, Irradiation, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Staining, Incubation, Generated, Cell Culture, Light Microscopy

    3) Product Images from "The protective role of MC1R in chromosome stability and centromeric integrity in melanocytes"

    Article Title: The protective role of MC1R in chromosome stability and centromeric integrity in melanocytes

    Journal: Cell Death Discovery

    doi: 10.1038/s41420-021-00499-9

    MC1R is a protective factor in chromosome stability and centromere integrity in melanocytes. In human melanocytes, α-MSH binds MC1R and activates MC1R signaling, thus the expression of MC1R downstream factor MITF is stimulated. As a result, the enhanced interaction between MITF and CENP-A protects chromosome stability and reduces the risk of tumor initiation.
    Figure Legend Snippet: MC1R is a protective factor in chromosome stability and centromere integrity in melanocytes. In human melanocytes, α-MSH binds MC1R and activates MC1R signaling, thus the expression of MC1R downstream factor MITF is stimulated. As a result, the enhanced interaction between MITF and CENP-A protects chromosome stability and reduces the risk of tumor initiation.

    Techniques Used: Expressing

    The role of α-MSH/MC1R in the prevention of UVR-induced chromosomal instability and centromere fragmentations in HPMs. A Chromosome was analyzed in HPMs stimulated with or without MSH ( n = 84). Low panel: centromeric fragments were counted as indicated. B Multiple cytogenetic abnormalities as indicated were counted in metaphase spread in HPMs.
    Figure Legend Snippet: The role of α-MSH/MC1R in the prevention of UVR-induced chromosomal instability and centromere fragmentations in HPMs. A Chromosome was analyzed in HPMs stimulated with or without MSH ( n = 84). Low panel: centromeric fragments were counted as indicated. B Multiple cytogenetic abnormalities as indicated were counted in metaphase spread in HPMs.

    Techniques Used:

    Lack of MC1R disrupts CENP-A/C binding to centromeric and pericentric DNAs. ChIP assays were performed to determine the binding of CENP-A ( A ) and CENP-C ( B ) to centromeric ( Satα ) and pericentric ( Sat2 ) DNAs in HPMs/shControl or HPMs/shMC1R, with the Ideal ChIP-seq kit (Diagenode). Anti-CENP-A, anti-CENP-C, or control IgG was used for each IP. After completion of the ChIP, samples were diluted 1:100 in ddH 2 O for qPCR. Satα and Sat2 DNAs were amplified. The value in IgG group was set as “1”. * p
    Figure Legend Snippet: Lack of MC1R disrupts CENP-A/C binding to centromeric and pericentric DNAs. ChIP assays were performed to determine the binding of CENP-A ( A ) and CENP-C ( B ) to centromeric ( Satα ) and pericentric ( Sat2 ) DNAs in HPMs/shControl or HPMs/shMC1R, with the Ideal ChIP-seq kit (Diagenode). Anti-CENP-A, anti-CENP-C, or control IgG was used for each IP. After completion of the ChIP, samples were diluted 1:100 in ddH 2 O for qPCR. Satα and Sat2 DNAs were amplified. The value in IgG group was set as “1”. * p

    Techniques Used: Binding Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Amplification

    Mitf is required in α - MSH/MC1R-controlled chromosome stability after UVR. A , B Metaphase spread was performed in HPMs with Mitf knockdown ( A ) or Mitf overexpression ( B ). The cells were then irradiated with 100 J/m 2 UVB. Representative images are displayed. C , D Graphs illustrating the percentage of cytogenetic abnormalities in A and B from three independent experiments. n = 50, Error bars represent SEM.
    Figure Legend Snippet: Mitf is required in α - MSH/MC1R-controlled chromosome stability after UVR. A , B Metaphase spread was performed in HPMs with Mitf knockdown ( A ) or Mitf overexpression ( B ). The cells were then irradiated with 100 J/m 2 UVB. Representative images are displayed. C , D Graphs illustrating the percentage of cytogenetic abnormalities in A and B from three independent experiments. n = 50, Error bars represent SEM.

    Techniques Used: Over Expression, Irradiation

    4) Product Images from "Diminishment of ?-MSH anti-inflammatory activity in MC1r siRNA-transfected RAW264.7 macrophages"

    Article Title: Diminishment of ?-MSH anti-inflammatory activity in MC1r siRNA-transfected RAW264.7 macrophages

    Journal:

    doi: 10.1189/jlb.0707463

    The effects of MC1r mRNA silencing on α-MSH suppression of LPS-stimulated NO and TNF-α production in macrophages. (A) Cultures of macrophages transfected 24 h before with MC1r siRNA were treated with 100 nM α-MSH and 100 ng/ml
    Figure Legend Snippet: The effects of MC1r mRNA silencing on α-MSH suppression of LPS-stimulated NO and TNF-α production in macrophages. (A) Cultures of macrophages transfected 24 h before with MC1r siRNA were treated with 100 nM α-MSH and 100 ng/ml

    Techniques Used: Transfection

    The effects of MC1r siRNA transfection of macrophages. The RAW264.7 macrophages were transfected with MC1r siRNA or with irrelevant siRNA. (A) After 24 and 48 h of incubation, MC1r mRNA expression was measured by real-time PCR, and the mRNA expression
    Figure Legend Snippet: The effects of MC1r siRNA transfection of macrophages. The RAW264.7 macrophages were transfected with MC1r siRNA or with irrelevant siRNA. (A) After 24 and 48 h of incubation, MC1r mRNA expression was measured by real-time PCR, and the mRNA expression

    Techniques Used: Transfection, Incubation, Expressing, Real-time Polymerase Chain Reaction

    The effects of MC1r mRNA silencing and LPS stimulation on MC3r expression and function. (A) Anti-MC3r immunoblotting of immunoprecipitated proteins from lysates of macrophages that were transfected 24 h before with MC1r siRNA or irrelevant (Irr) siRNA.
    Figure Legend Snippet: The effects of MC1r mRNA silencing and LPS stimulation on MC3r expression and function. (A) Anti-MC3r immunoblotting of immunoprecipitated proteins from lysates of macrophages that were transfected 24 h before with MC1r siRNA or irrelevant (Irr) siRNA.

    Techniques Used: Expressing, Immunoprecipitation, Transfection

    The effects of MC1r mRNA silencing on α-MSH suppression of LPS-stimulated NF-κB activation and p38 MAPK phosphorylation in macrophages. (A) Cultures of macrophages transfected 24 h before with MC1r siRNA were treated with 100 nM α-MSH
    Figure Legend Snippet: The effects of MC1r mRNA silencing on α-MSH suppression of LPS-stimulated NF-κB activation and p38 MAPK phosphorylation in macrophages. (A) Cultures of macrophages transfected 24 h before with MC1r siRNA were treated with 100 nM α-MSH

    Techniques Used: Activation Assay, Transfection

    Expression of MC1r, MC3r, and MC5r mRNA in RAW264.7 macrophage cells. Total RNA was isolated from RAW264.7 macrophages cultured for 36 h and analyzed by quantitative real-time PCR. The results are the mean relative mRNA expression ± sd . The results
    Figure Legend Snippet: Expression of MC1r, MC3r, and MC5r mRNA in RAW264.7 macrophage cells. Total RNA was isolated from RAW264.7 macrophages cultured for 36 h and analyzed by quantitative real-time PCR. The results are the mean relative mRNA expression ± sd . The results

    Techniques Used: Expressing, Isolation, Cell Culture, Real-time Polymerase Chain Reaction

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  • 93
    Thermo Fisher mouse mc1r
    (CKPV) 2 induces macrophage M1 to M2 polarization. Cytokine release profile (IL-1β, IL-6 and IL-10) and arginase activity after indicated treatment/s in primary cultured macrophages transfected with or without <t>MC1R</t> RNAi. LPS:5 ng/ml, IFN-γ:10 ng/ml, α-MSH: 10 µM, and (CKPV) 2 (0.1, 1 and 5 µM) (B–E). The supernatant of the above macrophages were collected and added to L929 cells, after 20 hours, cell viability was measured by MTT assay, the inhibitory rate was calculated by 100%-cell viability OD of treatment group/cell viability OD of untreated control group (A). * p
    Mouse Mc1r, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Price from $9.99 to $1999.99
    mouse mc1r - by Bioz Stars, 2022-10
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    99
    Thermo Fisher mc1r sirna duplex against mouse mc1r mrna duplex
    The effects of <t>MC1r</t> <t>mRNA</t> silencing on α-MSH suppression of LPS-stimulated NO and TNF-α production in macrophages. (A) Cultures of macrophages transfected 24 h before with MC1r <t>siRNA</t> were treated with 100 nM α-MSH and 100 ng/ml
    Mc1r Sirna Duplex Against Mouse Mc1r Mrna Duplex, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mc1r sirna duplex against mouse mc1r mrna duplex/product/Thermo Fisher
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    86
    Thermo Fisher gene exp mc1r hs00267167 s1
    Blood urea nitrogen (BUN) does not differ in untreated adriamycin mice compared with <t>MC1R</t> agonist treated mice. BUN was measured in plasma samples taken from mice 8–10 days after injection of adriamycin. There was no difference between the groups (n = 9–10, n.s.). Results are presented as mean ± SEM.
    Gene Exp Mc1r Hs00267167 S1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/gene exp mc1r hs00267167 s1/product/Thermo Fisher
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    gene exp mc1r hs00267167 s1 - by Bioz Stars, 2022-10
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    Image Search Results


    (CKPV) 2 induces macrophage M1 to M2 polarization. Cytokine release profile (IL-1β, IL-6 and IL-10) and arginase activity after indicated treatment/s in primary cultured macrophages transfected with or without MC1R RNAi. LPS:5 ng/ml, IFN-γ:10 ng/ml, α-MSH: 10 µM, and (CKPV) 2 (0.1, 1 and 5 µM) (B–E). The supernatant of the above macrophages were collected and added to L929 cells, after 20 hours, cell viability was measured by MTT assay, the inhibitory rate was calculated by 100%-cell viability OD of treatment group/cell viability OD of untreated control group (A). * p

    Journal: PLoS ONE

    Article Title: The Synthetic Melanocortin (CKPV)2 Exerts Anti-Fungal and Anti-Inflammatory Effects against Candida albicans Vaginitis via Inducing Macrophage M2 Polarization

    doi: 10.1371/journal.pone.0056004

    Figure Lengend Snippet: (CKPV) 2 induces macrophage M1 to M2 polarization. Cytokine release profile (IL-1β, IL-6 and IL-10) and arginase activity after indicated treatment/s in primary cultured macrophages transfected with or without MC1R RNAi. LPS:5 ng/ml, IFN-γ:10 ng/ml, α-MSH: 10 µM, and (CKPV) 2 (0.1, 1 and 5 µM) (B–E). The supernatant of the above macrophages were collected and added to L929 cells, after 20 hours, cell viability was measured by MTT assay, the inhibitory rate was calculated by 100%-cell viability OD of treatment group/cell viability OD of untreated control group (A). * p

    Article Snippet: MC1R RNA Interference (RNAi) The chemically synthesized MC1R siRNA (small interfering RNA) duplexes ( ) against mouse MC1R (s1, s2 and s3) were purchased from Ambion (GenePharm Co. Ltd. Shanghai, China).

    Techniques: Activity Assay, Cell Culture, Transfection, MTT Assay

    (CKPV) 2 promotes cAMP production via MC1R. (A) Upper panel: the effects of MC1R siRNA (S1, S2 and S3, see sequence on Tab. 1) on MC1R mRNA level in primary cultured macrophages. Lower panel: MC1R siRNA knockdown almost blocked (CKPV) 2 -induced cAMP production in macrophages. (B) Upper panel, RT-PCR results confirms (CKPV) 2 mRNA expression in MC1R cDNA-transfected COS-7 cells (COS-7/MC1R) or nonsense-cDNA-transfected control cells (COS-7). Lower panel: (CKPV) 2 -induced cAMP production in negative control-cDNA-transfected (NC) or MC1R cDNA-transfected COS-7 cells. * p

    Journal: PLoS ONE

    Article Title: The Synthetic Melanocortin (CKPV)2 Exerts Anti-Fungal and Anti-Inflammatory Effects against Candida albicans Vaginitis via Inducing Macrophage M2 Polarization

    doi: 10.1371/journal.pone.0056004

    Figure Lengend Snippet: (CKPV) 2 promotes cAMP production via MC1R. (A) Upper panel: the effects of MC1R siRNA (S1, S2 and S3, see sequence on Tab. 1) on MC1R mRNA level in primary cultured macrophages. Lower panel: MC1R siRNA knockdown almost blocked (CKPV) 2 -induced cAMP production in macrophages. (B) Upper panel, RT-PCR results confirms (CKPV) 2 mRNA expression in MC1R cDNA-transfected COS-7 cells (COS-7/MC1R) or nonsense-cDNA-transfected control cells (COS-7). Lower panel: (CKPV) 2 -induced cAMP production in negative control-cDNA-transfected (NC) or MC1R cDNA-transfected COS-7 cells. * p

    Article Snippet: MC1R RNA Interference (RNAi) The chemically synthesized MC1R siRNA (small interfering RNA) duplexes ( ) against mouse MC1R (s1, s2 and s3) were purchased from Ambion (GenePharm Co. Ltd. Shanghai, China).

    Techniques: Sequencing, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Expressing, Transfection, Negative Control

    MC1R is a protective factor in chromosome stability and centromere integrity in melanocytes. In human melanocytes, α-MSH binds MC1R and activates MC1R signaling, thus the expression of MC1R downstream factor MITF is stimulated. As a result, the enhanced interaction between MITF and CENP-A protects chromosome stability and reduces the risk of tumor initiation.

    Journal: Cell Death Discovery

    Article Title: The protective role of MC1R in chromosome stability and centromeric integrity in melanocytes

    doi: 10.1038/s41420-021-00499-9

    Figure Lengend Snippet: MC1R is a protective factor in chromosome stability and centromere integrity in melanocytes. In human melanocytes, α-MSH binds MC1R and activates MC1R signaling, thus the expression of MC1R downstream factor MITF is stimulated. As a result, the enhanced interaction between MITF and CENP-A protects chromosome stability and reduces the risk of tumor initiation.

    Article Snippet: The generation of MC1R expression plasmids and GST-MC1R fusion protein was described previously . shRNA constructs targeting human MC1R (Cat. No. RHS4533-EG4157) or mouse MC1R (Cat. No. RMM4534-EG17199) were purchased from Open Biosystems.

    Techniques: Expressing

    The role of α-MSH/MC1R in the prevention of UVR-induced chromosomal instability and centromere fragmentations in HPMs. A Chromosome was analyzed in HPMs stimulated with or without MSH ( n = 84). Low panel: centromeric fragments were counted as indicated. B Multiple cytogenetic abnormalities as indicated were counted in metaphase spread in HPMs.

    Journal: Cell Death Discovery

    Article Title: The protective role of MC1R in chromosome stability and centromeric integrity in melanocytes

    doi: 10.1038/s41420-021-00499-9

    Figure Lengend Snippet: The role of α-MSH/MC1R in the prevention of UVR-induced chromosomal instability and centromere fragmentations in HPMs. A Chromosome was analyzed in HPMs stimulated with or without MSH ( n = 84). Low panel: centromeric fragments were counted as indicated. B Multiple cytogenetic abnormalities as indicated were counted in metaphase spread in HPMs.

    Article Snippet: The generation of MC1R expression plasmids and GST-MC1R fusion protein was described previously . shRNA constructs targeting human MC1R (Cat. No. RHS4533-EG4157) or mouse MC1R (Cat. No. RMM4534-EG17199) were purchased from Open Biosystems.

    Techniques:

    Lack of MC1R disrupts CENP-A/C binding to centromeric and pericentric DNAs. ChIP assays were performed to determine the binding of CENP-A ( A ) and CENP-C ( B ) to centromeric ( Satα ) and pericentric ( Sat2 ) DNAs in HPMs/shControl or HPMs/shMC1R, with the Ideal ChIP-seq kit (Diagenode). Anti-CENP-A, anti-CENP-C, or control IgG was used for each IP. After completion of the ChIP, samples were diluted 1:100 in ddH 2 O for qPCR. Satα and Sat2 DNAs were amplified. The value in IgG group was set as “1”. * p

    Journal: Cell Death Discovery

    Article Title: The protective role of MC1R in chromosome stability and centromeric integrity in melanocytes

    doi: 10.1038/s41420-021-00499-9

    Figure Lengend Snippet: Lack of MC1R disrupts CENP-A/C binding to centromeric and pericentric DNAs. ChIP assays were performed to determine the binding of CENP-A ( A ) and CENP-C ( B ) to centromeric ( Satα ) and pericentric ( Sat2 ) DNAs in HPMs/shControl or HPMs/shMC1R, with the Ideal ChIP-seq kit (Diagenode). Anti-CENP-A, anti-CENP-C, or control IgG was used for each IP. After completion of the ChIP, samples were diluted 1:100 in ddH 2 O for qPCR. Satα and Sat2 DNAs were amplified. The value in IgG group was set as “1”. * p

    Article Snippet: The generation of MC1R expression plasmids and GST-MC1R fusion protein was described previously . shRNA constructs targeting human MC1R (Cat. No. RHS4533-EG4157) or mouse MC1R (Cat. No. RMM4534-EG17199) were purchased from Open Biosystems.

    Techniques: Binding Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Amplification

    Mitf is required in α - MSH/MC1R-controlled chromosome stability after UVR. A , B Metaphase spread was performed in HPMs with Mitf knockdown ( A ) or Mitf overexpression ( B ). The cells were then irradiated with 100 J/m 2 UVB. Representative images are displayed. C , D Graphs illustrating the percentage of cytogenetic abnormalities in A and B from three independent experiments. n = 50, Error bars represent SEM.

    Journal: Cell Death Discovery

    Article Title: The protective role of MC1R in chromosome stability and centromeric integrity in melanocytes

    doi: 10.1038/s41420-021-00499-9

    Figure Lengend Snippet: Mitf is required in α - MSH/MC1R-controlled chromosome stability after UVR. A , B Metaphase spread was performed in HPMs with Mitf knockdown ( A ) or Mitf overexpression ( B ). The cells were then irradiated with 100 J/m 2 UVB. Representative images are displayed. C , D Graphs illustrating the percentage of cytogenetic abnormalities in A and B from three independent experiments. n = 50, Error bars represent SEM.

    Article Snippet: The generation of MC1R expression plasmids and GST-MC1R fusion protein was described previously . shRNA constructs targeting human MC1R (Cat. No. RHS4533-EG4157) or mouse MC1R (Cat. No. RMM4534-EG17199) were purchased from Open Biosystems.

    Techniques: Over Expression, Irradiation

    The effects of MC1r mRNA silencing on α-MSH suppression of LPS-stimulated NO and TNF-α production in macrophages. (A) Cultures of macrophages transfected 24 h before with MC1r siRNA were treated with 100 nM α-MSH and 100 ng/ml

    Journal:

    Article Title: Diminishment of ?-MSH anti-inflammatory activity in MC1r siRNA-transfected RAW264.7 macrophages

    doi: 10.1189/jlb.0707463

    Figure Lengend Snippet: The effects of MC1r mRNA silencing on α-MSH suppression of LPS-stimulated NO and TNF-α production in macrophages. (A) Cultures of macrophages transfected 24 h before with MC1r siRNA were treated with 100 nM α-MSH and 100 ng/ml

    Article Snippet: Chemically synthesized, predesigned MC1r siRNA duplex against mouse MC1r mRNA duplex was obtained from Ambion (Austin, TX, USA).

    Techniques: Transfection

    The effects of MC1r siRNA transfection of macrophages. The RAW264.7 macrophages were transfected with MC1r siRNA or with irrelevant siRNA. (A) After 24 and 48 h of incubation, MC1r mRNA expression was measured by real-time PCR, and the mRNA expression

    Journal:

    Article Title: Diminishment of ?-MSH anti-inflammatory activity in MC1r siRNA-transfected RAW264.7 macrophages

    doi: 10.1189/jlb.0707463

    Figure Lengend Snippet: The effects of MC1r siRNA transfection of macrophages. The RAW264.7 macrophages were transfected with MC1r siRNA or with irrelevant siRNA. (A) After 24 and 48 h of incubation, MC1r mRNA expression was measured by real-time PCR, and the mRNA expression

    Article Snippet: Chemically synthesized, predesigned MC1r siRNA duplex against mouse MC1r mRNA duplex was obtained from Ambion (Austin, TX, USA).

    Techniques: Transfection, Incubation, Expressing, Real-time Polymerase Chain Reaction

    The effects of MC1r mRNA silencing and LPS stimulation on MC3r expression and function. (A) Anti-MC3r immunoblotting of immunoprecipitated proteins from lysates of macrophages that were transfected 24 h before with MC1r siRNA or irrelevant (Irr) siRNA.

    Journal:

    Article Title: Diminishment of ?-MSH anti-inflammatory activity in MC1r siRNA-transfected RAW264.7 macrophages

    doi: 10.1189/jlb.0707463

    Figure Lengend Snippet: The effects of MC1r mRNA silencing and LPS stimulation on MC3r expression and function. (A) Anti-MC3r immunoblotting of immunoprecipitated proteins from lysates of macrophages that were transfected 24 h before with MC1r siRNA or irrelevant (Irr) siRNA.

    Article Snippet: Chemically synthesized, predesigned MC1r siRNA duplex against mouse MC1r mRNA duplex was obtained from Ambion (Austin, TX, USA).

    Techniques: Expressing, Immunoprecipitation, Transfection

    The effects of MC1r mRNA silencing on α-MSH suppression of LPS-stimulated NF-κB activation and p38 MAPK phosphorylation in macrophages. (A) Cultures of macrophages transfected 24 h before with MC1r siRNA were treated with 100 nM α-MSH

    Journal:

    Article Title: Diminishment of ?-MSH anti-inflammatory activity in MC1r siRNA-transfected RAW264.7 macrophages

    doi: 10.1189/jlb.0707463

    Figure Lengend Snippet: The effects of MC1r mRNA silencing on α-MSH suppression of LPS-stimulated NF-κB activation and p38 MAPK phosphorylation in macrophages. (A) Cultures of macrophages transfected 24 h before with MC1r siRNA were treated with 100 nM α-MSH

    Article Snippet: Chemically synthesized, predesigned MC1r siRNA duplex against mouse MC1r mRNA duplex was obtained from Ambion (Austin, TX, USA).

    Techniques: Activation Assay, Transfection

    Expression of MC1r, MC3r, and MC5r mRNA in RAW264.7 macrophage cells. Total RNA was isolated from RAW264.7 macrophages cultured for 36 h and analyzed by quantitative real-time PCR. The results are the mean relative mRNA expression ± sd . The results

    Journal:

    Article Title: Diminishment of ?-MSH anti-inflammatory activity in MC1r siRNA-transfected RAW264.7 macrophages

    doi: 10.1189/jlb.0707463

    Figure Lengend Snippet: Expression of MC1r, MC3r, and MC5r mRNA in RAW264.7 macrophage cells. Total RNA was isolated from RAW264.7 macrophages cultured for 36 h and analyzed by quantitative real-time PCR. The results are the mean relative mRNA expression ± sd . The results

    Article Snippet: Chemically synthesized, predesigned MC1r siRNA duplex against mouse MC1r mRNA duplex was obtained from Ambion (Austin, TX, USA).

    Techniques: Expressing, Isolation, Cell Culture, Real-time Polymerase Chain Reaction

    Blood urea nitrogen (BUN) does not differ in untreated adriamycin mice compared with MC1R agonist treated mice. BUN was measured in plasma samples taken from mice 8–10 days after injection of adriamycin. There was no difference between the groups (n = 9–10, n.s.). Results are presented as mean ± SEM.

    Journal: PLoS ONE

    Article Title: Effects of Melanocortin 1 Receptor Agonists in Experimental Nephropathies

    doi: 10.1371/journal.pone.0087816

    Figure Lengend Snippet: Blood urea nitrogen (BUN) does not differ in untreated adriamycin mice compared with MC1R agonist treated mice. BUN was measured in plasma samples taken from mice 8–10 days after injection of adriamycin. There was no difference between the groups (n = 9–10, n.s.). Results are presented as mean ± SEM.

    Article Snippet: The following primers and probes, all verified by ABI, were used to detect mRNA of the following genes: GAPDH: Mm99999915_g1 (mouse), Rn01775763_g1 (rat), Hs99999905_m1 (human); MC1R: Mm00434851_s1 (mouse), custom made GenBankID AB306978.1 (rat), Hs00267167_s1 (human).

    Techniques: Mouse Assay, Injection

    Albuminuria was reduced in MC1R agonist treated PHN rats. After four weeks of MS05 treatment (n = 17), the urinary albumin- to-creatinine ratio (UACR) was significantly reduced compared to untreated PHN (n = 14; p

    Journal: PLoS ONE

    Article Title: Effects of Melanocortin 1 Receptor Agonists in Experimental Nephropathies

    doi: 10.1371/journal.pone.0087816

    Figure Lengend Snippet: Albuminuria was reduced in MC1R agonist treated PHN rats. After four weeks of MS05 treatment (n = 17), the urinary albumin- to-creatinine ratio (UACR) was significantly reduced compared to untreated PHN (n = 14; p

    Article Snippet: The following primers and probes, all verified by ABI, were used to detect mRNA of the following genes: GAPDH: Mm99999915_g1 (mouse), Rn01775763_g1 (rat), Hs99999905_m1 (human); MC1R: Mm00434851_s1 (mouse), custom made GenBankID AB306978.1 (rat), Hs00267167_s1 (human).

    Techniques:

    MC1R protein expression. Western blotting was performed in order to detect MC1R protein expression in mouse and human tissue. Left panel: incubation with a rabbit polyclonal MC1R antibody (Alomone Labs). Right panel: incubation with MC1R antibody and control blocking peptide antigen (BP) in order to show antibody specificity. A) mouse whole glomerular lysate, B) cultured wild type mouse podocytes and C) a human malignant melanoma cell line A375. MC1R protein expression was detected at the expected size of 37 kDa (left panel).

    Journal: PLoS ONE

    Article Title: Effects of Melanocortin 1 Receptor Agonists in Experimental Nephropathies

    doi: 10.1371/journal.pone.0087816

    Figure Lengend Snippet: MC1R protein expression. Western blotting was performed in order to detect MC1R protein expression in mouse and human tissue. Left panel: incubation with a rabbit polyclonal MC1R antibody (Alomone Labs). Right panel: incubation with MC1R antibody and control blocking peptide antigen (BP) in order to show antibody specificity. A) mouse whole glomerular lysate, B) cultured wild type mouse podocytes and C) a human malignant melanoma cell line A375. MC1R protein expression was detected at the expected size of 37 kDa (left panel).

    Article Snippet: The following primers and probes, all verified by ABI, were used to detect mRNA of the following genes: GAPDH: Mm99999915_g1 (mouse), Rn01775763_g1 (rat), Hs99999905_m1 (human); MC1R: Mm00434851_s1 (mouse), custom made GenBankID AB306978.1 (rat), Hs00267167_s1 (human).

    Techniques: Expressing, Western Blot, Incubation, Blocking Assay, Cell Culture

    MC1R agonists did not reduce albuminuria in adriamycin-treated mice. (A) Five different doses of adriamycin, in the range of 5–10 mg/kg, were intravenously injected and the level of albuminuria was measured after 7 days. The urinary albumin-to-creatinine ratio (UACR) increased with increasing levels of adriamycin. (B) Albuminuria at day 7. Treatment with the MC1R agonist BMS-470539 did not reduce the level of albuminuria for the 8 mg/kg adriamycin dose, on the contrary it slightly increased albuminuria for the 10 mg/kg dose (n = 17–19, p

    Journal: PLoS ONE

    Article Title: Effects of Melanocortin 1 Receptor Agonists in Experimental Nephropathies

    doi: 10.1371/journal.pone.0087816

    Figure Lengend Snippet: MC1R agonists did not reduce albuminuria in adriamycin-treated mice. (A) Five different doses of adriamycin, in the range of 5–10 mg/kg, were intravenously injected and the level of albuminuria was measured after 7 days. The urinary albumin-to-creatinine ratio (UACR) increased with increasing levels of adriamycin. (B) Albuminuria at day 7. Treatment with the MC1R agonist BMS-470539 did not reduce the level of albuminuria for the 8 mg/kg adriamycin dose, on the contrary it slightly increased albuminuria for the 10 mg/kg dose (n = 17–19, p

    Article Snippet: The following primers and probes, all verified by ABI, were used to detect mRNA of the following genes: GAPDH: Mm99999915_g1 (mouse), Rn01775763_g1 (rat), Hs99999905_m1 (human); MC1R: Mm00434851_s1 (mouse), custom made GenBankID AB306978.1 (rat), Hs00267167_s1 (human).

    Techniques: Mouse Assay, Injection