mc1r variants plasmid collection human mc1r trueorf clones  (OriGene)

Journal: NPJ precision oncology

Article Title: MC1R signaling through the cAMP-CREB/ATF-1 and ERK-NFκB pathways accelerates G1/S transition promoting breast cancer progression.

doi: 10.1038/s41698-023-00437-1

Figure Lengend Snippet: Fig. 1 MC1R expression is associated with breast cancer. a The MC1R R% allele frequency across cancers in The Cancer Genome Atlas (TCGA). The orange dashed line indicates the R% allele frequency in control ExAC non-TCGA non-finnish European (NFE) Ancestry population. *p < 0.05 (Fisher’s exact test with Benjamini–Hochberg (BH) multiple comparisons adjustment) compared to the control population. The cancer type abbreviations are expanded and listed in Supplementary Table 2. b MC1R mRNA expression in normal breast, primary, and metastatic breast cancers. Data obtained from TCGA. The dashed lines in the violin plot show the median and the 25th and 75th percentiles. *p < 0.05 (one-way ANOVA with Dunnett’s multiple comparisons). c–h Disease-free survival (DFS) (c, e, g) and progression-free survival (PFS) (d, f, h) of all patients with breast cancer in TCGA (DFS: low expression n = 462, high expression n = 477; PFS: low expression n = 539, high expression n = 541) (c, d), patients carrying active MC1R variants (DFS: low expression n = 395, high expression n = 408; PFS, low expression n = 461, high expression n = 462) (e, f), and patients carrying a disruptive MC1R variant (DFS, low expression n = 67, high expression n = 69; PFS, low expression n = 78, high expression n = 79) (g, h) based on MC1R expression. p-Value calculated using the Log-rank test.

Article Snippet: For transient MC1R overexpression, HEK 293 T cells, plated in 6-well plates in complete DMEM, were transfected with an MC1R overexpression plasmid (OriGene; Cat#: RC203218) using Lipofectamine 3000 (Thermo Fisher).

Techniques: Expressing, Control, Variant Assay

Journal: NPJ precision oncology

Article Title: MC1R signaling through the cAMP-CREB/ATF-1 and ERK-NFκB pathways accelerates G1/S transition promoting breast cancer progression.

doi: 10.1038/s41698-023-00437-1

Figure Lengend Snippet: Fig. 2 MC1R signaling is active in breast cancer cells and promotes breast cancer cell progression in vitro. a Mean ± SEM cAMP levels in T-47d and MCF7 cells treated with or without 0.2 μM NDP-MSH from three independent experiments. *p < 0.05 (unpaired Student’s t-test). b, c Mean ± SEM percentage fold change in cAMP levels in (b) T-47d and (c) MCF7 cells treated with or without 20 μM MSG-606 and stimulated with 0.2 μM NDP-MSH compared to those left untreated (UT) from three independent experiments. *p < 0.05 (one-way ANOVA with Tukey’s multiple comparisons). d T-47d and MCF7 cells were treated with 0.2 μM NDP-MSH or pretreated with 20 μM MSG-606, followed by stimulation with 0.2 μM NDP-MSH or left untreated. Representative western blot showing MC1R expression and downstream signaling in T-47d, MCF7 cells. GAPDH is shown as the loading control. Western blot quantification plots are shown in Supplementary Fig. 3a. e WT T-47d (WT) and MC1R knockdown (KD) T-47d (MC1R Sh1 and MC1R Sh2) cell lines were treated with 0.2 μM NDP-MSH. Representative western blot showing MC1R expression and downstream signaling. GAPDH is shown as the loading control. Western blot quantification plots are shown in Supplementary Fig. 3b. f Mean ± SEM fold change in cAMP levels in MC1R-KD T-47D cells treated with 0.2 μM NDP-MSH from 3 independent experiments. *p < 0.05 (one-way ANOVA with Tukey’s multiple comparisons). g, h Mean ± SEM fold change in cell number in (g) T-47d and (h) MCF7 cells treated either with 0.2 μM NDP-MSH or 20 μM MSG-606 or left untreated (UT) over time from three independent experiments. *p < 0.05 (two-way ANOVA with Dunnett’s multiple comparisons) compared to UT at the indicated time points. i Mean ± SEM fold change in cell number of WT T-47d cells and MC1R-KD T-47d cells (MC1R Sh1 and MC1R Sh2) treated with or without 0.2 μM NDP-MSH. (VC, vector control) over time from three independent experiments. *p < 0.05 (two-way ANOVA with Dunnett’s multiple comparisons) compared to T-47d WT at the indicated time points.

Article Snippet: For transient MC1R overexpression, HEK 293 T cells, plated in 6-well plates in complete DMEM, were transfected with an MC1R overexpression plasmid (OriGene; Cat#: RC203218) using Lipofectamine 3000 (Thermo Fisher).

Techniques: In Vitro, Western Blot, Expressing, Control, Knockdown, Plasmid Preparation

Journal: NPJ precision oncology

Article Title: MC1R signaling through the cAMP-CREB/ATF-1 and ERK-NFκB pathways accelerates G1/S transition promoting breast cancer progression.

doi: 10.1038/s41698-023-00437-1

Figure Lengend Snippet: Fig. 3 MC1R promotes tumorigenicity and breast cancer progression in vitro and in vivo. a, b Soft agar colony formation for WT T-47d and MC1R-KD T-47d (MC1R Sh1 and MC1R Sh2) cells. a Representative images of the soft agar wells. b Mean ± SEM fold change in the number of soft agar colonies from three independent experiments. *p < 0.05 (one-way ANOVA with Dunnett’s multiple comparisons). c, d Athymic nude mice carrying 17β-estradiol pellets were subcutaneously injected with WT T-47d or MC1R-KD T-47d cells, and tumor development was monitored. c Mice pictured on day 24 post-implant. d Tumor volume on day 24 post-implant in mice that received the WT T-47d (n = 7) or MC1R-KD T-47d (n = 6) cells are shown; the error bar shows mean ± SEM. *p < 0.05 (unpaired Student’s t-test). e Representative micrograph (40×) showing breast cancer tissue samples with different MC1R staining scores (0, 2, 6) and Ki67 expression in the breast cancer tissue microarray. Scale bar = 50 μm. f Plot comparing MC1R expression and Ki67 expression. The dashed lines in the violin plot show the median and the 25th and 75th percentiles. *p < 0.05 unpaired Student’s t-test.

Article Snippet: For transient MC1R overexpression, HEK 293 T cells, plated in 6-well plates in complete DMEM, were transfected with an MC1R overexpression plasmid (OriGene; Cat#: RC203218) using Lipofectamine 3000 (Thermo Fisher).

Techniques: In Vitro, In Vivo, Injection, Staining, Expressing, Microarray

Journal: NPJ precision oncology

Article Title: MC1R signaling through the cAMP-CREB/ATF-1 and ERK-NFκB pathways accelerates G1/S transition promoting breast cancer progression.

doi: 10.1038/s41698-023-00437-1

Figure Lengend Snippet: Fig. 4 MC1R downregulation delays G1–S progression in breast cancer cells. a–c Wild-type (WT) T-47d and MC1R-Knockdown (KD) T-47d cells were synchronized to the G1 phase by a double-thymidine block and then released. a Mean ± SEM percentage of cells in the G1, S, and G2 phases at 0, 3, 6, 9, and 12 h post-release. *p < 0.05 (unpaired Student’s t-test). b Mean ± SEM percentage of cells in the S phase across time. *p < 0.05 (one-way ANOVA with Dunnett’s multiple comparisons). c Representative western blot showing Cyclin D1, Cyclin E1, Ser780 p-RB, Rb, and GAPDH (loading control). Western blot quantification plots are shown in Supplementary Fig. 4a. d–f WT T-47d cells were treated with 0.2 μM NDP-MSH or 20 μM MSG-606 or left untreated after releasing from a double-thymidine block. d Mean ± SEM percentage of cells in the G1, S, and G2 phases at 0, 3, 6, 9, and 12 h post-release. *p < 0.05 (unpaired Student’s t-test). e Mean ± SEM percentage of cells in the S phase across time. *p < 0.05 (one-way ANOVA with Dunnett’s multiple comparisons). f Representative western blot showing Cyclin D1, Cyclin E1, Ser780 p-RB, Rb, and GAPDH (loading control). Western blot quantification plots are shown in Supplementary Fig. 4b.

Article Snippet: For transient MC1R overexpression, HEK 293 T cells, plated in 6-well plates in complete DMEM, were transfected with an MC1R overexpression plasmid (OriGene; Cat#: RC203218) using Lipofectamine 3000 (Thermo Fisher).

Techniques: Knockdown, Blocking Assay, Western Blot, Control

Journal: NPJ precision oncology

Article Title: MC1R signaling through the cAMP-CREB/ATF-1 and ERK-NFκB pathways accelerates G1/S transition promoting breast cancer progression.

doi: 10.1038/s41698-023-00437-1

Figure Lengend Snippet: Fig. 5 MC1R signaling through MC1R-cAMP-CREB contributes to the accelerated G1-S transition in breast cancer cells. a–c. MC1R-KD T- 47d cells were synchronized to the G1 phase by a double-thymidine block and then released with or without treatment with 25 μM FSK. (a) Mean ± SEM percentage of cells in the G1, S, and G2 phases at 0, 3, 6, 9, and 12 h post-release. *p < 0.05 (unpaired Student’s t-test). (b) Mean ± SEM percentage of cells in the S phase across time. *p < 0.05 (one-way ANOVA with Dunnett’s multiple comparisons). (c) Representative western blot showing Cyclin D1, Cyclin E1, Ser780 p-Rb, Rb, and GAPDH (loading control). Western blot quantification plots are shown in Supplementary Fig. 4d. d–f. WT T-47d cells were treated with 0.2 μM NDP-MSH or 0.2 μM NDP-MSH + 5 μM 666-15 (CREBi) or left untreated after releasing from a double-thymidine block. (d) Mean ± SEM percentage of cells in the G1, S, and G2 phases at 0, 3, 6, 9, and 12 h post-release. *p < 0.05 (unpaired Student’s t-test). (e) Mean ± SEM percentage of cells in the S phase across time. *p < 0.05 (one-way ANOVA with Dunnett’s multiple comparisons). (f) Representative western blot showing Cyclin D1, Cyclin E1, Ser780 p-Rb, Rb, and GAPDH (loading control). Western blot quantification plots are shown in Supplementary Fig. 4e.

Article Snippet: For transient MC1R overexpression, HEK 293 T cells, plated in 6-well plates in complete DMEM, were transfected with an MC1R overexpression plasmid (OriGene; Cat#: RC203218) using Lipofectamine 3000 (Thermo Fisher).

Techniques: Blocking Assay, Western Blot, Control

Journal: NPJ precision oncology

Article Title: MC1R signaling through the cAMP-CREB/ATF-1 and ERK-NFκB pathways accelerates G1/S transition promoting breast cancer progression.

doi: 10.1038/s41698-023-00437-1

Figure Lengend Snippet: Fig. 6 MC1R signaling through MC1R-cAMP-CREB/ATF and MC1R-ERK-NFκB axes promote G1/S transition. a WT T-47d (WT) and MC1R knockdown (KD) T-47d (MC1R Sh1 and MC1R Sh2) cell lines were stimulated with 0.2 μM NDP-MSH. Representative western blot showing p- ERK, t-ERK, p-p65 NFκB, and p65 NFκB, and GAPDH (loading control) in T-47d WT (wild-type), VC (vector control), and MC1R-KD T-47d (Sh1 and Sh2) cells. Western blot quantification plots are shown in Supplementary Fig. 5a. b T-47d and MCF7 cells were treated with different concentrations of MSG-606, as indicated, before stimulation with 0.2 μM NDP-MSH. Representative western blot showing p-CREB, p-ATF-1, t-CREB, p-ERK, t-ERK, p-p65 NFκB, p65 NFκB, and GAPDH (loading control). Western blot quantification plots are shown in Supplementary Fig. 5b. c HEK 293 T VC (vector control) and HEK 293 T cells transiently overexpressing MC1R (HEK 293 T MC1R) were serum-starved overnight and then released with 0.2 μM NDP-MSH with or without treatment with a MEK inhibitor, 5 μM U0126, a CREB inhibitor, 5 μM 666-15, or 20 μM MSG- 606. Representative western blot showing p-CREB, p-ATF-1, t-CREB, p-ERK, t-ERK, p-p65 NFκB, and p65 NFκB, Cyclin D1, Cyclin E1, Ser780 p-Rb, Rb, and GAPDH (loading control). Western blot quantification plots are shown in Supplementary Fig. 6c. d HEK 293 T VC (vector control) and HEK 293 T cells transiently overexpressing wild-type MC1R (MC1R WT) or the MC1R variants (MC1R R151C and MC1R R160W) were serum- starved overnight and then released with or without 0.2 μM NDP-MSH. Representative western blot showing p-CREB, p-ATF-1, t-CREB, p-ERK, t-ERK, p-p65 NFκB, p65 NFκB, and GAPDH (loading control). Western blot quantification plots are shown in Supplementary Fig. 6d.

Article Snippet: For transient MC1R overexpression, HEK 293 T cells, plated in 6-well plates in complete DMEM, were transfected with an MC1R overexpression plasmid (OriGene; Cat#: RC203218) using Lipofectamine 3000 (Thermo Fisher).

Techniques: Knockdown, Western Blot, Control, Plasmid Preparation

Journal: NPJ Precision Oncology

Article Title: MC1R signaling through the cAMP-CREB/ATF-1 and ERK-NFκB pathways accelerates G1/S transition promoting breast cancer progression

doi: 10.1038/s41698-023-00437-1

Figure Lengend Snippet: a The MC1R R% allele frequency across cancers in The Cancer Genome Atlas (TCGA). The orange dashed line indicates the R % allele frequency in control ExAC non-TCGA non-finnish European (NFE) Ancestry population. * p < 0.05 (Fisher’s exact test with Benjamini–Hochberg (BH) multiple comparisons adjustment) compared to the control population. The cancer type abbreviations are expanded and listed in Supplementary Table . b MC1R mRNA expression in normal breast, primary, and metastatic breast cancers. Data obtained from TCGA. The dashed lines in the violin plot show the median and the 25 th and 75 th percentiles. * p < 0.05 (one-way ANOVA with Dunnett’s multiple comparisons). c – h Disease-free survival (DFS) ( c , e , g ) and progression-free survival (PFS) ( d , f , h ) of all patients with breast cancer in TCGA (DFS: low expression n = 462, high expression n = 477; PFS: low expression n = 539, high expression n = 541) ( c , d ), patients carrying active MC1R variants (DFS: low expression n = 395, high expression n = 408; PFS, low expression n = 461, high expression n = 462) ( e , f ), and patients carrying a disruptive MC1R variant (DFS, low expression n = 67, high expression n = 69; PFS, low expression n = 78, high expression n = 79) ( g , h ) based on MC1R expression. p -Value calculated using the Log-rank test.

Article Snippet: For transient MC1R overexpression, HEK 293 T cells, plated in 6-well plates in complete DMEM, were transfected with an MC1R overexpression plasmid (OriGene; Cat#: RC203218) using Lipofectamine 3000 (Thermo Fisher).

Techniques: Control, Expressing, Variant Assay

Journal: NPJ Precision Oncology

Article Title: MC1R signaling through the cAMP-CREB/ATF-1 and ERK-NFκB pathways accelerates G1/S transition promoting breast cancer progression

doi: 10.1038/s41698-023-00437-1

Figure Lengend Snippet: a Mean ± SEM cAMP levels in T-47d and MCF7 cells treated with or without 0.2 μM NDP-MSH from three independent experiments. * p < 0.05 (unpaired Student’s t -test). b , c Mean ± SEM percentage fold change in cAMP levels in ( b ) T-47d and ( c ) MCF7 cells treated with or without 20 μM MSG-606 and stimulated with 0.2 μM NDP-MSH compared to those left untreated (UT) from three independent experiments. * p < 0.05 (one-way ANOVA with Tukey’s multiple comparisons). d T-47d and MCF7 cells were treated with 0.2 μM NDP-MSH or pretreated with 20 μM MSG-606, followed by stimulation with 0.2 μM NDP-MSH or left untreated. Representative western blot showing MC1R expression and downstream signaling in T-47d, MCF7 cells. GAPDH is shown as the loading control. Western blot quantification plots are shown in Supplementary Fig. . e WT T-47d (WT) and MC1R knockdown (KD) T-47d (MC1R Sh1 and MC1R Sh2) cell lines were treated with 0.2 μM NDP-MSH. Representative western blot showing MC1R expression and downstream signaling. GAPDH is shown as the loading control. Western blot quantification plots are shown in Supplementary Fig. . f Mean ± SEM fold change in cAMP levels in MC1R-KD T-47D cells treated with 0.2 μM NDP-MSH from 3 independent experiments. * p < 0.05 (one-way ANOVA with Tukey’s multiple comparisons). g , h Mean ± SEM fold change in cell number in ( g ) T-47d and ( h ) MCF7 cells treated either with 0.2 μM NDP-MSH or 20 μM MSG-606 or left untreated (UT) over time from three independent experiments. * p < 0.05 (two-way ANOVA with Dunnett’s multiple comparisons) compared to UT at the indicated time points. i Mean ± SEM fold change in cell number of WT T-47d cells and MC1R-KD T-47d cells (MC1R Sh1 and MC1R Sh2) treated with or without 0.2 μM NDP-MSH. (VC, vector control) over time from three independent experiments. * p < 0.05 (two-way ANOVA with Dunnett’s multiple comparisons) compared to T-47d WT at the indicated time points.

Article Snippet: For transient MC1R overexpression, HEK 293 T cells, plated in 6-well plates in complete DMEM, were transfected with an MC1R overexpression plasmid (OriGene; Cat#: RC203218) using Lipofectamine 3000 (Thermo Fisher).

Techniques: Western Blot, Expressing, Control, Knockdown, Plasmid Preparation

Journal: NPJ Precision Oncology

Article Title: MC1R signaling through the cAMP-CREB/ATF-1 and ERK-NFκB pathways accelerates G1/S transition promoting breast cancer progression

doi: 10.1038/s41698-023-00437-1

Figure Lengend Snippet: a , b Soft agar colony formation for WT T-47d and MC1R-KD T-47d (MC1R Sh1 and MC1R Sh2) cells. a Representative images of the soft agar wells. b Mean ± SEM fold change in the number of soft agar colonies from three independent experiments. * p < 0.05 (one-way ANOVA with Dunnett’s multiple comparisons). c , d Athymic nude mice carrying 17β-estradiol pellets were subcutaneously injected with WT T-47d or MC1R-KD T-47d cells, and tumor development was monitored. c Mice pictured on day 24 post-implant. d Tumor volume on day 24 post-implant in mice that received the WT T-47d ( n = 7) or MC1R-KD T-47d ( n = 6) cells are shown; the error bar shows mean ± SEM. * p < 0.05 (unpaired Student’s t -test). e Representative micrograph (40×) showing breast cancer tissue samples with different MC1R staining scores (0, 2, 6) and Ki67 expression in the breast cancer tissue microarray. Scale bar = 50 μm. f Plot comparing MC1R expression and Ki67 expression. The dashed lines in the violin plot show the median and the 25th and 75th percentiles. * p < 0.05 unpaired Student’s t -test.

Article Snippet: For transient MC1R overexpression, HEK 293 T cells, plated in 6-well plates in complete DMEM, were transfected with an MC1R overexpression plasmid (OriGene; Cat#: RC203218) using Lipofectamine 3000 (Thermo Fisher).

Techniques: Injection, Staining, Expressing, Microarray

Journal: NPJ Precision Oncology

Article Title: MC1R signaling through the cAMP-CREB/ATF-1 and ERK-NFκB pathways accelerates G1/S transition promoting breast cancer progression

doi: 10.1038/s41698-023-00437-1

Figure Lengend Snippet: a – c Wild-type (WT) T-47d and MC1R-Knockdown (KD) T-47d cells were synchronized to the G1 phase by a double-thymidine block and then released. a Mean ± SEM percentage of cells in the G1, S, and G2 phases at 0, 3, 6, 9, and 12 h post-release. * p < 0.05 (unpaired Student’s t -test). b Mean ± SEM percentage of cells in the S phase across time. * p < 0.05 (one-way ANOVA with Dunnett’s multiple comparisons). c Representative western blot showing Cyclin D1, Cyclin E1, Ser780 p-RB, Rb, and GAPDH (loading control). Western blot quantification plots are shown in Supplementary Fig. . d – f WT T-47d cells were treated with 0.2 μM NDP-MSH or 20 μM MSG-606 or left untreated after releasing from a double-thymidine block. d Mean ± SEM percentage of cells in the G1, S, and G2 phases at 0, 3, 6, 9, and 12 h post-release. * p < 0.05 (unpaired Student’s t -test). e Mean ± SEM percentage of cells in the S phase across time. * p < 0.05 (one-way ANOVA with Dunnett’s multiple comparisons). f Representative western blot showing Cyclin D1, Cyclin E1, Ser780 p-RB, Rb, and GAPDH (loading control). Western blot quantification plots are shown in Supplementary Fig. .

Article Snippet: For transient MC1R overexpression, HEK 293 T cells, plated in 6-well plates in complete DMEM, were transfected with an MC1R overexpression plasmid (OriGene; Cat#: RC203218) using Lipofectamine 3000 (Thermo Fisher).

Techniques: Knockdown, Blocking Assay, Western Blot, Control

Journal: NPJ Precision Oncology

Article Title: MC1R signaling through the cAMP-CREB/ATF-1 and ERK-NFκB pathways accelerates G1/S transition promoting breast cancer progression

doi: 10.1038/s41698-023-00437-1

Figure Lengend Snippet: a–c . MC1R-KD T-47d cells were synchronized to the G1 phase by a double-thymidine block and then released with or without treatment with 25 μM FSK. ( a ) Mean ± SEM percentage of cells in the G1, S, and G2 phases at 0, 3, 6, 9, and 12 h post-release. * p < 0.05 (unpaired Student’s t -test). ( b ) Mean ± SEM percentage of cells in the S phase across time. * p < 0.05 (one-way ANOVA with Dunnett’s multiple comparisons). ( c ) Representative western blot showing Cyclin D1, Cyclin E1, Ser780 p-Rb, Rb, and GAPDH (loading control). Western blot quantification plots are shown in Supplementary Fig. . d–f . WT T-47d cells were treated with 0.2 μM NDP-MSH or 0.2 μM NDP-MSH + 5 μM 666-15 (CREBi) or left untreated after releasing from a double-thymidine block. ( d ) Mean ± SEM percentage of cells in the G1, S, and G2 phases at 0, 3, 6, 9, and 12 h post-release. * p < 0.05 (unpaired Student’s t -test). ( e ) Mean ± SEM percentage of cells in the S phase across time. * p < 0.05 (one-way ANOVA with Dunnett’s multiple comparisons). ( f ) Representative western blot showing Cyclin D1, Cyclin E1, Ser780 p-Rb, Rb, and GAPDH (loading control). Western blot quantification plots are shown in Supplementary Fig. .

Article Snippet: For transient MC1R overexpression, HEK 293 T cells, plated in 6-well plates in complete DMEM, were transfected with an MC1R overexpression plasmid (OriGene; Cat#: RC203218) using Lipofectamine 3000 (Thermo Fisher).

Techniques: Blocking Assay, Western Blot, Control

Journal: NPJ Precision Oncology

Article Title: MC1R signaling through the cAMP-CREB/ATF-1 and ERK-NFκB pathways accelerates G1/S transition promoting breast cancer progression

doi: 10.1038/s41698-023-00437-1

Figure Lengend Snippet: a WT T-47d (WT) and MC1R knockdown (KD) T-47d (MC1R Sh1 and MC1R Sh2) cell lines were stimulated with 0.2 μM NDP-MSH. Representative western blot showing p-ERK, t-ERK, p-p65 NFκB, and p65 NFκB, and GAPDH (loading control) in T-47d WT (wild-type), VC (vector control), and MC1R-KD T-47d (Sh1 and Sh2) cells. Western blot quantification plots are shown in Supplementary Fig. . b T-47d and MCF7 cells were treated with different concentrations of MSG-606, as indicated, before stimulation with 0.2 μM NDP-MSH. Representative western blot showing p-CREB, p-ATF-1, t-CREB, p-ERK, t-ERK, p-p65 NFκB, p65 NFκB, and GAPDH (loading control). Western blot quantification plots are shown in Supplementary Fig. . c HEK 293 T VC (vector control) and HEK 293 T cells transiently overexpressing MC1R (HEK 293 T MC1R) were serum-starved overnight and then released with 0.2 μM NDP-MSH with or without treatment with a MEK inhibitor, 5 μM U0126, a CREB inhibitor, 5 μM 666-15, or 20 μM MSG-606. Representative western blot showing p-CREB, p-ATF-1, t-CREB, p-ERK, t-ERK, p-p65 NFκB, and p65 NFκB, Cyclin D1, Cyclin E1, Ser780 p-Rb, Rb, and GAPDH (loading control). Western blot quantification plots are shown in Supplementary Fig. . d HEK 293 T VC (vector control) and HEK 293 T cells transiently overexpressing wild-type MC1R (MC1R WT) or the MC1R variants (MC1R R151C and MC1R R160W) were serum-starved overnight and then released with or without 0.2 μM NDP-MSH. Representative western blot showing p-CREB, p-ATF-1, t-CREB, p-ERK, t-ERK, p-p65 NFκB, p65 NFκB, and GAPDH (loading control). Western blot quantification plots are shown in Supplementary Fig. .

Article Snippet: For transient MC1R overexpression, HEK 293 T cells, plated in 6-well plates in complete DMEM, were transfected with an MC1R overexpression plasmid (OriGene; Cat#: RC203218) using Lipofectamine 3000 (Thermo Fisher).

Techniques: Knockdown, Western Blot, Control, Plasmid Preparation

Journal: Molecular Neurodegeneration

Article Title: Melanocortin 1 receptor activation protects against alpha-synuclein pathologies in models of Parkinson’s disease

doi: 10.1186/s13024-022-00520-4

Figure Lengend Snippet: MC1R disruption exacerbates synucleinopathies in the nigrostriatal pathway in an αSyn AAV mouse model. MC1R e/e and WT mice were injected unilaterally with human WT αSyn AAV into the SN and sacrificed 8 weeks later: A Immunoblot of human αSyn species in Triton X-100-soluble and -insoluble SDS-soluble fractions in ipsilateral ventral midbrain and striatum and B quantification of αSyn monomers and oligomers. n = 3 mice/group. Measurements were normalized by dividing values by the mean of WT and multiplying by 100. One-way ANOVA followed by Tukey's post hoc test. C p-αSyn staining and D quantification of p-αSyn aggregates in the ipsilateral SN. n = 3 mice/group. Measurements were normalized by dividing values by the mean of WT and multiplying by 100. Two-tail Student’s t -test. Scale bar, 50 µm. MC1R e/e and WT mice were injected unilaterally with BiFC human WT αSyn AAV into the SN and sacrificed 8 weeks later: E VenusYFP fluorescence before and after proteinase K treatment and F quantification of venusYFP fluorescence density in the ipsilateral SN. n = 4 mice/group. Measurements were normalized by dividing values by the mean of WT and multiplying by 100. One-way ANOVA followed by Tukey's post hoc test. Scale bar, 50 µm. G Thioflavin-S staining and H quantification of thioflavin-S fluorescence density in the ipsilateral SN. n = 4 mice/group. Measurements were normalized by dividing values by the mean of WT and multiplying by 100. Two-tail Student’s t -test. Scale bar, 50 µm. * P < 0.05, ** P < 0.01

Article Snippet: MC1R-Tango expressing human MC1R tagged with FLAG and vector control GPRC5A-Tango were gifts from Bryan Roth (Addgene plasmid #66,427 and #66,382). αSyn and MC1R and their respective controls were transfected into cells using Lipofectamine® 2000 (ThermoFisher Scientific) according to the manufacturer’s instructions.

Techniques: Disruption, Injection, Western Blot, Staining, Fluorescence

Journal: Molecular Neurodegeneration

Article Title: Melanocortin 1 receptor activation protects against alpha-synuclein pathologies in models of Parkinson’s disease

doi: 10.1186/s13024-022-00520-4

Figure Lengend Snippet: MC1R disruption amplifies microglia activation and alters Nrf2 response to αSyn overexpression in the nigrostriatal pathway. MC1R e/e and WT mice were injected unilaterally with human WT αSyn AAV into the SN and sacrificed 8 weeks later: A Iba1 staining and B morphological classification and quantification of iba1-positive cells in the SN. n = 4 mice/group. Two-way ANOVA followed by Tukey’s post hoc test. Scale bar, 30 µm. C IL-1a, IL-6, TNFα, and ICAM1 mRNA levels in ventral midbrain. Measurements were normalized by dividing values by the mean of the WT contralateral side. One-way ANOVA followed by Tukey’s post hoc test. n = 5 mice/group. D Representative oxyblots for protein carbonyls and the corresponding Ponceau S staining in the ipsilateral ventral midbrain and E quantification of band density. Measurements were normalized by dividing values by the mean of WT control and multiplying by 100. Two-tail Student’s t -test. n = 3 mice/group. F Immunoblot for Nrf2 using ventral midbrain tissue and G quantification of Nrf2 band density in original values or H normalized to contralateral side by dividing values by the mean of the contralateral side and multiplying by 100. One-way ANOVA followed by Tukey’s post hoc test. n = 3 mice/group. I SN sections double-stained for Nrf2 and TH and J quantification of nuclear and cytoplasmic Nrf2. One-way ANOVA followed by Tukey’s post hoc test. n = 3 mice/group. Scale bar, 20 µm. K mRNA levels of Nrf2 target genes HO-1, NQO1, GCLM, and GCLC in the ipsilateral ventral midbrain. Measurements were normalized by dividing values by the mean of WT control. One-way ANOVA followed by Tukey’s post hoc test. n = 5 mice/group. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: MC1R-Tango expressing human MC1R tagged with FLAG and vector control GPRC5A-Tango were gifts from Bryan Roth (Addgene plasmid #66,427 and #66,382). αSyn and MC1R and their respective controls were transfected into cells using Lipofectamine® 2000 (ThermoFisher Scientific) according to the manufacturer’s instructions.

Techniques: Disruption, Activation Assay, Over Expression, Injection, Staining, Control, Western Blot

Journal: Molecular Neurodegeneration

Article Title: Melanocortin 1 receptor activation protects against alpha-synuclein pathologies in models of Parkinson’s disease

doi: 10.1186/s13024-022-00520-4

Figure Lengend Snippet: αSyn-induced dopaminergic neurotoxicity is exacerbated by MC1R disruption and reversed by human MC1R transgene. MC1R e/e and WT mice were injected unilaterally with human WT αSyn AAV into the SN. A Contralateral and ipsilateral turns induced by amphetamine 12 weeks post-AAV injection. Two-way ANOVA followed by Tukey’s post hoc test. n = 12 mice/group. B Striatal dopamine content 16 weeks post-AAV injection. Two-way ANOVA followed by Tukey’s post hoc test. n = 12 mice/group. C TH staining and D stereological quantification of TH-positive and -negative cells in the SN 16 weeks post-AAV injection. Two-way ANOVA followed by Tukey’s post hoc test. n = 12 mice/group. Scale bar, 100 µm. MC1R e/e Tg and MC1R e/e mice were injected unilaterally with human WT αSyn AAV into the SN. E Striatal dopamine content 16 weeks post-AAV injection. Two-way ANOVA followed by Tukey’s post hoc test. n = 6–7 mice/group. F TH staining and G stereological quantification of TH-positive and negative cells in the SN 16 weeks post-AAV injection. Two-way ANOVA followed by Tukey’s post hoc test. n = 6–7 mice/group. Scale bar, 100 µm. H p-αSyn staining and I quantification of p-αSyn aggregates in the ipsilateral SNpc 12 weeks post-AAV injection. Measurements were normalized by dividing values by the mean of the MC1R e/e mice and multiplying by 100. Two-tail Student’s t test. n = 4 mice/group. Scale bar, 50 µm. J Iba1 staining and morphological classification and K quantification of iba1-positive cells in the SNpc 12 weeks post-AAV injection. Two-way ANOVA followed by Tukey’s post hoc test. n = 4 mice/group. Scale bar, 30 µm. L Nrf2 and TH double-labeling and (M) quantification of nuclear and cytoplasmic Nrf2 in the SNpc 12 weeks post-AAV injection. Two-way ANOVA followed by Tukey’s post hoc test. n = 4 mice/group. Scale bar, 20 µm. N mRNA levels of Nrf2 target genes HO-1, NQO1, GCLC, and GCLM in the ipsilateral ventral midbrain 12 weeks post-AAV injection. Measurements were normalized by dividing values by the mean of the MC1R e/e mice. One-way ANOVA followed by Tukey’s post hoc test. n = 5 mice/group. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: MC1R-Tango expressing human MC1R tagged with FLAG and vector control GPRC5A-Tango were gifts from Bryan Roth (Addgene plasmid #66,427 and #66,382). αSyn and MC1R and their respective controls were transfected into cells using Lipofectamine® 2000 (ThermoFisher Scientific) according to the manufacturer’s instructions.

Techniques: Disruption, Injection, Staining, Labeling

Journal: Molecular Neurodegeneration

Article Title: Melanocortin 1 receptor activation protects against alpha-synuclein pathologies in models of Parkinson’s disease

doi: 10.1186/s13024-022-00520-4

Figure Lengend Snippet: MC1R agonists protect against αSyn-induced dopaminergic neurotoxicity. C57Bl/6 J mice were injected with αSyn or empty vector (Vec) AAV, treated subcutaneously with 20 mg/kg BMS-470539 (BMS-) or saline for 4 weeks, and sacrificed 16 weeks post-AAV injection. A Striatal dopamine content. Two-way ANOVA followed by Tukey’s post hoc test. n = 6–8 mice/group. B Stereological quantification of TH-positive cells in the SN. Two-way ANOVA followed by Tukey’s post hoc test. n = 6–8 mice/group. C p-αSyn staining and D quantification of p-αSyn aggregates in the ipsilateral SN. Measurements were normalized by dividing values by the mean of the vehicle-treated group and multiplying by 100. Two-tailed Student’s t test. n = 4 mice/group. Scale bar, 50 µm. WT and MC1R e/e mice were injected unilaterally with 3 nmol NDP-MSH into the striatum and then with αSyn AAV into the SN. Mice were sacrificed 16 weeks post-AAV injection. E Contralateral and ipsilateral turns induced by amphetamine 12 weeks post-AAV injection. Two-way ANOVA followed by Tukey’s post hoc test. n = 7 mice/group. F Striatal dopamine content. Two-way ANOVA followed by Tukey’s post hoc test. n = 7 mice/group. G Stereological quantification of TH-positive cells in the SN. Two-way ANOVA followed by Tukey’s post hoc test. n = 7 mice/group. H p-αSyn staining and I quantification of p-αSyn aggregates in the ipsilateral SN. Measurements were normalized by dividing values by the mean of the vehicle-treated group and multiplying by 100. Two-tail Student’s t test. n = 4 mice/group. Scale bar, 50 µm. J Morphological classification and quantification of iba1-positive cells in the SN in WT mice. Two-way ANOVA followed by Tukey’s post hoc test. n = 4 mice/group. K Nuclear and cytoplasmic Nrf2 ratio in the SN in WT mice. Two-way ANOVA followed by Tukey’s post hoc test. n = 4 mice/group. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: MC1R-Tango expressing human MC1R tagged with FLAG and vector control GPRC5A-Tango were gifts from Bryan Roth (Addgene plasmid #66,427 and #66,382). αSyn and MC1R and their respective controls were transfected into cells using Lipofectamine® 2000 (ThermoFisher Scientific) according to the manufacturer’s instructions.

Techniques: Injection, Plasmid Preparation, Saline, Staining, Two Tailed Test

Journal: Molecular Neurodegeneration

Article Title: Melanocortin 1 receptor activation protects against alpha-synuclein pathologies in models of Parkinson’s disease

doi: 10.1186/s13024-022-00520-4

Figure Lengend Snippet: MC1R activation alleviates αSyn oligomerization and neurotoxicity by activating Nrf2 in vitro. HEK293T cells were co-transfected with αSyn and MC1R or vector (GPRC5A-Tango). Non-transfected cells served as controls (NC): A Immunoblot and quantification of MC1R and Nrf2. One-way ANOVA followed by Tukey's post hoc test. B Nrf2 mRNA levels. One-way ANOVA followed by Tukey's post hoc test. C Immunoblot and quantification of pCREB and CBP. One-way ANOVA followed by Tukey's post hoc test. D ChIP-qPCR analysis of pCREB binding in the Nrf2 promoter. Chromatins were immunoprecipitated using pCREB or IgG as negative control (Neg). Values were calculated by subtracting the cycle threshold (Ct) values for immunoprecipitated DNA from adjusted Ct of input DNA to get delta Ct, followed by raising 2 to the power of the delta Ct and multiplying by 100. One-way ANOVA followed by Tukey's post hoc test. E Nrf2 staining and quantification of nuclear and cytoplasmic Nrf2. Nuclei were stained with DAPI. One-way ANOVA followed by Tukey's post hoc test. Scale bar, 10 µm. F Nrf2 target gene HO-1 mRNA levels. One-way ANOVA followed by Tukey's post hoc test. G and H Immunoblot and quantification of αSyn species. Measurements were normalized by dividing values by the mean of vector control and multiplying by 100. One-way ANOVA followed by Tukey's post hoc test. HEK293T cells were transfected with αSyn, MC1R or vector control, and shNrf2 RNA or scRNA control: G Immunoblot and I and J quantification of MC1R and Nrf2. One-way ANOVA followed by Tukey's post hoc test. G Immunoblot and K quantification of αSyn species. Measurements were normalized by dividing values by the mean of vector control and multiplying by 100. One-way ANOVA followed by Tukey's post hoc test. Primary cortical neurons were prepared from embryonic day 16–17 WT mice: L MAP2 and MC1R staining at DIV5. Scale bar, 10 µm. (M) Nrf2 mRNA levels in primary neurons transduced with αSyn AAV and lentiviral scRNA or shNrf2 RNA. Two-tail Student’s t test. N LDH release in primary neurons transduced with αSyn AAV and lentiviral scRNA or shNrf2 RNA and treated with PBS or NDP-MSH. Two-way ANOVA followed by Tukey’s post hoc test. O MAP2 staining and N quantification of MAP2-postive cells in primary neurons transduced with αSyn AAV and lentiviral scRNA or shNrf2 RNA. Two-way ANOVA followed by Tukey’s post hoc test. Scale bar, 10 µm. Visual field area = 0.13508 mm 2 . * P < 0.05, ** P < 0.01, *** P < 0.001, ns = not statistically significant. n = 3 replicates. Experiments were repeated ≥ 3 times

Article Snippet: MC1R-Tango expressing human MC1R tagged with FLAG and vector control GPRC5A-Tango were gifts from Bryan Roth (Addgene plasmid #66,427 and #66,382). αSyn and MC1R and their respective controls were transfected into cells using Lipofectamine® 2000 (ThermoFisher Scientific) according to the manufacturer’s instructions.

Techniques: Activation Assay, In Vitro, Transfection, Plasmid Preparation, Western Blot, ChIP-qPCR, Binding Assay, Immunoprecipitation, Negative Control, Staining, Control, Transduction

Journal: Molecular Neurodegeneration

Article Title: Melanocortin 1 receptor activation protects against alpha-synuclein pathologies in models of Parkinson’s disease

doi: 10.1186/s13024-022-00520-4

Figure Lengend Snippet: MC1R is present in dopaminergic neurons in humans and is reduced in patients with PD. A Immunohistochemistry for MC1R and TH in the SN of a 60-year-old control individual, PMI 15 h. Control sections were processed in the same manner, except primary or secondary antibodies were omitted. Scale bars, top 100 µm, bottom 25 µm. B Fluorescence double-staining for MC1R and TH or GFAP in the SN of a 63-year-old control individual, PMI 16 h. Scale bar, 50 µm. C Immunohistochemistry for MC1R and TH in the SN of a 91-year-old control individual, PMI 8 h, and an 84-year-old PD patient, PMI 6 h. Scale bar, 50 µm. D Fluorescence double-staining of MC1R and TH in the SN of an 87-year-old control individual, PMI 48 h, and a 91-year-old PD patient, PMI 32 h. Scale bar, 50 µm. E Immunoblot for MC1R and TH using SN tissue from control individuals and PD patients and F quantification of band density, n = 4/group; G Immunoblot for Nrf2 using SN tissue from control individuals and PD patients and H quantification of band density, n = 3/group. Actin as a loading control. Two-tail Student’s t test. Age (years)/PMI (h): 63/16, 87/48, 91/8, and 60/15 for control 1–4; 62/33, 91/32, 84/6, and 69/17 for PD 1–4. * P < 0.05

Article Snippet: MC1R-Tango expressing human MC1R tagged with FLAG and vector control GPRC5A-Tango were gifts from Bryan Roth (Addgene plasmid #66,427 and #66,382). αSyn and MC1R and their respective controls were transfected into cells using Lipofectamine® 2000 (ThermoFisher Scientific) according to the manufacturer’s instructions.

Techniques: Immunohistochemistry, Control, Fluorescence, Double Staining, Western Blot

Journal: Molecular Neurodegeneration

Article Title: Melanocortin 1 receptor activation protects against alpha-synuclein pathologies in models of Parkinson’s disease

doi: 10.1186/s13024-022-00520-4

Figure Lengend Snippet: MC1R disruption exacerbates synucleinopathies in the nigrostriatal pathway in an αSyn AAV mouse model. MC1R e/e and WT mice were injected unilaterally with human WT αSyn AAV into the SN and sacrificed 8 weeks later: A Immunoblot of human αSyn species in Triton X-100-soluble and -insoluble SDS-soluble fractions in ipsilateral ventral midbrain and striatum and B quantification of αSyn monomers and oligomers. n = 3 mice/group. Measurements were normalized by dividing values by the mean of WT and multiplying by 100. One-way ANOVA followed by Tukey's post hoc test. C p-αSyn staining and D quantification of p-αSyn aggregates in the ipsilateral SN. n = 3 mice/group. Measurements were normalized by dividing values by the mean of WT and multiplying by 100. Two-tail Student’s t -test. Scale bar, 50 µm. MC1R e/e and WT mice were injected unilaterally with BiFC human WT αSyn AAV into the SN and sacrificed 8 weeks later: E VenusYFP fluorescence before and after proteinase K treatment and F quantification of venusYFP fluorescence density in the ipsilateral SN. n = 4 mice/group. Measurements were normalized by dividing values by the mean of WT and multiplying by 100. One-way ANOVA followed by Tukey's post hoc test. Scale bar, 50 µm. G Thioflavin-S staining and H quantification of thioflavin-S fluorescence density in the ipsilateral SN. n = 4 mice/group. Measurements were normalized by dividing values by the mean of WT and multiplying by 100. Two-tail Student’s t -test. Scale bar, 50 µm. * P < 0.05, ** P < 0.01

Article Snippet: Blots were then probed by anti-human MC1R (LSBio LS-A1040) at 1:700, anti TH (Sigma, T1299) at 1:1000, and anti Nrf2 (D1Z9C) (Cell Signaling 12721S) at 1:1000, overnight at 4 ºC, followed by an extra one-hr incubation at 37 ºC for MC1R probing to enhance the binding.

Techniques: Injection, Western Blot, Staining, Fluorescence

Journal: Molecular Neurodegeneration

Article Title: Melanocortin 1 receptor activation protects against alpha-synuclein pathologies in models of Parkinson’s disease

doi: 10.1186/s13024-022-00520-4

Figure Lengend Snippet: MC1R disruption amplifies microglia activation and alters Nrf2 response to αSyn overexpression in the nigrostriatal pathway. MC1R e/e and WT mice were injected unilaterally with human WT αSyn AAV into the SN and sacrificed 8 weeks later: A Iba1 staining and B morphological classification and quantification of iba1-positive cells in the SN. n = 4 mice/group. Two-way ANOVA followed by Tukey’s post hoc test. Scale bar, 30 µm. C IL-1a, IL-6, TNFα, and ICAM1 mRNA levels in ventral midbrain. Measurements were normalized by dividing values by the mean of the WT contralateral side. One-way ANOVA followed by Tukey’s post hoc test. n = 5 mice/group. D Representative oxyblots for protein carbonyls and the corresponding Ponceau S staining in the ipsilateral ventral midbrain and E quantification of band density. Measurements were normalized by dividing values by the mean of WT control and multiplying by 100. Two-tail Student’s t -test. n = 3 mice/group. F Immunoblot for Nrf2 using ventral midbrain tissue and G quantification of Nrf2 band density in original values or H normalized to contralateral side by dividing values by the mean of the contralateral side and multiplying by 100. One-way ANOVA followed by Tukey’s post hoc test. n = 3 mice/group. I SN sections double-stained for Nrf2 and TH and J quantification of nuclear and cytoplasmic Nrf2. One-way ANOVA followed by Tukey’s post hoc test. n = 3 mice/group. Scale bar, 20 µm. K mRNA levels of Nrf2 target genes HO-1, NQO1, GCLM, and GCLC in the ipsilateral ventral midbrain. Measurements were normalized by dividing values by the mean of WT control. One-way ANOVA followed by Tukey’s post hoc test. n = 5 mice/group. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Blots were then probed by anti-human MC1R (LSBio LS-A1040) at 1:700, anti TH (Sigma, T1299) at 1:1000, and anti Nrf2 (D1Z9C) (Cell Signaling 12721S) at 1:1000, overnight at 4 ºC, followed by an extra one-hr incubation at 37 ºC for MC1R probing to enhance the binding.

Techniques: Activation Assay, Over Expression, Injection, Staining, Western Blot

Journal: Molecular Neurodegeneration

Article Title: Melanocortin 1 receptor activation protects against alpha-synuclein pathologies in models of Parkinson’s disease

doi: 10.1186/s13024-022-00520-4

Figure Lengend Snippet: αSyn-induced dopaminergic neurotoxicity is exacerbated by MC1R disruption and reversed by human MC1R transgene. MC1R e/e and WT mice were injected unilaterally with human WT αSyn AAV into the SN. A Contralateral and ipsilateral turns induced by amphetamine 12 weeks post-AAV injection. Two-way ANOVA followed by Tukey’s post hoc test. n = 12 mice/group. B Striatal dopamine content 16 weeks post-AAV injection. Two-way ANOVA followed by Tukey’s post hoc test. n = 12 mice/group. C TH staining and D stereological quantification of TH-positive and -negative cells in the SN 16 weeks post-AAV injection. Two-way ANOVA followed by Tukey’s post hoc test. n = 12 mice/group. Scale bar, 100 µm. MC1R e/e Tg and MC1R e/e mice were injected unilaterally with human WT αSyn AAV into the SN. E Striatal dopamine content 16 weeks post-AAV injection. Two-way ANOVA followed by Tukey’s post hoc test. n = 6–7 mice/group. F TH staining and G stereological quantification of TH-positive and negative cells in the SN 16 weeks post-AAV injection. Two-way ANOVA followed by Tukey’s post hoc test. n = 6–7 mice/group. Scale bar, 100 µm. H p-αSyn staining and I quantification of p-αSyn aggregates in the ipsilateral SNpc 12 weeks post-AAV injection. Measurements were normalized by dividing values by the mean of the MC1R e/e mice and multiplying by 100. Two-tail Student’s t test. n = 4 mice/group. Scale bar, 50 µm. J Iba1 staining and morphological classification and K quantification of iba1-positive cells in the SNpc 12 weeks post-AAV injection. Two-way ANOVA followed by Tukey’s post hoc test. n = 4 mice/group. Scale bar, 30 µm. L Nrf2 and TH double-labeling and (M) quantification of nuclear and cytoplasmic Nrf2 in the SNpc 12 weeks post-AAV injection. Two-way ANOVA followed by Tukey’s post hoc test. n = 4 mice/group. Scale bar, 20 µm. N mRNA levels of Nrf2 target genes HO-1, NQO1, GCLC, and GCLM in the ipsilateral ventral midbrain 12 weeks post-AAV injection. Measurements were normalized by dividing values by the mean of the MC1R e/e mice. One-way ANOVA followed by Tukey’s post hoc test. n = 5 mice/group. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Blots were then probed by anti-human MC1R (LSBio LS-A1040) at 1:700, anti TH (Sigma, T1299) at 1:1000, and anti Nrf2 (D1Z9C) (Cell Signaling 12721S) at 1:1000, overnight at 4 ºC, followed by an extra one-hr incubation at 37 ºC for MC1R probing to enhance the binding.

Techniques: Injection, Staining, Labeling

Journal: Molecular Neurodegeneration

Article Title: Melanocortin 1 receptor activation protects against alpha-synuclein pathologies in models of Parkinson’s disease

doi: 10.1186/s13024-022-00520-4

Figure Lengend Snippet: MC1R agonists protect against αSyn-induced dopaminergic neurotoxicity. C57Bl/6 J mice were injected with αSyn or empty vector (Vec) AAV, treated subcutaneously with 20 mg/kg BMS-470539 (BMS-) or saline for 4 weeks, and sacrificed 16 weeks post-AAV injection. A Striatal dopamine content. Two-way ANOVA followed by Tukey’s post hoc test. n = 6–8 mice/group. B Stereological quantification of TH-positive cells in the SN. Two-way ANOVA followed by Tukey’s post hoc test. n = 6–8 mice/group. C p-αSyn staining and D quantification of p-αSyn aggregates in the ipsilateral SN. Measurements were normalized by dividing values by the mean of the vehicle-treated group and multiplying by 100. Two-tailed Student’s t test. n = 4 mice/group. Scale bar, 50 µm. WT and MC1R e/e mice were injected unilaterally with 3 nmol NDP-MSH into the striatum and then with αSyn AAV into the SN. Mice were sacrificed 16 weeks post-AAV injection. E Contralateral and ipsilateral turns induced by amphetamine 12 weeks post-AAV injection. Two-way ANOVA followed by Tukey’s post hoc test. n = 7 mice/group. F Striatal dopamine content. Two-way ANOVA followed by Tukey’s post hoc test. n = 7 mice/group. G Stereological quantification of TH-positive cells in the SN. Two-way ANOVA followed by Tukey’s post hoc test. n = 7 mice/group. H p-αSyn staining and I quantification of p-αSyn aggregates in the ipsilateral SN. Measurements were normalized by dividing values by the mean of the vehicle-treated group and multiplying by 100. Two-tail Student’s t test. n = 4 mice/group. Scale bar, 50 µm. J Morphological classification and quantification of iba1-positive cells in the SN in WT mice. Two-way ANOVA followed by Tukey’s post hoc test. n = 4 mice/group. K Nuclear and cytoplasmic Nrf2 ratio in the SN in WT mice. Two-way ANOVA followed by Tukey’s post hoc test. n = 4 mice/group. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Blots were then probed by anti-human MC1R (LSBio LS-A1040) at 1:700, anti TH (Sigma, T1299) at 1:1000, and anti Nrf2 (D1Z9C) (Cell Signaling 12721S) at 1:1000, overnight at 4 ºC, followed by an extra one-hr incubation at 37 ºC for MC1R probing to enhance the binding.

Techniques: Injection, Plasmid Preparation, Staining, Two Tailed Test

Journal: Molecular Neurodegeneration

Article Title: Melanocortin 1 receptor activation protects against alpha-synuclein pathologies in models of Parkinson’s disease

doi: 10.1186/s13024-022-00520-4

Figure Lengend Snippet: MC1R activation alleviates αSyn oligomerization and neurotoxicity by activating Nrf2 in vitro. HEK293T cells were co-transfected with αSyn and MC1R or vector (GPRC5A-Tango). Non-transfected cells served as controls (NC): A Immunoblot and quantification of MC1R and Nrf2. One-way ANOVA followed by Tukey's post hoc test. B Nrf2 mRNA levels. One-way ANOVA followed by Tukey's post hoc test. C Immunoblot and quantification of pCREB and CBP. One-way ANOVA followed by Tukey's post hoc test. D ChIP-qPCR analysis of pCREB binding in the Nrf2 promoter. Chromatins were immunoprecipitated using pCREB or IgG as negative control (Neg). Values were calculated by subtracting the cycle threshold (Ct) values for immunoprecipitated DNA from adjusted Ct of input DNA to get delta Ct, followed by raising 2 to the power of the delta Ct and multiplying by 100. One-way ANOVA followed by Tukey's post hoc test. E Nrf2 staining and quantification of nuclear and cytoplasmic Nrf2. Nuclei were stained with DAPI. One-way ANOVA followed by Tukey's post hoc test. Scale bar, 10 µm. F Nrf2 target gene HO-1 mRNA levels. One-way ANOVA followed by Tukey's post hoc test. G and H Immunoblot and quantification of αSyn species. Measurements were normalized by dividing values by the mean of vector control and multiplying by 100. One-way ANOVA followed by Tukey's post hoc test. HEK293T cells were transfected with αSyn, MC1R or vector control, and shNrf2 RNA or scRNA control: G Immunoblot and I and J quantification of MC1R and Nrf2. One-way ANOVA followed by Tukey's post hoc test. G Immunoblot and K quantification of αSyn species. Measurements were normalized by dividing values by the mean of vector control and multiplying by 100. One-way ANOVA followed by Tukey's post hoc test. Primary cortical neurons were prepared from embryonic day 16–17 WT mice: L MAP2 and MC1R staining at DIV5. Scale bar, 10 µm. (M) Nrf2 mRNA levels in primary neurons transduced with αSyn AAV and lentiviral scRNA or shNrf2 RNA. Two-tail Student’s t test. N LDH release in primary neurons transduced with αSyn AAV and lentiviral scRNA or shNrf2 RNA and treated with PBS or NDP-MSH. Two-way ANOVA followed by Tukey’s post hoc test. O MAP2 staining and N quantification of MAP2-postive cells in primary neurons transduced with αSyn AAV and lentiviral scRNA or shNrf2 RNA. Two-way ANOVA followed by Tukey’s post hoc test. Scale bar, 10 µm. Visual field area = 0.13508 mm 2 . * P < 0.05, ** P < 0.01, *** P < 0.001, ns = not statistically significant. n = 3 replicates. Experiments were repeated ≥ 3 times

Article Snippet: Blots were then probed by anti-human MC1R (LSBio LS-A1040) at 1:700, anti TH (Sigma, T1299) at 1:1000, and anti Nrf2 (D1Z9C) (Cell Signaling 12721S) at 1:1000, overnight at 4 ºC, followed by an extra one-hr incubation at 37 ºC for MC1R probing to enhance the binding.

Techniques: Activation Assay, In Vitro, Transfection, Plasmid Preparation, Western Blot, Binding Assay, Immunoprecipitation, Negative Control, Staining, Transduction

Journal: Molecular Neurodegeneration

Article Title: Melanocortin 1 receptor activation protects against alpha-synuclein pathologies in models of Parkinson’s disease

doi: 10.1186/s13024-022-00520-4

Figure Lengend Snippet: MC1R is present in dopaminergic neurons in humans and is reduced in patients with PD. A Immunohistochemistry for MC1R and TH in the SN of a 60-year-old control individual, PMI 15 h. Control sections were processed in the same manner, except primary or secondary antibodies were omitted. Scale bars, top 100 µm, bottom 25 µm. B Fluorescence double-staining for MC1R and TH or GFAP in the SN of a 63-year-old control individual, PMI 16 h. Scale bar, 50 µm. C Immunohistochemistry for MC1R and TH in the SN of a 91-year-old control individual, PMI 8 h, and an 84-year-old PD patient, PMI 6 h. Scale bar, 50 µm. D Fluorescence double-staining of MC1R and TH in the SN of an 87-year-old control individual, PMI 48 h, and a 91-year-old PD patient, PMI 32 h. Scale bar, 50 µm. E Immunoblot for MC1R and TH using SN tissue from control individuals and PD patients and F quantification of band density, n = 4/group; G Immunoblot for Nrf2 using SN tissue from control individuals and PD patients and H quantification of band density, n = 3/group. Actin as a loading control. Two-tail Student’s t test. Age (years)/PMI (h): 63/16, 87/48, 91/8, and 60/15 for control 1–4; 62/33, 91/32, 84/6, and 69/17 for PD 1–4. * P < 0.05

Article Snippet: Blots were then probed by anti-human MC1R (LSBio LS-A1040) at 1:700, anti TH (Sigma, T1299) at 1:1000, and anti Nrf2 (D1Z9C) (Cell Signaling 12721S) at 1:1000, overnight at 4 ºC, followed by an extra one-hr incubation at 37 ºC for MC1R probing to enhance the binding.

Techniques: Immunohistochemistry, Fluorescence, Double Staining, Western Blot