Journal: International Journal of Molecular Medicine

Article Title: Orcinol glucoside ameliorates pulmonary fibrosis by suppressing hyaluronic acid synthesis and macrophage M2 polarization via targeting hyaluronic acid synthase 2

doi: 10.3892/ijmm.2026.5764

Figure Lengend Snippet: Elevated HAS2 expression and HA accumulation in murine and cellular fibrosis models. (A) Schematic of the experimental design for the time-course study. C57BL/6J mice received a single intratracheal dose of BLM or saline and were sacrificed at the indicated time points (days 0, 3, 5, 7, 10, 14 and 21) for sample collection (n=5 for per group). (B) Representative western blot images (left panel) and densitometric quantification (right panel) showing the protein expression levels of COL1A1 and HAS2 in lung tissues across the time course. β-tubulin served as the loading control (n=4 for per group). (C) ELISA quantification of HA levels in BALF at different days post-BLM injury (n=5 for per group). (D) Schematic diagram illustrating the workflow for the isolation and culture of primary mouse lung fibroblasts from BLM-treated fibrotic mice. (E) Western blotting analysis of HAS2 and COL1A1 protein expression in primary lung fibroblasts isolated from control (saline) and BLM-induced fibrotic mice. Representative blot images (left panel) and densitometric quantification of protein levels normalized to β-tubulin are shown (n=3 for each group). (F) Schematic diagram depicting TGF-β1-induced transition of NIH/3T3 fibroblasts into myofibroblasts. (G) COL1A1, ACTA2, and HAS2 mRNA expression in NIH/3T3 cells was detected using PCR after 5 ng/ml TGF-β1 administration for 12 h (n=3-4 for each group). (H) HAS2 protein expression in NIH/3T3 was detected using western blotting after 5 ng/ml TGF-β1 administration for 24 h (n=3 for each group). (I) ELISA was used to quantify HA concentrations in the culture media of myofibroblasts (n=6 for each group). * P<0.05, ** P<0.01, *** P<0.001. HAS2, hyaluronic acid synthase 2; HA, hyaluronic acid; BLM, bleomycin; BALF, bronchoalveolar lavage fluid; COL1A1, Collagen type I α 1 chain; ACTA2, actin alpha 2, smooth muscle.

Article Snippet: The HAS2 (cat. no. bs-11290R; 1:1,000) antibody was purchased from BIOSS and HRP-conjugated β-tubulin (cat. no. HRP-66240: 1:20,000) and STAT6 (cat. no. 51073-1-AP; 1:5,000) antibodies were obtained from Proteintech Group, Inc. Membranes were incubated for 1 h at room temperature with an HRP-conjugated secondary antibody (Cell Signaling Technology, Inc.; 1:2,000) following TBS-T (containing 0.05% Tween-20) washing.

Techniques: Expressing, Saline, Western Blot, Control, Enzyme-linked Immunosorbent Assay, Isolation

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: Frontiers in physiology

Article Title: Agouti Signaling Protein and Its Receptors as Potential Molecular Markers for Intramuscular and Body Fat Deposition in Cattle.

doi: 10.3389/fphys.2018.00172

Figure Lengend Snippet: FIGURE 7 | Localization of ASIP in bovine tissues. The polyclonal rabbit anti-ASIP antibody was detected with an Alexa Fluor 488 labeled goat anti rabbit IgG secondary antibody in bovine liver (A), MLD (C), and SCF (E). Nuclei were stained with Hoechst 33248 and overlaid to the brightfield image of liver (B), MLD (D), and SCF (F), respectively. Arrows indicate specific staining in stellate cells (A) or in and around adipocytes (C,E). SCF, subcutaneous fat; MLD, M. longissimus dorsi.

Article Snippet: The polyclonal antibodies against MC1R (ELA-ENT2673, Elabscience) and ATRN (BYT-ORB155769, Biorbyt) were also generated in rabbit and purchased from Biozol (Eching, Germany).

Techniques: Labeling, Staining

Journal: Scientific Reports

Article Title: Oxyresveratrol suppressed melanogenesis, dendrite formation, and melanosome transport in melanocytes via regulation of the MC1R/cAMP/MITF pathway

doi: 10.1038/s41598-025-05248-x

Figure Lengend Snippet: Oxyresveratrol inhibited melanin production through down-regulating the MC1R/cAMP/MITF signaling pathway in B16F10 cells. ( a ) The effect of oxyresveratrol on the viability of B16F10 cells was investigated by a CCK-8 assay. N = 3, Student’s t test, *** P < 0.001 vs. 0 µg/mL oxyresveratrol treatment. ( b ) The melanin content of B16F10 cells with or without oxyresveratrol treatment. CK, solvent control. OXY-5, 5 µg/mL oxyresveratrol group. OXY-10, 10 µg/mL oxyresveratrol group. OXY-15, 15 µg/mL oxyresveratrol group. Control cells (CK) were treated with the solvent (0.1% DMSO) equivalent to the highest concentration used in treatment groups. N = 3, Student’s t test, * P < 0.05, ** P < 0.01 vs. CK. ( c ) The cAMP levels of B16F10 cells with or without oxyresveratrol treatment were assessed by ELISA. ( d – i ) The effects of oxyresveratrol on the expression of six melanogenesis-related genes were evaluated by RT-qPCR in B16F10 cells. The mRNA expression levels of Mc1r , Mitf , Pmel , Tyr , Trp-1 , and Trp-2 were normalized to β-actin gene ( Actb ) levels. ( j ) The MITF protein levels of B16F10 cells with or without oxyresveratrol treatment were assessed by ELISA. ( k ) The effects of oxyresveratrol on the protein expression of PMEL, TRP-2, and MC1R in B16F10 cells. The protein levels of PMEL, TRP-2, and MC1R were normalized to GAPDH levels.

Article Snippet: For western blot assay, antibodies CDC42 (10155-1-AP, 1:1000), RAC1 (24072-1-AP, 1:1000), MLPH (10338-1-AP, 1:1000), KIF5B (21632-1-AP, 1:1000), RAB11B (67780-1-Ig, 1:1000) and TRP-2 (13095-1-AP, 1:1000) were purchased from proteintech (China), RAB17 (TA350622S, 1:1000) and MC1R (TA321478S, 1:1000) were purchased from OriGene (USA), PMEL/gp100 (ab137078, 1:1000) was purchased from Abcam (UK), and GAPDH (KC-5G5, 1:10000) was purchased from Aksomics (China).

Techniques: CCK-8 Assay, Solvent, Control, Concentration Assay, Enzyme-linked Immunosorbent Assay, Expressing, Quantitative RT-PCR

Journal: Journal of controlled release : official journal of the Controlled Release Society

Article Title: Receptor-mediated transcytosis: a mechanism for active extravascular transport of nanoparticles in solid tumors.

doi: 10.1016/j.jconrel.2012.05.014

Figure Lengend Snippet: Fig. 1. (A) Chemical structure of MC1R agonist (Ago) and antagonist (Ant) and schemes of bioconjugation. (B) Competitive binding assay comparing 125I-NDP-α-MSH with unlabeled Ago or Ant. (C) Competitive binding assay comparing 125I-NDP-α-MSH with unlabeled Ago- or Ant-conjugated pegylated HAuNS. Ago39 and Ago85 had an average of 39 and 85 agonists, respectively, conjugated per nanoparticle. Ant98 and Ant367 had an average of 98 and 367 antagonists, respectively, conjugated per nanoparticle.

Article Snippet: For visualization of intracellular translocation of MC1R, transient transfection of plasmid construct encoding GFP-tagged open reading frame clone of Homo sapiens MC1R (Origene) was carried out using Lipofectamine 2000 reagent (Invitrogen) as recommended by the manufacturer.

Techniques: Competitive Binding Assay

Journal: Journal of controlled release : official journal of the Controlled Release Society

Article Title: Receptor-mediated transcytosis: a mechanism for active extravascular transport of nanoparticles in solid tumors.

doi: 10.1016/j.jconrel.2012.05.014

Figure Lengend Snippet: Fig. 2. Intracellular localization of tetramethylrhodamine-labeled PEG-HAuNS, Ago39- PEG-HAuNS, or Ant367-PEG-HAuNS (red) in HEK 293 cells transfected with MC1R- GFP coexpression plasmid (green) after 20 min incubation at 37 °C, with or without the presence of 200 μg/ml free Ant (blocking). Arrows, overlap of green and red fluo- rescence in cytoplasm; arrowheads, overlap of green and red fluorescence on cell membrane. Bar, 20 μm.

Article Snippet: For visualization of intracellular translocation of MC1R, transient transfection of plasmid construct encoding GFP-tagged open reading frame clone of Homo sapiens MC1R (Origene) was carried out using Lipofectamine 2000 reagent (Invitrogen) as recommended by the manufacturer.

Techniques: Labeling, Transfection, Plasmid Preparation, Incubation, Blocking Assay, Membrane

Journal: Journal of controlled release : official journal of the Controlled Release Society

Article Title: Receptor-mediated transcytosis: a mechanism for active extravascular transport of nanoparticles in solid tumors.

doi: 10.1016/j.jconrel.2012.05.014

Figure Lengend Snippet: Fig. 4. (A) Schematic drawing of in vitro B16/F10 melanoma cell multilayer for evalua- tion of HAuNS transcytosis. (B) Permeability of 64Cu-labeled PEG-HAuNS, Ago39-PEG- HAuNS, or Ant367-PEG-HAuNS across the B16/F10 multilayer (Pc) with or without the presence of 200 μg/ml of free Ant (blocking), n=3. (C) Transmission electron micro- graphs of intracellular distribution of nontargeted PEG-HAuNS and PEG-HAuNS with different MC1R targeting moieties. M represents cell culture membrane. MP represents membrane pore. Asterisks represent endocytosis of nanoparticles at luminal cytoplasmic membrane. Arrows represent transcytosis of nanoparticle-incorporated vesicles toward basolateral cytoplasmic membrane. Arrowheads represent exocytosis of nanoparticles.

Article Snippet: For visualization of intracellular translocation of MC1R, transient transfection of plasmid construct encoding GFP-tagged open reading frame clone of Homo sapiens MC1R (Origene) was carried out using Lipofectamine 2000 reagent (Invitrogen) as recommended by the manufacturer.

Techniques: In Vitro, Permeability, Labeling, Blocking Assay, Transmission Assay, Cell Culture, Membrane

Journal: Journal of controlled release : official journal of the Controlled Release Society

Article Title: Receptor-mediated transcytosis: a mechanism for active extravascular transport of nanoparticles in solid tumors.

doi: 10.1016/j.jconrel.2012.05.014

Figure Lengend Snippet: Fig. 5. Targeting B16/F10 melanoma in a mouse model. (A) Representative microPET images at 1, 4, and 24 h after intravenous injection of 64Cu-labeled PEG-HAuNS, Ago39-PEG-HAuNS, or Ant367-PEG-HAuNS with or without the presence of 500 μg of pegylated Ant (blocking), n=3. Arrows, tumor. (B) Quantitative analysis of tumor up- take of 64Cu-labeled HAuNS, n=3. (C) Immunofluorescence micrographs of tumor tis- sue following nanoparticle injection. Bars, 20 μm. Arrows, colocalization of Ago39-PEG- HAuNS with internalized MC1R.

Article Snippet: For visualization of intracellular translocation of MC1R, transient transfection of plasmid construct encoding GFP-tagged open reading frame clone of Homo sapiens MC1R (Origene) was carried out using Lipofectamine 2000 reagent (Invitrogen) as recommended by the manufacturer.

Techniques: Injection, Labeling, Blocking Assay

Journal: Journal of controlled release : official journal of the Controlled Release Society

Article Title: Receptor-mediated transcytosis: a mechanism for active extravascular transport of nanoparticles in solid tumors.

doi: 10.1016/j.jconrel.2012.05.014

Figure Lengend Snippet: Fig. 6. (A) Scheme for transcytosis of MC1R agonist-conjugated HAuNS. (B) Schematic illustration showing the mechanism of tumor targeting through delivery of nanoparticles with different homing ligands on surface.

Article Snippet: For visualization of intracellular translocation of MC1R, transient transfection of plasmid construct encoding GFP-tagged open reading frame clone of Homo sapiens MC1R (Origene) was carried out using Lipofectamine 2000 reagent (Invitrogen) as recommended by the manufacturer.

Techniques:

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Melanocortin 1 Receptor Regulates Pathological and Physiological Cardiac Remodeling

doi: 10.1161/JAHA.124.037961

Figure Lengend Snippet: A , Immunostaining of MC1R in cardiac cross‐section of C57Bl/6J mouse. In THE control section, anti‐MC1R antibody was replaced by purified normal rabbit IgG (isotype control). Scale bar 20 μm. B , Immunofluorescence staining of MC1R (red) and sarcomeric α‐actinin, α‐SMA or CD31 (green) in cardiac cross‐section of C57Bl/6J mouse. Scale bar 20 μm. C and D , Representative western blots and quantification of MC1R protein expression in the left ventricle of C57Bl/6J mice subjected to TAC for 4 or 8 wks. n=6–7 mice per group. *** P <0.001 and **** P <0.0001 vs sham by unpaired Student's t test. E and F , Quantitative real‐time polymerase chain reaction analysis of MC1R mRNA expression (normalized to the geometric mean of GAPDH and RPS18 ) in human induced pluripotent stem cell‐derived cardiomyocytes that were mechanically stretched for 24 or 48 h or treated with ET‐1 (100 nM) for 24 h. n=3–4 individual experiments/batches of differentiation. * P <0.05 and ** P <0.01 vs control by 2‐way ANOVA and Šídák's post hoc test ( E ) or Mann–Whitney U test ( F ). Data are mean±SEM. α‐SMA indicates α‐smooth muscle actin; ET‐1, endothelin 1; MC1R, melanocortin 1 receptor; and TAC, transverse aortic constriction.

Article Snippet: For isotype control, a consecutive heart section was treated similarly except that the primary MC1R antibody was replaced by purified normal rabbit IgG (Novus Biologicals, Littleton, CO; No. NB810‐56910).

Techniques: Immunostaining, Control, Purification, Immunofluorescence, Staining, Western Blot, Expressing, Real-time Polymerase Chain Reaction, Derivative Assay, MANN-WHITNEY

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Melanocortin 1 Receptor Regulates Pathological and Physiological Cardiac Remodeling

doi: 10.1161/JAHA.124.037961

Figure Lengend Snippet: A , Representative hematoxylin and eosin–stained cross‐sections of the heart of sham‐ and TAC‐operated WT and Mc1r e/e mice. Scale bar, 1 mm. B , Ventricular weight in WT and Mc1r e/e mice at euthanasia after 8 wks of sham or TAC operation. C and D , Echocardiographic analysis of left ventricular ejection fraction and LVPW thickness. n=7 in sham WT, n=8 in sham Mc1r e/e , n=7 in TAC WT, and n=13 in TAC Mc1r e/e . E , Quantification of cardiomyocyte CSA in the left ventricle of sham‐ and TAC‐operated WT and Mc1r e/e mice. n=6 mice per group. F through I , Quantitative real‐time polymerase chain reaction analysis of Nppa (atrial natriuretic peptide), Nppb (B‐type natriuretic peptide), Ctgf (connective tissue growth factor) and Mmp2 (matrix metalloproteinase 2) in the left ventricle of indicated groups n=5 to 7 mice per group in each graph. Gene expression is normalized against the geometric mean of Actb and Mrps18a . Data are mean±SEM; each dot represents an individual mouse. * P <0.05 for the indicated comparisons by 2‐way ANOVA and Šídák's post hoc tests. ## P <0.01, ### P <0.001 and #### P <0.0001 for the main effect of TAC by 2‐way ANOVA. CSA indicates cross‐sectional area; LVPW, left ventricular posterior wall; MC1R, melanocortin 1 receptor; Mc1r e/e , melanocortin 1 receptor recessive yellow; TAC, transverse aortic constriction; and WT, wild‐type.

Article Snippet: For isotype control, a consecutive heart section was treated similarly except that the primary MC1R antibody was replaced by purified normal rabbit IgG (Novus Biologicals, Littleton, CO; No. NB810‐56910).

Techniques: Staining, Real-time Polymerase Chain Reaction, Gene Expression

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Melanocortin 1 Receptor Regulates Pathological and Physiological Cardiac Remodeling

doi: 10.1161/JAHA.124.037961

Figure Lengend Snippet: A , Representative hematoxylin and eosin–stained cross‐sections of the heart of sham‐ and TAC‐operated control (Myh6‐MCM) and Mc1r‐cKO mice. Scale bar: 1 mm (upper) and 20 μm (lower panel). B and C , Ventricular weight and VW/BW. D through F , Quantification of cardiomyocyte CSA, length and length‐to‐CSA ratio in the LV. G through J , Echocardiographic analyses of LVEDD and ejection fraction as well as mitral valve E/A ratio and E/e′ ratio in Myh6‐MCM and Mc1r‐cKO after 8 wks of sham or TAC operation. K through N , Quantitative real‐time polymerase chain reaction analysis of Nppa , Acta1 (Actin, α‐skeletal muscle), Myh6 (myosin heavy chain‐α) and Myh7 (myosin heavy chain‐β) in the left ventricle of indicated groups. Gene expression is normalized against the geometric mean of Actb and Mrps18a . Data are mean±SEM; each dot represents an individual mouse. n=3–5 in sham Myh6‐MCM mice, n=3–7 in sham Mc1r‐cKO mice, n=7–8 in TAC Myh6‐MCM mice, and n=7–9 in TAC Mc1r‐cKO mice. * P <0.05, ** P <0.01, *** P <0.001 and **** P <0.001 for the indicated comparisons by 2‐way ANOVA and Šídák's post hoc tests. ## P <0.01, ### P <0.001 and #### P <0.0001 for the main effect of TAC by 2‐way ANOVA. CSA indicates cross‐sectional area; LVEDD, left ventricular end‐diastolic dimension; MC1R, melanocortin 1 receptor; Mc1r‐cKO, cardiomyocyte‐specific melanocortin 1 receptor knockout; Myh6‐MCM, Myh6‐MerCreMer transgenic; TAC, transverse aortic constriction; and VW/BW, ventricular weight‐to‐body weight ratio.

Article Snippet: For isotype control, a consecutive heart section was treated similarly except that the primary MC1R antibody was replaced by purified normal rabbit IgG (Novus Biologicals, Littleton, CO; No. NB810‐56910).

Techniques: Staining, Control, Real-time Polymerase Chain Reaction, Gene Expression, Knock-Out, Transgenic Assay

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Melanocortin 1 Receptor Regulates Pathological and Physiological Cardiac Remodeling

doi: 10.1161/JAHA.124.037961

Figure Lengend Snippet: A through D , Quantitative real‐time polymerase chain reaction analysis of Ctgf , Acta2 (α‐smooth muscle actin), Mmp2 , and Col1a1 (collagen type I, α1) in the left ventricle of Myh6‐MCM and Mc1r‐cKO mice after 8 wks of sham or TAC operation. Gene expression is normalized against the geometric mean of Actb and Mrps18a . E , Representative Picrosirius Red–stained cardiac cross‐sections showing the extent of perivascular and interstitial fibrosis in the indicated groups. Scale bar, 100 μm. F , Quantification of fibrosis in the indicated groups. Data are mean±SEM; each dot represents an individual mouse. n=2–5 in sham Myh6‐MCM mice, n=3–7 in sham Mc1r‐cKO mice, n=7 in TAC Myh6‐MCM mice, and n=7–9 in TAC Mc1r‐cKO mice. * P <0.05, ** P <0.01 and *** P <0.001 for the indicated comparisons by 2‐way ANOVA and Šídák's post hoc tests. # P <0.05, ## P <0.01 and #### P <0.0001 for the main effect of TAC by 2‐way ANOVA. MC1R indicates melanocortin 1 receptor; Mc1r‐cKO, cardiomyocyte‐specific melanocortin 1 receptor knockout; Myh6‐MCM, Myh6‐MerCreMer transgenic; and TAC, transverse aortic constriction.

Article Snippet: For isotype control, a consecutive heart section was treated similarly except that the primary MC1R antibody was replaced by purified normal rabbit IgG (Novus Biologicals, Littleton, CO; No. NB810‐56910).

Techniques: Real-time Polymerase Chain Reaction, Gene Expression, Staining, Knock-Out, Transgenic Assay

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Melanocortin 1 Receptor Regulates Pathological and Physiological Cardiac Remodeling

doi: 10.1161/JAHA.124.037961

Figure Lengend Snippet: A , Representative H&E‐ and Picrosirius Red–stained cardiac cross‐sections in Myh6‐MCM and Mc1r‐cKO mice subjected to sham operation or 4 wks of Ang II infusion. Scale bars: 1 mm (upper) and 20 μm (lower panel) in H&E; 100 μm in Picrosirius Red. B and C , Ventricular weight and VW/BW ratio in Myh6‐MCM and Mc1r‐cKO mice at euthanasia after 4 weeks of angiotensin II infusion or sham operation. D , Quantification of cardiomyocyte CSA in the indicated groups. E , Quantification of the extent of left ventricular fibrosis in the indicated groups. F through I , Quantitative real‐time polymerase chain reaction analysis of Nppa , Nppb , Acta2 and Tgfb1 (transforming growth factor β) in the left ventricle of the indicated groups. Gene expression is normalized against the geometric mean of Actb and Mrps18a . Data are mean±SEM; each dot represents an individual mouse. n=5 to 7 in sham Myh6‐MCM mice, n=4 to 6 in sham Mc1r‐cKO mice, n=5 to 7 in angiotensin II Myh6‐MCM mice, and n=5 to 8 in angiotensin II Mc1r‐cKO mice. ** P <0.01, *** P <0.001 and ****P <0.0001 for the indicated comparisons by 2‐way ANOVA and Šídák's post hoc tests. ## P <0.01 and #### P <0.0001 for the main effect of angiotensin II infusion by 2‐way ANOVA. Ang II indicates angiotensin II; CSA, cross‐sectional area; H&E, hematoxylin and eosin; MC1R, melanocortin 1 receptor; Mc1r‐cKO, cardiomyocyte‐specific melanocortin 1 receptor knockout; Myh6‐MCM, Myh6‐MerCreMer transgenic; TAC, transverse aortic constriction; and VW/BW, ventricular weight‐to‐body weight ratio.

Article Snippet: For isotype control, a consecutive heart section was treated similarly except that the primary MC1R antibody was replaced by purified normal rabbit IgG (Novus Biologicals, Littleton, CO; No. NB810‐56910).

Techniques: Staining, Real-time Polymerase Chain Reaction, Gene Expression, Knock-Out, Transgenic Assay

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Melanocortin 1 Receptor Regulates Pathological and Physiological Cardiac Remodeling

doi: 10.1161/JAHA.124.037961

Figure Lengend Snippet: A , Cumulative running distance of Myh6‐MCM and Mc1r‐cKO mice during the 5‐wk running wheel experiment. The data were analyzed using 2‐way repeated measures ANOVA, P =0.88 for genotype effect. B , Ventricular weight in Myh6‐MCM and Mc1r‐cKO mice at euthanasia after the 5‐wk voluntary running (exercise group) or nonexercising period (sedentary group). C and D , Echocardiographic analysis of ejection fraction and LVEDD at the end of the experiment. E through H , Echocardiographic analysis of ejection fraction, LVEDD, LVPW thickness, and LVAW thickness at baseline and after 5 wks of voluntary wheel running. I , RWT at the end of the experiment. J through L , Quantitative real‐time polymerase chain reaction analysis of Nppb , Myh6 and Myh7 in the left ventricle of the indicated groups. Gene expression is normalized against the geometric mean of Actb and Mrps18a . Data are mean±SEM; each dot represents an individual mouse. n=4 in sedentary/Myh6‐MCM mice, n=5 in sedentary/Mc1r‐cKO mice, n=8 in exercise/Myh6‐MCM mice, and n=11 in exercise/Mc1r‐cKO mice. * P <0.05, ** P <0.01 and *** P <0.001 for the indicated comparisons by 2‐way ANOVA and Šídák's post hoc tests. # P <0.05, ## P <0.01 and #### P <0.0001 for the main effect of exercise by 2‐way ANOVA (B–D , I–L ). * P <0.05 vs Myh6‐MCM mice at 5 wks by 2‐way repeated measures ANOVA and Šídák's post hoc test ( E–H ). LVAW indicates left ventricular anterior wall; LVEDD, left ventricular end‐diastolic dimension; LVPW, left ventricular posterior wall; MC1R, melanocortin 1 receptor; Mc1r‐cKO, cardiomyocyte‐specific melanocortin 1 receptor knockout; Myh6‐MCM, Myh6‐MerCreMer transgenic; and RWT, relative wall thickness.

Article Snippet: For isotype control, a consecutive heart section was treated similarly except that the primary MC1R antibody was replaced by purified normal rabbit IgG (Novus Biologicals, Littleton, CO; No. NB810‐56910).

Techniques: Real-time Polymerase Chain Reaction, Gene Expression, Knock-Out, Transgenic Assay

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Melanocortin 1 Receptor Regulates Pathological and Physiological Cardiac Remodeling

doi: 10.1161/JAHA.124.037961

Figure Lengend Snippet: A , [ 3 H]‐Leucine incorporation assay in H9c2 cells treated with different concentrations of LD211 for 24 h. B , [ 3 H]‐Leucine incorporation assay in H9c2 cells treated with LD211 (10 nM) or control (PBS) for 24 h in the absence or presence of angiotensin II (1 μM). C , Quantitative real‐time polymerase chain reaction analysis of Nppb mRNA expression in H9c2 cells treated with LD211 (10 nM) for 1, 3, 6 and 24 h. Gene expression is normalized against the geometric mean of Gapdh and Rn18s . D through H , Representative Western blots and quantification of p‐ERK, p‐p38, p‐JNK and p‐CREB in H9c2 cells treated with LD211 (10 nM) for 5, 15, 30, or 60 min. Data are expressed as a percentage of control. I and J , Representative western blots and quantification of p‐CREB and p‐p38 in H9c2 cells treated with different concentrations of LD211 for 15 min. Data are expressed as percentage of control. K , [ 3 H]‐Leucine incorporation assay in H9c2 cells pretreated with or without the CREB inhibitor 666–15 (1 μM) for 2 h followed by treatment with LD211 (10 nM) or control (PBS) for 24 h. Data are mean±SEM, n=4–6 per group. * P <0.05, ** P <0.01 and **** P <0.0001 for the indicated comparisons by 1‐way ANOVA and Dunnett's post hoc tests ( B ), Kruskal–Wallis and Dunn's post hoc tests ( A , C–J ) or 2‐way ANOVA and Šídák's post hoc tests ( K ). Ang II indicates angiotensin II; CREB, cAMP response element–binding protein; Ctrl, control; ERK, extracellular signal‐related kinase; JNK, c‐Jun N‐terminal kinase; and MC1R, melanocortin 1 receptor.

Article Snippet: For isotype control, a consecutive heart section was treated similarly except that the primary MC1R antibody was replaced by purified normal rabbit IgG (Novus Biologicals, Littleton, CO; No. NB810‐56910).

Techniques: Control, Real-time Polymerase Chain Reaction, Expressing, Gene Expression, Western Blot, Binding Assay