Fig. 2: Cellular senescence induced by selective MC1 agonism.
a SF cells were treated for 7 days with 10 μM αMSH or 1 μM BMS and proliferation assessed by cell counting. Data are mean ± SE (n = 13, Student’s t-test vs. control; **p < 0.01, ***p < 0.001). b Cells were treated with BMS or αMSH for 7 days and SA-βGal+ cells quantified. Scale bars indicate 100 μm. Data are mean ± SE (n = 2, two-way ANOVA; **p < 0.01). c p53 protein levels were analyzed by western blot after 7-day treatment of SF cells with 3 or 10 μM BMS (B) or 10 μM αMSH and bands quantified (fold change with respect to vehicle). Data are mean ± SE (n = 6, Student’s t-test vs. control, C; *p < 0.05). d Immunofluorescence for p16INK4 in SF cells treated with 1 μM BMS was performed on SA-βGal-stained cells to determine their association. Scale bars indicate 200 μm. e SF cells were grown on Matrigel®-based 3D spheroids for 3 weeks with or without 1 μM BMS. p16INK4 was determined by immunofluorescence. Scale bars indicate 200 μm. f Effect of BMS on SA-βGal and p16INK4 staining in human macrophages incubated with 1 µM BMS for 7 days. Data are mean ± SE (n = 5, Student’s t-test vs. control, Ctrl). g Senescence on B16-F10 mouse melanocytes (1 µM BMS for 7 days) was determined by SA-βGal staining. Scale bars indicate 1000 μm. h SF were isolated from the joints of wild type (WT), Mc1re/e, or Mc3r−/− mice and treated with 1 μM BMS (B) and/or the ERK1/2 inhibitor FR180204 (ERK−; 1 µM) for 7 days. Senescence was quantified by SA-βGal staining. Data are mean ± SE (n = 4–6, Student’s t-test vs. control, C). i Senescence was induced in SF with 1 μM BMS and determined by SA-βGal staining. The MC1 antagonist ASIP or MC3/MC4 antagonist AGRP were used at 50 nM. Data are mean ± SE (n = 3, Student’s t-test vs. control, C; **p < 0.01, ***p < 0.001). j BMS (10 µM) was tested on the PathHunter β-Arrestin assay. Data represent % activation respect to positive controls: αMSH for melanocortin receptors and ghrelin for GHS receptor. k RT-PCR for the reference control HPRT1, and the ghrelin receptor GHSR on three different SF cells lines with expected bands at 130 and 148 bp, respectively. Source data are provided as Source Data file.
