Fig. 4

c-Myc regulates proliferation and differentiation in SG organoids. a Blimp1-YFP⁺-derived sebaceous gland organoids cultured for 6 days and treated for 4 days with c-Myc inhibitor 10058-F4 or DMSO as control. Cross-section of control and treated organoids stained for actin. b Treated organoids display decreased organoid size in comparison to control organoids. c Cell size analysis of treated and control organoids measuring cell diameter of inner cells and cells at the outer layer [n = 80 individual organoids in three independent wells]. d, e Confocal analysis of treated and control organoids stained with (d) Keratin-5 (K-5) and Ki67 and (e) MCM2, showing dramatic decrease in proliferating cells upon c-Myc inhibition. f Control and treated organoids stained for Oil Red O (ORO), showing diminished differentiation upon c-Myc inhibition. g Quantitative real time (RT)–PCR analysis of treated organoids. mRNA levels of known sebocyte differentiation markers including androgen receptor (AR), fatty acid synthase (FASN), and peroxisome proliferator-activated receptors beta and gamma (PPAR-β,ɣ) are significantly decreased upon c-Myc inhibition. Changes in cycle threshold values were normalized to Rplp0. Data are represented as mean relative to control organoids [n = 3 independent wells analyzed in triplicates]. Error bars show ± s.e.m. Significance was determined using two-tailed unpaired Student’s t-test, where *p < 0.05, **p < 0.005, ***p < 0.001. All images are representative of organoids grown from a mix of at least three mice and cultured in three independent biological repeats. All experiments were repeated at least three times with similar results. Scale bars: 50 μm (b, d–f), 100 μm (a)