Selective Rho Kinase Inhibitor Allows for Expansion of Human Primary Sebocytes In Vitro  Amanda M. Nelson, Melanie C. Dispenza, Zhaoyuan Cong, Matthew Moll, Kathryn L. Gilliland, Diane M. Thiboutot  Journal of Investigative Dermatology  Volume 136, Issue 6, Pages 1278-1280 (June 2016) DOI: 10.1016/j.jid.2016.02.019 Copyright © 2016 The Authors Terms and Conditions

Figure 1 Primary sebocytes proliferate in culture in the presence of Y-27632. (a) Growth characteristics of seven independent primary sebocyte cultures. Cell cultures were intentionally stopped at the passage listed (see Supplemental Material online). (b) P-Seb A sebocytes proliferate for over 200 days in culture (blue line). Removal of either Y-27632 (red line) or 3T3 fibroblasts (green line) triggers growth arrest after a few passages. (c) Left: representative image of primary sebocyte outgrowths from sebaceous gland. Gland outlined in dashed line (original magnification ×20; scale bar = 50 μm). Right: (top) morphology of P-Seb A at passage 10 shows that primary sebocytes grow in small round colonies (original magnification ×20; scale bar = 50 μm) and (bottom) show characteristic yellow lipid droplets in the cytoplasm (arrows; ×40 magnification; scale bar = 50 μm). (d) Karyotype of P-Seb A at passage 13. M, male; p, passage; P-Seb, primary sebocyte; y, years; Y, Y-27632. Journal of Investigative Dermatology 2016 136, 1278-1280DOI: (10.1016/j.jid.2016.02.019) Copyright © 2016 The Authors Terms and Conditions

Figure 2 Sebocyte lipogenesis is significantly increased after removal of Y-27632. (a) Oil Red O staining (lipid droplets) of primary sebocytes (p15) ± Y-27632 for 5 days (original magnification ×40; scale bar = 50 μm). (b) Total lipogenesis in P-Seb A sebocytes (p27) after Y-27632 withdrawal for 5 days. n = 3; statistical significance determined by paired t test, ∗p < .05. (c) Individual sebaceous lipids in P-Seb A sebocytes (p40–42) after Y-27632 withdrawal for 5 days. n = 3; statistical significance determined by paired t test, ∗p < .05. (d) Lipogenic enzyme messenger RNA expression in P-Seb A sebocytes (p19) after Y-27632 withdrawal for 5 days. n = 3; ∗p < .05. ACSS, acetyl CoA synthetase; C, cholesterol; CO, cholesterol oleate; CPM, counts per minute; FA, fatty acid; FASN, fatty acid synthase; FOH, fatty alcohol; HMGCR, 3-hydroxy-3-methyl-glutaryl-CoA reductase; SCD, stearyol CoA desaturase; SQ, squalene; SQLE, squalene epoxidase; TAG, triglycerides; WE, wax esters; Y, Y-27632. Journal of Investigative Dermatology 2016 136, 1278-1280DOI: (10.1016/j.jid.2016.02.019) Copyright © 2016 The Authors Terms and Conditions