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. 2020 Jun 1;11:2711. doi: 10.1038/s41467-020-16475-3

Fig. 1. p16 induces cell-cycle arrest and cell hypertrophy, and disrupts hair-follicle growth.

Fig. 1

a Immunostaining of human p16 (brown) in back skin sections of K5-rtTA/tet-p16 and control tet-p16 mice treated with doxycycline (dox) for 2 days (2d) starting at 3 weeks of age. b Skin sections from the same mice stained for p16 (red) and K14 (green), which labels the basal epidermis. c Percentage of p16+ keratinocytes in the interfollicular epidermis (IFE) in mice treated with dox for 2 days (2d), 2 weeks (2w), or 6 months (6 m). Dots indicate individual mice, n = 5, 5, 4 in respective groups. d Skin sections from mice treated with dox for 2 days, stained for Rb phosphorylated on Thr-821/826 (p-Rb) (red) and K14 (green). e Immunostaining of the proliferation marker Ki67 (brown) in sections from mice treated with dox for 2 days (2d) or 2 weeks (2w). f Percentage of IFE keratinocytes expressing Ki67 in the same mice (dots). n = 4, 6, 4, 4. g SA-βGal staining (blue) of skin sections from indicated mice, treated with dox for 2 weeks. h Percentage of SA-βGal positive IFE cells from indicated mice (dots) treated with dox for 2 weeks. n = 9, 10. i Skin sections from indicated mice stained for E-Cadherin (brown) to indicate cell circumference. j Area of epidermal keratinocytes from indicated mice (dots). Shown are values for control mice (tet-p16), and for p16− and p16+ keratinocytes in K5-rtTA/tet-p16 mice. n = 3, 6, 6 mice, >60 cells were scored per mouse. k H&E-stained skin sections from the indicated mice treated with dox for 2 weeks, showing hair-follicle morphology. l Number of hair follicles per field in indicated mice (dots), following dox treatment for 6 months. n = 9 per group. m FACS analysis of epidermal cells from indicated mice treated with dox for 6 months, stained for CD34, CD49f, and Sca1. Charts show Sca1− (follicular) cells only. Gate indicates percentage of CD34+/CD49fhigh hair-follicle stem cells. Cells were pooled from three mice per group. All graphs indicate mean across mice ± S.E.M, all scores were conducted visually from images. Blue labels DNA in all fluorescence images. ***P < 0.0001, t test. ns non-significant. Scale bars—20 μm, except k—100 μm.