Figure 3: DOXO induces senescence and a SASP in human keratinocytes and mouse skin.

(A-C) Human keratinocytes were treated with DMSO or DOXO (250 nM) for 24 h. 10 d later, the cells were stained for SA-ß-gal (A-B) and assessed for cell proliferation (C). N=3 independent experiments. Shown are means ±SD, *p<0.5, ***p<0.001, (Student t test). (D) Total RNA was isolated from the indicated cells and analyzed for p16, p21 and SASP mRNAs, normalized to actin. N=3 independent experiments. Shown are means ±SD; **p<0.01, ***p<0.001. (E) Protein lysates from human keratinocytes were evaluated for p16 and LMNB1 by immunoblotting. (F) Total RNA was isolated from the skin of PBS- or DOXO-treated mice and analyzed for the indicated mRNAs, normalized to actin and tubulin. N=6 per each treatment group. Shown are means ±SEM; *p<0.05, **p<0.01 (Student t-test). (G-H) Representative image (G) and quantification in arbitrary units (A.U.) of whole body luminescence of p16–3MR mice one mo after PBS or DOXO treatments. N=6 per each treatment group. Shown are means ±SEM; *p<0.05 (Student t test).