Figure 2: Nuclear Cathepsin L mediates H3 tail processing and promotes SAHF.

(a) Immunoblot for CTSL using whole-cell extract from growing and senescent IMR90s. Actin used for loading. (b) (Top) time course of CTSL1 induction as assessed by reverse transcription (RT)-quantitative (q)PCR. Values are normalized to expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH); mean±s.d. (n=3 biological replicates); two-tailed unpaired Student’s t-test; P values as indicated. (Bottom) immunoblot of the chromatin fraction from growing and senescent IMR90 cells with the indicated antibodies. In all panels, core histones were stained with Amido Black for loading. Samples for RT-qPCR and immunoblot analysis were obtained at identical time points. Note correlation of CTSL1 transcriptional induction, presence of the active form of CTSL1 in the chromatin fraction and H3 tail cleavage. (c) (Top) relative expression of CTSL1 in growing and senescent melanocytes assessed by RT-qPCR. Values are normalized to expression of GAPDH; mean±s.d. (n=3 technical replicates); two-tailed unpaired Student’s t-test; P values as indicated. (Bottom) immunoblot of CTSL using the chromatin fraction of growing and senescent melanocytes. (d) Chromatin extracts from growing and senescent IMR90 cells cultured in the presence of DMSO or a CTSL1 inhibitor probed with the indicated antibodies. Note the loss of H3 cleavage products in the CTSLi-treated cells, particularly at day 8. (e) Chromatin extracts from growing and senescent IMR90 cells expressing shLuc (control) or shCTSL1 lentiviral constructs probed with the indicated antibodies. Note the loss of H3 cleavage products in shCTSL1 senescent cells. (f) Quantification of SAHF in growing and senescent IMR90 cultured under the specified conditions obtained at day 8 post induction; mean±s.d. (n=3 biological replicates); two-tailed unpaired Student’s t-test; DMSO versus CTSLi *P=0.0147; DMSO versus shCTSL *P=0.0147.