Research Paper Volume 16, Issue 13 pp 10749—10764

Figure 5. CTSL is an upstream regulator of CUX1 and p16^INK4a, inducing DNA damage-induced senescence. (A, B) Western blot and qPCR analyses showing that bleomycin (Bleo)-induced activation of CTSL induces CUX1 and p16^INK4a expression, which can be reversed by treating the cells with Z-FY-CHO. (C, D) SA-β-gal and γ-H2AX staining and the expression of the SASP genes IL6, IL-1β, and ICAM-1 verify that bleomycin-induced activation of CTSL induces cellular senescence, which can also be reversed by treating the cells with Z-FY-CHO. Quantitative plots for both β-gal⁺ cells (%) in SA-β-gal staining and γ-H2AX foci/cells (%) with γ-H2AX staining were shown on the right side of the panel C. DAPI staining visualizes the presence of nuclei as a control for the cells in this analysis. Data for Western blots represent three biologically independent samples (n = 3). Data for qPCR represent a combination of three (n = 3) biologically independent experiments. Data for both SA-β-gal staining and γ-H2AX staining represent three biologically independent samples (n = 3).