Research Paper Volume 16, Issue 13 pp 10749—10764

Figure 4. CTSL is an upstream regulator of CUX1 and p16^INK4a, inducing replicative senescence. (A, B) Western blot and qPCR analyses showing that overexpression of CTSL (pLVX-CTSL) activates CUX1 and p16^INK4a expression, and that shRNA-mediated CUX1 knockdown (shCUX1) reverses the increased expression of CUX1 and p16^INK4a in CTSL-overexpressing (pLVX-CTSL) human ECs. (C, D) SA-β-gal and γ-H2AX staining and the expression of SASP genes IL6, IL-1β and ICAM-1 verify that overexpression of CTSL (pLVX-CTSL) induces cellular senescence, and that shRNA-mediated CUX1 knockdown (shCUX1) reverses the increased senescence in the CTSL-overexpressing (pLVX-CTSL) human ECs. 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).