Research Paper Volume 16, Issue 13 pp 10749—10764

Figure 3. Inhibition of CTSL protease activity decreases the expression of CUX1 and p16^INK4a, and inhibits cellular senescence in human ECs and VSMCs. (A, B) Western blot and qPCR analyses showing that inhibition of CTSL protease activity by Z-FY-CHO downregulates CUX1 and p16^INK4a in human ECs. (C, D) inhibition of CTSL protease activity by Z-FY-CHO suppresses cellular senescence in human ECs as evidenced by SA-β-gal and γ-H2AX staining as well as by the expression of the SASP genes IL6, IL-1β, and ICAM-1. 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. (E, F) Western blot and qPCR analyses showing that inhibition of CTSL protease activity by Z-FY-CHO downregulates CUX1 and p16^INK4a in human VSMCs. (G, H) inhibition of CTSL protease activity by Z-FY-CHO suppresses cellular senescence in human VSMCs as evidenced by SA-β-gal and γ-H2AX staining as well as by the expression of the SASP genes IL6, IL-1β, and ICAM-1. 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 G. 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).