Journal: Molecular Therapy Oncolytics

Article Title: CDC20 Knockdown and Acidic Microenvironment Collaboratively Promote Tumorigenesis through Inhibiting Autophagy and Apoptosis

doi: 10.1016/j.omto.2020.03.015

Figure Lengend Snippet: CDC20 Silencing in Acidic Environment Suppressed Autophagy and Apoptosis Mock RPE1 cells and shCDC20 RPE1 cells were cultured in various acidic media for 5 passages. Cells cultured in pH 7.0 media were normalized to 2M and 2C. Cells cultured in pH 6.6 media were normalized to 3M and 3C. Cells cultured in pH 6.2 media were normalized to 4M and 4C. (A) Levels of LC3, p62, ATG5, p-S6K, and S6K in mock and shCDC20 RPE1 cells cultured in different media were detected by western blotting. (B and C) The levels of (B) LC3 and (C) p62 were analyzed by western blotting and normalized to cells cultured in pH 6.7 medium (2 g/L MES). All data are presented as mean ± standard error of the mean (SEM). (D) Pro-caspase-3, cleaved caspase-3, and p53 levels in 2M, 2C, 3M, 3C, 4M, and 4C RPE1 cells were detected by western blotting. (E and F) Levels of (E) cleaved caspase-3 and (F) p53 were analyzed and normalized. All blots shown are representative of at least three independent experiments. All data are presented as mean ± standard error of the mean (SEM). (G) Potential mechanism by which cells survive acid and mitotic stress. In the presence of acid stress, autophagy and apoptosis may be induced to promote cell death. The survival of cells with CIN induced by CDC20 knockdown cultured in acidic media may be mediated by the inhibition of autophagy and apoptosis.

Article Snippet: Membranes were incubated with a rabbit polyclonal anti-mouse/human CDC20 antibody (1:1,000; Proteintech, Rosemont, IL, USA), a rabbit polyclonal anti-mouse/human LC3 antibody (1:1,000; Novus, Shanghai, China), a mouse monoclonal anti-human p62 antibody (1:1,000; Abcam, Cambridge, MA, USA), a rabbit monoclonal anti-human ATG5 antibody (1:1,000; Abcam, Cambridge, MA, USA), a rabbit polyclonal anti-human p-S6K antibody (1:1,000; Cell Signaling Technology, Beverly, MA, USA), a rabbit polyclonal anti-human S6K antibody (1:1,000; Cell Signaling Technology, Beverly, MA, USA), a rabbit monoclonal anti-active caspase-3 antibody (1:1000; Abcam, Cambridge, MA, USA), a rabbit polyclonal anti-caspase-3 antibody (1:1,000; Abcam, Cambridge, MA, USA), or a mouse monoclonal anti-human β-actin antibody (1:1,000; Proteintech, Wuhan, China).

Techniques: Cell Culture, Western Blot, Inhibition