Fig. 3: Downregulation of CEMIP attenuates autophagy and cell viability in anoikis-resistant PCa cells.

A qRT-PCR assay analysis of the mRNA level of Beclin1 and multiple ATGs. B Western blot analysis of the Beclin1, p-Beclin1, ATG5, ATG7, ATG9, ATG12, and LC3BII/LC3BI ratio in CEMIP-silenced PC-3-AR and DU145-AR cells. C The autophagic flux in CEMIP-silenced PC-3-AR cells and negative control cells was certified by immunofluorescent staining, and endogenous LC3B tagged with a tandem fluorescent-mCherry-GFP as a reporter. Autophagy flux was detected in 2D culture, and a total of more than 20 cells under each condition were counted for quantification of autophagy (Scale bar, 50 μm). D Transmission electron microscopy manifested that stable downregulation of CEMIP in PC-3-AR cells attenuated the number of double-membrane autophagosomes (Original magnification, ×1000, ×1600, respectively). E The detachment-induced apoptosis in CEMIP-silenced PC-3-AR cells and negative control cells after the treatment of Rapa (200 nM) or 3-MA (10 μM) in suspension situation for 48 h was displayed by flow cytometry assay. F Cell viabilities of knockdown CEMIP in PCa-AR cells after the treatment Rapa or 3-MA for 48 h were presented by the CCK-8 assay. PCa-P cells as negative control. G H&E staining indicated that knockdown of CEMIP led to decreased number of lung metastatic colonies (n = 5 per group, Original magnification, ×10, ×100, respectively). H Bioluminescence in vivo imaging showed that knockdown of CEMIP significantly decreased the number of pulmonary metastasis focuses (n = 5 per group). Data are presented as the means ± SD of three independent experiments; *p < 0.05, **p < 0.01, ***p < 0.001.