Autophagy Induced by Muscarinic Acetylcholine Receptor 1 Mediates Migration and Invasion Targeting Atg5 via AMPK/mTOR Pathway in Prostate Cancer

Figure 3

The activation of CHRM1 positively regulates autophagy in prostate cancer cells. (a) TEM for PCa cell after adding different concentrations of CAR. Red arrows pointed to autophagosomes with materials to be digested (CTRL: 4000X; CAR:15000X). (b) Western blotting of protein expression of p62, LC3-II or GAPDH treated with 2 μM carbachol or 200 μM pirenzepine for up to 24 h in the condition of serum-free medium or complete medium (CTRL) for up to 36 h. (c) Statistics of LC3-II/LC3-I ratio were obtained from Western blot results in Figure 3(b). Data in C are means ± SD. ; ; and . (d) Western blotting of protein expression of p62, LC3-II, or GAPDH incubated in medium lacking serum for 36 h after the addition of carbachol or pirenzepine compared to nonspecific drugs in PC-3 cells. (e) LC3 puncta were analyzed by immunofluorescence after treatment with 2 μM carbachol for 24 h under starvation conditions. Nuclei were stained with DAPI. Scale bar: 10 μm. (f) PC-3 cells were transfected with Ad-mCherry-GFP-LC3B and cultured without serum for 24 h and treated with pirenzepine (200 μM) and carbachol (2 μM) for 18 h. A fluorescence microscope was used to determine the direction of autophagy flow by the expression of LC3-II. (g) Western blotting of the expression of CHRM1 after knocking down CHRM1 in PCa cell and the expression of LC3 in PC-3-sh-CHRM1 cell after adding CAR. (h) LC3 puncta were analyzed by immunofluorescence after knocking down CHRM1 in PC-3 for 24 h. DAPI was used to stain nuclei. sh-CHRM1: green fluorescence. Scale bar: 10 μm.

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