Fig. 6

TM9SF4 activates autophagy via inhibiting mTOR phosphorylation to promote PCa cells anoikis-resistance and metastasis. A The protein levels of mTOR pathway proteins (mTOR, p-mTOR, S6K1, p-S6K1, 4E-BP1, and p-4E-BP1) detected by western-blotting in TM9SF4 silenced PC-3 cells. B Western-blotting showed that circ_0004585 and miR-1248 co-regulate the protein level of mTOR, p-mTOR, pS6, p-pS6, 4E-BP1, and p-4E-BP1. C The protein levels of mTOR, p-mTOR, pS6, p-pS6, 4E-BP1, and p-4E-BP1 detected by western-blotting in PC-3 cells transfected with knocking down TM9SF4 alone or cotransfected with miR-1248. D Autophagic flux was monitored in stable down-regulated TM9SF4 PC-3 cells expressing endogenous LC3BII/I tagged with tandem fluorescent-mCherry-GFP as a reporter. GFP and mCherry signal colocalization (yellow dots) indicated the lack of phagophore or autophagosome fusion with lysosomes (scale bar,50 μm). E Transmission electron microscopy (TEM) revealed the number of double-membrane autophagosomes in stable down-regulated TM9SF4 PC-3 cells. F Western blot assay detected the expression levels of TM9SF4 and autophagy related proteins (mTOR, p-mTOR, P62, and LC3BII/I) after the addition of Rapa or 3-MA for 24 h. G The cell viability rate was detected using CCK-8 after individually adding Rapa or 3-MA to the PCa cells displaying stable TM9SF4 knockdown. H Flow cytometry was used to detect the apoptosis of PC-3 cells with stable overexpression of TM9SF4 after exogenous addition of Rapa or 3-MA for 24 h. Bar graphs show the statistical analysis of three independent experiments (* P < 0.05; ** P < 0.01)