Fig. 6

AAA237 induced autophagy through mTOR-mediated pathway regulation. A The representative images of transmission electron microscopy (TEM) of U251 cells after treatment of 3 μM AAA237 for 48 h. Scale bar = 500 nm. B The representative images of transmission electron microscopy (TEM) of LN229 cells after treatment of 3 μM AAA237 for 48 h. Scale bar = 500 nm. C U251 cells with stably expressing mRFP-GFP-LC3 were treated with AAA237 (3 μM) for 48 h and autophagosomes were observed under the fluorescence microscope. Scale bar = 5 μm. D LN229 cells with stably expressing mRFP-GFP-LC3 were treated with AAA237 (3 μM) for 48 h and autophagosomes were observed under the fluorescence microscope. Scale bar = 5 μm. E Expression of p-mTOR, mTOR, P62, Beclin 1, ATG5 and LC3BII in U251 cells was checked by Western blot under treatment with different concentrations of AAA237(0, 1, 3 and 10 μM) after 24 h, 48 h, 72 h. F Expression of p-mTOR, mTOR, P62, Beclin 1, ATG5 and LC3BII in LN229 cells was checked by Western blot under treatment with different concentrations of AAA237(0, 1, 3 and 10 μM) after 24 h, 48 h, 72 h. G IC50 of 3-MA on U251. H IC50 of 3-MA on LN229. I The CCK8 assay was used to show 3-MA could reverse the inhibition of cell proliferation caused by AAA237 in U251. J The CCK8 assay was used to show 3-MA could reverse the inhibition of cell proliferation caused by AAA237 in LN229. K After incubation with AAA237 and 3-MA for 48 h, the inhibition of cell proliferation caused by AAA237 was reversed. Scale bar = 100 μm. L, M The EdU-DNA synthesis assay was used to show 3-MA could reverse the inhibition of cell proliferation caused by AAA237 in U251 andLN229. Scale bar = 100 μm. N, O Expression of p-mTOR, mTOR, P62, Beclin 1, ATG5 and LC3BII in U251 cells was checked by Western blot under treatment with AAA237 and 3-MA