Fig. 3

Inhibition of gefitinib-induced autophagy by GPR119 ligands in breast cancer cells. a Autophagy induction by gefitinib in human breast cancer cells. LC3B I/II were measured by immunoblottings in breast cancer cells (MCF-7 and MDA-MB-231 cells). Cells were incubated with 1-30 μM gefitinib for 24 h. b Autophagosome formations in gefitinib-treated MCF-7. Autophagosome formation was visualized by TEM in MCF-7 cells. Cells were incubated with 10 μM gefitinib for 24 h. Star marks indicate double lipid layer vesicle structures. c Effect of ATG7 siRNA on anti-proliferative effect of gefitinib. ATG7 expression was detected by western blotting after siATG7 transfection (upper) and cell proliferation was monitored by Incucyte® ZOOM basic analyzer in MCF-7 cells (lower). Data represent the mean ± S.D. (n=6). d Inhibition of gefitinib-induced autophagy formation by MBX-2982 (MBX). LC3B I/II were measured by western blottings in breast cancer cells incubated with 10 μM gefitinib in the presence or absence of 1-10 μM MBX or 3 μM chloroquine. e Inhibition of gefitinib-induced autophagy formation by GSK1292263 (GSK). LC3B I/II were measured by western blottings in MCF-7 cells incubated with 10 μM gefitinib, 10 μM MBX, 10 μM GSK or 3 μM chloroquine for 24 h. f Inhibition of autophagosome puncture formation by MBX. Green fluorescence puncta were detected by fluorescence microscopy after LC3-GFP transfection in MCF-7 cells (left). Red fluorescence was calculated in MCF-7 cells after mCherry-GFP-LC3 plasmid transfection by Incucyte® ZOOM basic analyzer (right). MCF-7 cells were incubated with 10 μM gefitinib in the presence or absence of 10 μM MBX. Data represent the mean ± S.D. (n=3). g Tumor growth of MCF-7 xenograft was monitored for 40 days. Data represent the mean ± S.E. (n=5). * p < 0.05, significant difference between the indicated two groups