Fig. 5

GPR119 signaling in autophagy inhibition by MBX-2982. a GPR119 mRNA expression in GPR119 shRNA-infected cells. MCF-7 cells were infected with shGPR119 or shNonTarget lentivirus particle, and GPR119 mRNA expression was determined by real-time qPCR. Data represent the mean ± S.D. (n = 3)(*** p < 0.005, significant difference versus shNonTarget-infected control). b Reversal of MBX-2982 (MBX)-induced autophagy inhibition in GPR119 knock-down cells. LC3B I/II were measured by western blotting in shGPR119 or shNonTarget-infected MCF-7 cells. c Xenograft analysis. Tumor growth of shGPR119-MCF-7 xenograft was monitored for 41 days. Balb/c-nu mice were orally administered with gefitinib (1 mg/kg), MBX (10 mg/kg) or gefitinib with MBX (5 times a week). Data represent the mean ± S.E. (n = 3). d CRE reporter activity. MCF-7 cells were transiently transfected with CRE-luciferase reporter plasmid (CRE-luc) and its reporter activity was measured using luminometer. 10 μM forskolin, an adenylyl cyclase activator, was used as positive control. Data represent the mean ± S.D. (n = 3)(* p < 0.05, ** p < 0.01, significant difference versus control group). e Effect of PKA inhibitor on autophagy inhibition by MBX. MCF-7 cells were preincubated with 10 μM of TK5720, a PKA inhibitor for 30 min and then treated with 3 μM chloroquine in the presence or absence of 10 μM MBX. f Effect of MBX on mTOR signaling pathway. mTOR signaling pathway was estimated by immunoblottings for phosphorylated p70S6 kinase, phosphorylated 4EBP1 and phosphorylated ULK1 in MCF-7 cells incubated with 10 μM MBX-2982 for 24 h. g Effects of MBX on the protein levels of ATG5, ATG7 and ATG12. MCF-7 cells were incubated with MBX (1–10 μM) for 24 h