ARHI induces autophagy in breast cancer cells. (A) The detection of LC3 punctae. SKBr3 cells were co-transfected with GFP-LC3 and ARHI or pcDNA3 vector. The cells transfected with GFP-LC3 alone and treated with rapamycin served as a positive control. The cells were observed by fluorescence microscopy. The white arrows indicate LC3 punctae. (B) The autophagosomes were detected by TEM in xenografts after treatment with ARHI-liposomes (N, nucleus; L, lysosome). The white arrows indicate autophagosomes. (C) ARHI re-expression increased autophagy induction. SKBr3 cells were transfected with ARHI expression vector (left), MDA-MB-231 cells were transfected with ARHI expression vector (middle) or SKBr3 cells were treated with TSA (right), and flow cytometry-AVO analysis was performed to examine autophagy induction. The data were obtained from three independent experiments. (D) LC3 conversion and signaling. (Left) SKBr3 cells were stably transfected with GFP-LC3 and subsequently transfected with ARHI or pcDNA3 vector constructs. LC3-I and LC3-II were detected by western blot. (Right) SKBr3 cells were transfected with ARHI expression vector. The pcDNA3 vector was used as the negative control. The pAKT, AKT, pmTOR, mTOR and ARHI proteins were detected by western blot.