The cytotoxic effect of Huaier extract combined with 3-MA and CQ treatment was measured by MTT

The experiments were done in triplicate and info is introduced as the suggest SD of a few The two reagents also showed great performance in enhanced chemiluminescence detection, and only the anti-IgY antibody exhibited delicate cross reactivity with IgM beneath non-minimizing circumstances individual experiments. (G) AO/EB staining of T47D cells was performed to detect apoptosis and necrosis induced by Huaier extract. Bars, 50 m. (H) Representative TUNEL staining (red fluorescence) of MDA-MB-231 cells treated with or without Huaier extract. Bars, 50 m. of autophagy. We next investigated the expression of several autophagy-related genes using the immunoblot assay. The data revealed a significant increase of processed LC3B-, Atg7, Beclin1 and a decrease of selective autophagy target p62/SQSTM1 in a dose-dependent way (Fig 2D). Furthermore, the induction of autophagy by Huaier extract was confirmed by flow cytometry using acridine orange staining in order to detect acidic vesicular organelles (AVOs). As shown in Fig 2E, Huaier treatment resulted in increased formation of AVOs. These results suggested that autophagy was activated in response to Huaier extract.Next, we used the autophagy inhibitors 3-methyladenine (3-MA) and chloroquine (CQ) to investigate whether the induction of autophagy contributed to Huaier-induced cell death. 3-MA is a well-known inhibitor of autophagosome formation, whereas CQ inhibits lysosome Fig 2. Huaier extract induced autophagy in breast cancer cells. (A) Representative electron micrographs of breast cancer cells treated with or without 4 mg/ml Huaier extract for 48 h. Bars, 100nm. (B) Acidic vesicular organelles induced by Huaier extract were stained with MDC. Bars, 10 m. (C) Aggregation of LC3B in Huaier-treated cells. Cells were treated with or without 4 mg/ml Huaier extract and stained with the LC3B antibodies using immunofluorescence staining. Puncta represent the autophagosome formation. Bars, 10 m. (D) Cell lysates were harvested after incubation with different concentrations of Huaier extract for 48h. -actin was used as a loading control. (E) Cells in suspension were labeled with acridine orange and quantified using flow cytometry. FL1-H indicates green color intensity (cytoplasm and nucleus), whereas FL3-H shows red color intensity (AVO). Cells in up quadrants were considered AVO-positive. Results shown are representative of three independent experiments.acidification and degradation [26]. The cytotoxic effect of Huaier extract combined with 3-MA and CQ treatment was measured by MTT. Pretreatment with 4 M 3-MA resulted in rescued cell viability (Fig 3A, 3C and 3E). In addition, 20 M CQ could significantly reduce the cell death in all three cell lines (Fig 3B, 3D and 3F). We then applied a genetic approach to confirm the effect of autophagy inhibition. We used siRNA to specifically knock down LC3B in MDA-MB-231 and MCF7 cells (Fig 4A). Using flow cytometry-AVO analysis, we observed that Huaier treatment enhanced autophagy induction, but transfection with siLC3B blocked the effect of Huaier extract (Fig 4B). As shown in Fig 4C and 4D, gene silencing with small interfering LC3B RNA (siLC3B) suppressed the cytotoxic effect of Huaier extract. These data suggested that inhibition of Huaier-induced autophagy recovered the reduced cell viability in MDA-MB-231, MDA-MB-468 and MCF7 cells.Recent studies have indicated that inhibition of mTOR/S6K pathway is associated with the triggering of autophagy in cancer cells [27, 28]. Therefore, we investigated whether the mTOR/ S6K pathway was involved in Huaier-induced autophagic cell death in all three breast cancer cell lines using Western blotting.