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 μm. (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 μm. (D) Cell lysates were harvested after incubation with different concentrations of Huaier extract for 48h. β-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.