Figure 1.

Doxorubicin (ADR) resistance is related to a high level of autophagy in ADR-resistant MCF-7 (MCF-7/ADR) cells. (A) MCF-7 and MCF-7/ADR cells were fixed in 2% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) and then post-fixed in 1% OsO4 for 2 h at 4°C. The cells were dehydrated via a graded ethanol series and embedded in LR White resin. The solidified blocks were cut into ultrathin sections and stained with uranyl acetate and lead citrate. Samples were observed under a transmission electron microscope. MCF-7/ADR cells showed higher autophagosome levels than MCF-7 cells. Yellow arrows indicate Autophagosome. (B) Total protein was extracted using a combination of RIPA buffer (150 mM NaCl, 50 mM Tris-HCl, 0.5% sodium deoxycholate, 200 mM NaF, 200 mM PMSF, 1.0% NP40, and 1 mM EDTA), PMSF, and phosphatase inhibitors. Lysates (10 uL) were subjected to SDS–PAGE and then transferred to PVDF membranes. Membranes were analyzed using ImageJ software. Western blotting analysis showed that MCF-7/ADR cells exhibit higher expression of the autophagy-associated protein LC3II/I, and lower expression of p62 protein, than MCF-7 cells. (C) MCF-7/ADR cells were treated with the autophagy inhibitor 3-MA (5 mM). Western blotting analysis showed that 3-MA treatment inhibited the expression of LC3II/I and MDR1. (D) MCF-7/ADR cells were treated with ADR, 3-MA, or a combination of both. MTT was added after 48 h. After 4 h of incubation, the MTT solution was discarded and DMSO was added for 10 min with slow shaking. Absorbance was measured at 550 nm using a spectrophotometer. The results showed that 3-MA treatment increased the sensitivity of MCF-7/ADR cells to ADR. N/A, not significant; ***p<0.001 (Student’s t-test).