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. 2016 Aug 19;8(51):88386–88400. doi: 10.18632/oncotarget.11393

Figure 4. Rapamycin enhances SH003-induced autophagy-mediated apoptosis.

Figure 4

(A) MDA-MB-231 cells were treated with different concentrations of SH003 (0, 100, 250 and 500 μg/ml) for 24 hours and then subjected to western blots with the antibodies indicated (anti-p-mTOR, -p-p70S6K and -p70S6K). Actin was used as internal control. (B) Cells were treated with 10 μM of rapamycin (Rapa) and 500 μg/ml of SH003 and then autophagosome vacuoles were measured by Cyto-ID fluorescence. Data analyzed using a FACSCalibur. *P < 0.05. (C) MDA-MB-231 cells were treated with rapamycin and SH003 and then stained with anti-LC3B and LAMP2 antibodies. Colocalization with LC3B and LAMP2 were analyzed using Olympus FV10i Self Contained Confocal Laser System. The object was 20× and scale bar indicates 10 μm. *P < 0.05. (D) Cells were treated with rapamycin and SH003 for 48 hours and then stained with Annexin V and 7AAD at room temperature in the dark. Annexin V-positive apoptotic cells were detected using FACSCalibur. *P < 0.05. Representative data were presented as the means and standard deviations (SD).