Fig. 5

Autophagy inhibition in combination with chemotherapy was more effective than monotherapy in inducing apoptosis in TNBC. A Cells were transfected with a tandem fluorescent-tagged LC3 (tfLC3), and were exposed to TSN (1 μM), SN-38 (0.1 μM) or combination. The co-localization of GFP-LC3 and RFP-LC3 puncta was examined by confocal microscopy, in at least 20 cells. Scale bars: 10 μm. B MDA-MB-231 cells were treated with 0.1 μM of SN-38 for 24 h, then 0.1 μM TSN was added for another 24 h. Cell morphology was collected by Incucyte S3 Live-Cell System. Scale bars: 200 μm. C MDA-MB-231 cells were treated with the indicated concentrations of TSN (0.1 μM) with or without SN-38 (0.1 μM) for 24 h, and the supernatants were collected for the LDH release measurement. D MDA-MB-231 cells were treated with various concentrations of SN-38 for 24 h, then TSN was added for another 24 h. MTT assays were performed to assess cell viability. E MDA-MB-231 cells were treated with SN-38 (0.1 μM) for 24 h. Then, TSN (0.1 μM) was added into the plate in the presence of SN-38 for another 24 h. PI positive late apoptotic/necrotic cells were used for quantitative analysis. F Western blot analysis of cleaved caspase 3 in MDA-MB-231 cells treated with or without SN-38 (0.1 μM), TSN (0.1 μM) for 24 h. G MDA-MB-231 cells were treated with or without SN-38 (0.1 μM), TSN (0.1 μM) for 24 h. Then the cells were incubated with DCFH-DA for 20 min at 37 °C, and the fluorescence was observed by confocal laser-scanning microscope (488 nm excitation and 525 nm emission). The results are expressed as the mean ± SD values obtained from three independent experiments (**p < 0.01, ***p < 0.001 vs. SN-38 alone; ^##p < 0.01, ^###p < 0.001 vs. CTL)