Fig. 5

T-96 initiated autophagy but blocked autophagic flux in human CRC cells. A The punctate staining pattern of LC3 was detected using high content analysis system-operetta CLS™ in DU145 and PC3 cells after treatment with increasing concentrations of T-96 for 48 h. Scale bar, 10 μm. B The expression levels of proteins involved in initiation of autophagy were detected after exposure to T-96 using western blot. β-Tubulin was used as a loading control. C T-96 suppressed the autophagic flux in PC3 cells overexpressing mCherry-EGFP-LC3. Both cells were treated with indicated concentrations of T-96 for 24 h and then observed with fluorescent microscopy. Scale bar, 10 μm. Quantitative analysis of the autophagosome accumulation in T-96-treated cells. Yellow dots representing the autophagosomes were counted in three independent experiments. Red fluorescence indicates autophagolysosomes. D The suppression of Autophagic flux significantly increased apoptosis. DU145 and PC3 cell lines were treated with rapamycin, chloroquine, T-96, rapamycin in combination with T-96, and chloroquine in combination with T-96. The rate of apoptosis was measured using flow cytometry. Data represent the mean ± SD of three independent experiments. **P < 0.01 and ***P < 0.001 compared with the control group