Cell cycle assay was performed using the DNA Content Quantitation Assay (Solarbio, Beijing, China) [77 (link)]. MDA-MB-231 cells—after 48 h of PTX (0.1 nM) or PTX-TTHA (0, 0.1, 1 nM) treatment—were harvested and fixed with 70% ethanol at 4 °C overnight. Then, the fixed cells were incubated with RNase A solution at 37 °C for 30 min and stained with PI in the dark for 30 min at 4 °C. The red fluorescence was detected using a flow cytometer (BD FACSCalibur) at 488 nm and analyzed by FlowJo Software (Tree Star Inc, Ashland, OR, USA). The PTX-treated group was set as a positive control.
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