Airyscan laser confocal microscope

HepG2 cells were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Mycoplasma contamination of cell line were tested via Hoechst33258 staining, and no contamination was observed. HepG2 cells were grown on glass coverslips in 6-well plates (at a density of 1 × 10⁶ per well) overnight in a 5% CO₂ incubator at 37 °C and then treated with 30 nM taccalonolide AJ (solubilized in dimethylsulfoxide (DMSO)) or same amount of DMSO as control for 16 h. After treatment, cells were fixed with 4% paraformaldehyde and then washed two times with PBS, and then treated with PBS containing 0.5% Triton X-100. After treatment with blocking buffer (PBST containing 5% BSA) for 1 h at room temperature, microtubules were marked using anti-α-tubulin (Santa Cruz; sc-5286) antibody (1:100 dilution in blocking buffer). Then, cells were incubated overnight at 4 °C. After rinsing three times with PBST, cells were stained with FITC-conjugated fluorescence second antibody (Proteintech; SA00003-1) and labelled with 0.1 g ml⁻¹ 4,6-diamidino-2-phenylindole (Beyotime; C1002) and then washed five times with PBST. Samples were visualized and photographed using a Zeiss LSM 880 with Airyscan laser confocal microscope (laser 405 nm, 488 nm).