Crystal violet staining solution

HCC cells were seeded into 12‐well plates at a density of 1.5 × 10⁵ cells with fresh cell culture medium. Then, plates with cells were treated with related chemo‐drugs after 24 h in 1% O₂ hypoxic conditions. After another 24 h with medication, the plates were treated with polyformaldehyde fixing solution and crystal violet staining solution (Beyotime, China). The colony pictures were captured with Nikon (Japan).
For CCK8 assay, cells were seeded at 1,000 cells per well in 96‐well plates with fresh medium. Cell viability was assayed using Cell Counting Kit‐8 (CK04, Dojindo, Japan) at the time in 0, 48, 72, and 96 h. The microplates were incubated at 37°C for additional 2 h. Absorbance was read at 450 nm using a microplate reader (Thermo Fisher, USA).
For clonogenic assay, cells were seeded into 6‐well plates at a density of 5,000 cells with fresh cell culture medium and maintain the related drug concentration for 7 days. Then, the plates were treated with polyformaldehyde fixing solution and crystal violet staining solution (Beyotime, China) and the colony pictures were captured separately.

The artificial basement membrane (Matrigel; Corning, New York, USA) was thawed in advance and then diluted in DMEM medium (serum-free) at a 1 : 8 ratio. After the Transwell chamber (8 μm pore size; Corning, New York, USA) was evenly overlaid with Matrigel on the microporous membrane, the Matrigel was then kept for incubation for 2 h at 37°C. The cells of each group were converted to cell suspension of 1 × 10⁴ cells/mL. 100 μL of each cell suspension was picked and added to the upper chamber of the Transwell chamber. In the lower chamber, DMEM (700 μL) was added. The cells were then allowed to culture for 48 h in an incubator. After incubation, cells present in the upper layer of the microporous membrane were gently wiped out using a cotton swab and then washed 2 times with PBS. For cells in the lower layer of the microporous membrane, 95% ethanol was used for fixation. The cells were then stained with a 0.5% crystal violet staining solution (Beyotime Biotech, China), washed with PBS, viewed, and counted under an inverted microscope (average of 6 fields of view for each group).

Cells were seeded in six-well plates and exposed to conditioned medium until the cell density reached 80% confluence. Before evaluation, the cells were washed with PBS and fixed with 4% paraformaldehyde for 15 min at room temperature. Then, the plate was washed with tap water and stained with 500 µL of 0.5% crystal violet staining solution (Beyotime, #C0121) for 45 min at room temperature. The plate was then washed with tap water three times and air-dried before scanning.