Crystal violet

Bacterial biofilm formation was assessed using crystal violet as previously described, with some modifications [98 (link)]. The wells of a sterile 96-well microplate were filled with 200 μl of LB medium; 2 μl of overnight culture was added to each well. Each strain was tested in triplicate. Wells containing sterile LB medium were treated as negative controls. The plate was covered and incubated at 37 °C for 24 h without shaking. Planktonic cells in wells were aspirated, and washed twice with 200 μl of sterile PBS. Attached cells were stained with 200 μl of 1 % (w/v) crystal violet (Biosharp, Hefei, China) for 15 min at room temperature. Wells were rinsed twice with 200 μl of sterile PBS, and plates dried at 37 °C for 30 min. Stained adherent biofilms were solubilized with 200 μl of 33 % (v/v) glacial acetic acid. To quantify, the optical density (OD) at 600 nm (OD₆₀₀) of each well was determined using a Synergy™ HT Multi-Detection Reader (BioTed Instruments, Winooski, VT). All tests were independently performed three times and the results averaged. The cutoff value for determining a biofilm producer was defined as 2-fold greater than the negative control [97 (link)].

For the colony-formation assays in monolayer cultures, LLC and SPC-A1 cells (150 cells/well) were seeded in 100 mm dishes for 24 h and then treated with TAX at different concentrations for 10 days. Then, the cells were fixed in 4% paraformaldehyde (Servicebio, Wuhan, China) at 4 °C and stained with crystal violet (Biosharp, Hefei, China). Details can be found in reference [17 (link)]. Further details are also shown in the Supplementary Materials.

Cell proliferation was detected using colony formation assay. The cells at a density of 5 × 10²/ml were plated in six‐well plates (NEST, China) and allowed to grow. The medium was changed every three days. After a 14‐day incubation, the colonies were fixed with the methanol for 15 min and observed by staining with 1% crystal violet (Biosharp, China) for 30 min at room temperature and washed again. At last, the colonies were observed and counted under a microscope (Olympus, Japan).

Three small interfering RNAs (siRNAs) specific for human FOXO1 were designed and synthesized by Ribbio Co. Ltd (Q000002308-1-A, Guangzhou, China). Two of them were selected for their efficiency of knockdown. Scrambled non-targeting siRNA was used as a negative control. HOS cells were transfected with X-tremeGENE siRNA Transfection Reagent (Roche, Mannheim, Germany) according to the manufacturer's instructions. After transfection, cells were cultured for 12 h and then seeded in 12-well plates at a density of 50, 100, 200 cells per well. The transfection was performed every 4 days. Twelve days later, the plates were stained with 0.1% crystal violet (Biosharp, Hefei, China) and the colony numbers were counted under a microscope.

Cell colony formation assay was performed to detect the proliferation ability of BMSCs. First, BMSCs in single‐cell suspension were seeded into six‐well plates (Nest) at a density of 100 per well and cultured in normal growth medium. The cells were cultured for 2 weeks, and the medium was changed every 3 days. Then the colonies were washed by PBS and fixed in 4% PFA solution, and the cells were incubated in 0.4% crystal violet (Biosharp, Hefei, China) for 20 min. Finally, the number of colonies was observed and calculated under an inverted light microscope (Olympus, Hachioji, Japan).