6 well cell plates

A Cell Counting Kit-8 (CCK-8) assay (Dojindo, Kumamoto, Japan) was used according to the manufacturer’s instructions to measure the effects of drugs on the proliferation of papillary thyroid carcinoma cells. Briefly, BCPAP cells were seeded in 96-well cell plates (Corning Inc., Corning, USA) at a density of 1000 cells/well in 200 μL of culture medium and grown overnight. The next day, vemurafenib or/and E3330 were added to each well at an appropriate concentration, and cells were incubated for 0, 24, 48, 72, and 96 h. Then, 20 μL of CCK-8 reagent was added to each well and incubated for 3 h at 37 °C. Finally, the absorbance was measured with an enzyme-labeled instrument (Thermo) at 450/650 nm. The experiments were repeated at least three times.
For colony formation analysis, BCPAP and K-1 cells were seeded in 6-well cell plates (Corning Inc., Corning, USA) at a density of 500 and 1000 cells/well in 2 mL of culture medium, respectively, and then treated with vemurafenib or/and E3330 at an appropriate concentration for 24 h. In the drug treatment groups, the medium was replaced with a fresh medium after 14 days. Colonies were washed with phosphate-buffered saline (PBS) 3 times, fixed with 4% paraformaldehyde for 20 min, stained with 0.5% crystal violet for 15 min at room temperature, and then counted.

Subgingival plaque was collected from hopeless elements extracted for periodontal reasons and with probing greater than 7 mm. Plaque was incubated in 2 mL of LB Broth (Lennox L-broth base, Invitrogen, MA, USA), a generic culture medium for bacteria, for 24 h at 37 °C in oscillating incubator to obtain the inoculum for biofilm cultivation. Discs were placed into wells of 6-well cell plates (Corning Life Sciences, Woburn, MA, USA), covered with 3 mL of subgingival human plaque suspension + 2 mL of fresh LB broth medium. Bacteria and discs finally were incubated for 7 days, at 37 °C. The LB medium was replaced every 24 h. After 7 days of cultivation, the medium rich in bacteria was removed, and the biofilm-covered discs were transferred into new wells of a sterile 6-well plate. All discs were washed with PBS 1X, then discs used for Cell culture were leaved in PBS 1X, whereas discs used for microscopy were fixed in formalin solution neutral buffered 10% (Sigma Aldrich, MO, USA) for 15 min at room temperature and dehydrated using solutions with an increased concentration of alcohol (50%, 5 min—75%, 5 min—80%, 5 min, 95%, 5 min and 100%, 30 min). Fixed and non-fixed discs were stored at 4 °C.

IPEC-J2 cells were seeded into 6-well cell plates (Corning Inc., Corning, NY, USA) at a concentration of 1 × 10⁶ cells/mL. The cells were cultured in Dulbecco’s modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS, Gibco, Grand Island, NY, USA) in a cell incubator (37 °C, 5% CO₂). After the cells grew to reach 80% confluency, the cells were incubated for 4 h with Salmonella enterica (1 × 10⁷ CFU/mL) [17 (link)] or LPS (1 µg/mL), without antibiotics [18 (link)]. For the control group, a comparable amount of PBS was replaced, and then gentamicin (10 μg/ml) was added to the media to kill extracellular bacteria [19 (link)]. The workflow of this experiment is described in Figure 1A.