96 well cell plate

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.

SM from Silybum marianum seeds was prepared according to Kahol et al. [49 ]. The SM did not contain any solvent residue. The same bioactive flavonolignans were determined as in the standards with the help of a HPLC-MS method [4 (link)]. P60 was obtained from Sigma-Aldrich Buchs (St. Gallen, Switzerland). TC was a kind gift from Gattefossé (Lyon, France). Cremophor A6, A25 was obtained from BASF (Ludwigshafen, Germany). Sucrose esters (SP 50, SP 70, PS 750) were kind gifts from Sisterna (Roosendaalc, The Netherlands). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Dulbecco’s Modified Eagle’s Medium (DMEM), Hank’s Balanced Salt Solution (HBSS), phosphate buffered saline (PBS), Trypsin-EDTA, Heat-inactivated fetal bovine serum (FBS), l-glutamine, non-essential amino acids solution, penicillin-streptomycin, were purchased from Sigma-Aldrich). 96-Well cell plates, 12 well cell plates and culturing flasks were obtained from Corning (Corning, New York, NY, USA). Cetostearyl alcohol, stearic acid, propylene glycol, IPM, nipagin M were supplied by Hungaropharma Ltd., (Budapest, Hungary). HeLa cells (human cervical cancer cells) were obtained from the European Collection of Cell Cultures (ECACC, Public Health England, Salisbury, UK). HaCaT cells (human keratinocyte cells) were obtained from Cell Lines Service (CLS, Heidelberg, Germany).

All primers of four shRNAs (shRNA-1, -2, -3, -4) targeting S4 were listed in Table 1 and cloned into pcDNA 6.2-GW/EmGFP-miR vectors (Invitrogen, Carlsbad, USA). CIK cells were transfected with each shRNA as described above and infected with GCRV at MOI of 1. Following incubation at 28°C for 30 min, the inoculum was removed and rinsed three times with cell culture medium, subsequently incubated in complete medium. Infected cells were collected and subjected to RT-PCR and WB analysis. Meanwhile, cell supernatant (extracellular virus) was harvested and virus titers were determined by 50% tissue culture infective dose (TCID50) assay in CIK cells. TCID50 assays were performed in 96-well cell plates (Corning, USA) with 8 replicates per dilution from 10⁻¹ to 10⁻⁸ in MEM for 5 days. Viral infectivity was calculated by the formula of Reed and Muench [52 ].

The production of viral progeny was measured in RK‐13 cells using the Reed–Muench method as described previously (1 (link)). Briefly, the cells were grown to approximately 80%–90% confluence in 96-well cell plates (Corning, USA). The viral supernatants were serially diluted 10-fold, and 100 µL of each dilution was added to each well. After 1 h of virus adsorption, the cells were washed with PBS and incubated in 3% FBS DMEM. Five days post-infection, the TCID₅₀ was calculated and analyzed using GraphPad 8.0.