Cck 8 assay

The cell migration and matrigel invasion assays were performed as described in our previous report [20 (link)]. For the cell proliferation assay, 2 × 10³ cells were incubated in 96-well plates, and at the prescribed time, the OD value was determined by a cell counting kit-8 (CCK-8) assay (Yeasen, Shanghai, China).

Commercial L929 cells were obtained from Chinese Academy of Sciences, Shanghai, China, and were employed to determine the biocompatibility of all the CS-PVA and CS-PVA-US nanofiber mats. The samples were immersed into commercial cell culture medium for 24 h, and the extract-contained media were harvested. L929 cells were seeded in a 96-well plate with a density of 1.0 × 10⁴ cells per well. After 24 h of culture, 100 μL of the as-harvested extract-contained medium was utilized to further culture cells for another 24 h. Then, a CCK-8 assay (Yeasen Biotechnology Co., Ltd., Shanghai, China) was conducted to quantitatively test the cell viability according to the manufacturer’s protocol. Moreover, a live–dead assay was performed to observe the cell viability from the qualitative perspective. Specifically, calcein-AM dye (Yeasen Biotechnology Co., Ltd., Shanghai, China) and propidium iodide dye (Yeasen Biotechnology Co., Ltd., Shanghai, China) were utilized to stain the live and dead cells, respectively. A confocal microscope (CLSM, Zeiss 900 CLSM, Oberkochen, Germany) was utilized to take photos of live–dead staining. The excitation wavelength and emission wavelength of Calcein AM were 490 nm and 515 nm, respectively. The excitation wavelength and emission wavelength of propidium iodide were 490 nm and 630 nm, respectively.

Cell viability was measured using the CCK-8 assay (YEASEN Biotech). HeLa cells stably expressing the FLAG-tagged WT, 3KQ or 3KR RPA1 allele (siRNA resistant) were seeded at a density of 5 × 10³ cells/well in 96-well plates. After 24hrs, cells were transfected with siRNA against RPA1 using the Lipofectamine 2000 reagent. Cells were then incubated for 24 h before treating with iniparib (MCE) at indicated doses (0, 10, 25, 50 and 100 μM). After 24hr treatment, 10 μl CCK-8 reagent was added to each well, and the plates were incubated for 1 h at 37°C. Finally, the absorbance was measured at 450 nm using a scanning microplate reader (Cytation3). Cell viability at individual time points was normalized to the untreated group.

mTEC1 cell viability was assessed by CCK-8 assay (Yeasen, Shanghai, China). mTEC1 cells (5 × 10⁵ cells/mL) were suspended in a 96-well plate with enriched RPMI-1640 medium containing 1% penicillin/streptomycin and 10% fetal bovine serum (Gibco, Grand Island, NY, USA). The cells were maintained at 37 °C in a humidified atmosphere with 5% CO₂ and stimulated with/without FAC (ferric ammonium citrate) (Sigma, St. Louis, MI, USA) or L-cit (Solarbio, Beijing, China). After 24 h or 48 stimulation, 10 μL CCK-8 solution was added to each well and then incubated for 60 min at 37 °C with 5% CO₂. A microplate reader (Thermo Fisher, Waltham, MA, USA) was used to measure the absorbance at 450 nm.