Cell counting kit

Cells in logarithmic growth phase were seeded in 96-well Falcon Petri dishes (3,000 cells/well) the day before the assays were performed. The cell viability was assessed using a Cell Counting Kit (Dojindo, Kumamoto, Japan) based on sensitive colorimetric assays with the absorbance measured at 450 nm (OD 450 nm), according to the instructions of the manufacturer. The results were confirmed with three independent experiments.

Cells from the human kidney HEK293‐T cell line (denoted as HEK293‐T cells; Cell Bank of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences), the human lung cancer A549 cell line (denoted as A549 cells; Cell Bank of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) and the human liver cancer SMMC‐7721 cell line (denoted as SMMC‐7721 cells; Cell Bank of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) were used for the in vitro cytotoxicity assay of ZrC NSs. Cells were cultured with Dulbecco's modified Eagle's medium (DMEM, Gibco, Invitrogen) under 5% CO₂ and supplemented with 10% fetal bovine serum (FBS, Gibco, Invitrogen) and 1% penicillin/streptomycin in a humidified incubator at 37 °C. After harvesting with 0.25% trypsin‐EDTA solution (cat. no. C0201, Beyotime, China), cells were seeded in 96‐well culture plates (Corning) at a density of 1 × 10⁵ cells per well (n = 4) for 24 h, and the culture medium was then replaced with fresh culture medium containing ZrC NSs at different concentrations (0, 25, 50, 100, and 200 µg mL⁻¹). After 24 h of incubation, the in vitro cytotoxicity of ZrC NSs was determined by the CCK‐8 viability assay (Cell Counting Kit, Dojindo Laboratories, Kumamoto, Japan).

Using the Cell Counting Kit, the influence of H/R on cell viability was determined at the indicated time points (CCK8, Dojindo, Kumamoto, Japan). The cells were plated at 5000 cells/well into 96-well plates. After H/R treatment, 5 µl of CCK8 reagent was added to each well, followed by incubation for 60 min in a humidified incubator. The optical density (OD) value at the wavelength of 450 nm was then measured using a microplate reader (Varioskan LUX, Thermo Fisher, Massachusetts, USA).

Following transfected with siRNA or overexpressing plasmid for 2 days, cells were seeded in 6-well plate with 800 cells in each well and cultured in a CO₂-incubator at 37°C for 2 weeks. Cells were stained with crystal violet and counted. Approximately 1,000 cells/well were seeded in 96-well plate after transfected with siRNA or overexpressing plasmid for 2 days. Cell viability was detected at regular intervals by Cell counting kit (Dojindo) following the manufacturer’s protocol. After incubating with 10 μl regent for 2 h, the absorbance at 450 nm was detected by a microplate reader. Also, HCC cells were treated with decitabine for 3 days and followed experiments.

The cell viability of each layered construct was evaluated by using a Cell Counting Kit (Dojindo Laboratories, Kumamoto, Japan). The layered construct was soaked in PBS and the Cell Counting Kit, and incubated in a CO₂ incubator for 2 h. The absorbance was measured by a spectrophotometric plate reader (2040 ARVO™ X2, Perkin Elmer Co., Yokohama, Japan) at a wavelength of 450 nm.