Mtt dye

A549 cells (cat. no. CCL-185; American Type Culture Collection) and H460 cells (kindly provided by Stem Cell Bank, Chinese Academy of Sciences) were cultured in DMEM (Gibco; Invitrogen; Thermo Fisher Scientific, Inc.) containing 10% FBS (Thermo Fisher Scientific, Inc.). Cells were transfected with either Ad-RGC32 or Ad-shRGC32 for overexpression or knockdown of RGC32. NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC) was used to evaluate the effect of RGC32 overexpression. The transfection of adenoviral vector (Null) was used as a control. Briefly, the cells were seeded into a 96-well plate at a density of 2×10³ cells/well and incubated for 24 h at 37°C in 5% CO₂ incubator. After transfection, cells were cultured for 24, 48 or 72 h for a time-dependent study. Subsequently, the culture medium was discarded and the wells were washed twice with PBS, followed by the addition of 10 µl Cell Counting Kit-8 reagent (cat. no. CK04; Dojindo Molecular Technologies, Inc.) or 10 µl MTT dye (0.5 mg/ml; Beijing Solarbio Science & Technology, Co., Ltd.) to each well, respectively. The cells were incubated for a further 2 h at 37°C. The samples were then detected at 450 nm using a Microplate reader (Bio-Rad Laboratories, Inc.). Each treatment was replicated and each experiment was repeated 3–5 times.

The cell viability was detected in this study with MTT assay. MTT dye (Cat No.: M1025, Solarbio, China) was added to the 96-well plate with ARPE-19 cells in different groups and incubated for four hours at 37°C. After replacing the culture media with 150 µL of DMSO to each well, the 96-cell plates were shaken thoroughly until the crystals dissolved. Absorbance was measured at a wavelength of 490 nm using the Thermo Scientific Fluorescent microplate reader (Thermo Fisher Scientific, USA). Three independent experiments were conducted for each group and data were used for advanced analyses.