High content imaging pathway 855

Cell proliferation was determined using the Cell Counting Kit-8 assay kit (Dojindo, Kumamato, Japan) and the Cell Titer 96 assay kit (Promega, Madison, WI, USA) according to the manufacturer's instructions. The EdU incorporation assay was carried out according to the manufacturer's instructions (Guangzhou RiboBio). Images were obtained and analyzed by High Content Imaging Pathway 855 (BD Biosciences, San Jose, CA USA). The fraction (%) of EdU-positive cells was calculated as (EdU add-in cells/Hoechst stained cells) ×100.

The EdU proliferation assay was used to test lung cancer cell proliferation according to the Kit protocol (Cell Light EdU DNA imaging Kit, Guangzhou RiboBio, China). In brief, A549 and 95D cells were transfected with miRNA mimics in 96-well plates. Forty-eight hours after transfection, 50 mM 5-ethynyl-2′-deoxyuridine (EdU) was added and the cells were cultured for an additional 2 hours. The cells were then stained according to the Kit protocol: discard the EdU medium mixture, wash cells with PBS, add 4% paraformaldehyde (PFA) to fix cells at room temperature for 30 minutes, wash with glycine (2 mg/ml) for 5 minutes in a shaker, add 0.2% Trion X-100 for 10 minutes, wash with PBS for two times, add click reaction buffer (Tris–HCl, pH 8.5, 100mM; CuSO₄, 1mM; Apollo 550 fluorescent azide,100 mM; ascorbic acid, 100mM) for 30 minutes while protecting from light, wash with 0.5% Triton X-100 for three times, stain with DAPI (0.5 mg/L) for 30 minutes at room temperature, wash with 0.5% Triton X-100 for five times, and finally add 150 ul PBS. Images were taken and analyzed using High Content Imaging Pathway 855 (BD, USA). EdU positive cells were calculated with EdU% = (EdU-add-in cells/Hoechst stained cells)∗ 100%.