Facscan flow cytometry

After transfection for 48 h, the cells were harvested and resuspended in 1×Annexin V binding buffer (500 μL). Next, the cells were incubated with Annexin V-FITC (5 μL) and propidium iodide (5 μL) for 15 min at room temperature without light. Finally, the apoptosis ratio was analyzed using FACScan flow cytometry (BD Biosciences).

SMMC-7721 and HepG2 cells were transfected with shHOXA10#3 or shNC. MHCC-97H cells were divided into four groups: Group 1, transfected with pcDNA3.1 and shNC; Group 2, transfected with HOXA10-pcDNA3.1 and shNC; Group 3, transfected with pcDNA3.1 and shHDAC1; and Group 4, transfected with HOXA10-pcDNA3.1 and shHDAC1. After 48 h, cells were harvested. For cell cycle analysis, cells were fixed in 70% ethanol overnight at −20 °C and washed once in ice-cold PBS. Cells were then stained with propidium iodide (PI)-RNase solution (Sigma-Aldrich, St. Louis, MO, USA). For cell apoptosis analysis, cells were stained with Annexin V-FITC Kit (Beyotime) following the manufacturer’s instructions. Cells in early apoptosis were Annexin V-FITC+ and PI-. DNA content and apoptosis were analyzed by FACScan flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA).

CC cells were harvested and treated with trypsin, and then washed with phosphate buffer solution. For cell cycle distribution analysis, the cells were fixed with ethanol (70%) at 4°C overnight. Thereafter, the cells were incubated with propidium iodide (PI) (50 μg/mL, Solarbio) and RNase A (100 μg/mL, Solarbio). For cell apoptosis evaluation, the cells were stained with the Annexin V-fluorescein isothiocyanate (FITC)/PI Apoptosis Detection Kit (BD Biosciences, San Jose, CA, USA). The distribution of the cells was analyzed by a FACScan flow cytometry (BD Biosciences) with FACS Diva Software (BD Biosciences).

After being cultured in either 1 or 10% FBS for 24 h, the cells were treated with a concentration gradient of recombinant CCN3 (rCCN3; Sino Biological Inc., Beijing, China; 0, 0.5, 1 and 2 µg/ml) for 24 h. Subsequently, apoptosis was determined by staining with annexin V fluorescein isothiocyanate (FITC) and propidium iodide (PI) (BD Pharmingen, San Diego, CA, USA), according to the manufacturer's protocol. To quantify apoptosis, the cells were washed with cold PBS and suspended in binding buffer. The cells were then stained with 5 µl annexin V-FITC and 5 µl PI at 4°C for 15 min, and were analyzed using FACScan flow cytometry (BD Biosciences, San Jose, CA, USA) (22 (link)). The cells were quantified as follows: i) Annexin V negative/PI negative (viable cells); ii) annexin V positive/PI negative (cells in the initial stages of apoptosis); iii) annexin V positive/PI positive (cells in the advanced stages of apoptosis); and iv) annexin V negative/PI positive (necrotic cells).