Apoptosis analysis kit

The cell cycle was analyzed according to the protocol of the cell cycle and apoptosis analysis kit (Beyotime, Shanghai, China). VSMCs were grown to 80% confluence in a 6-well plate. After starvation for 24 h, VSMCs were pretreated by PDGF-BB or JB or both. Digested with trypsin, the cells were centrifuged (12,000 g, 5 min) and washed with 1 ml pre-cooled PBS. All samples were fixed with 75% ethanol at 4°C overnight and then washed with 1 ml of pre-cooled PBS. After cells were incubated with propidium iodide (PI) staining buffer (100 ug/mL PI and 100 ug/mL RNase A) at 37°C for 30 min, the samples were analyzed by flow cytometer (BD Biosciences, Franklin Lakes, NJ, United States).

Briefly, HaCaT cells were seeded in a 6-well plate at 1 × 10⁶ cells/well and cultured to the density of 70–80%. In a pretreatment protocol to examine the protective effect of PTD-CcTrx1 on H₂O₂-induced apoptosis, the cells were preincubated with PBS, 10 μM CcTrx1, and 10 μM PTD-CcTrx1 for 2 h, respectively, rinsed with PBS, and then exposed to 400 μM H₂O₂ for 30 min. In a posttreatment protocol, HaCaT cells were exposed to 400 μM H₂O₂ for 30 min, rinsed with PBS, and then incubated with PBS, 10 μM PTD-CcTrx1, and 10 μM CcTrx1 for 2 h, respectively. Afterward, cells were washed with ice-cold PBS, gently collected into sterile centrifuge tubes, and resuspended in 200 μL of the binding buffer from an apoptosis analysis kit (Beyotime, Shanghai, China). Then, 5 μL of FITC-Annexin V and 5 μL of propidium iodide (PI) were added into each tube for 15 min of staining. The cells were subjected to flow cytometry analysis with a flow cytometer (ThermoFisher Scientific, Waltham, MA, USA).

After transfection, HEC-1A and Ishikawa cells were splashed with phosphate-buffered saline (PBS) and gathered into the centrifuge pipe. After centrifugation, cells were stained using Apoptosis Analysis Kit (Beyotime Biotechnology, Shanghai, China). Flow cytometry was performed using a Flow cytometer (BD Biosciences, Detroit, MI, U.S.A.), and the apoptosis was estimated.

Treated cells were collected and then fixed in precooled 75% ethanol at −20 °C overnight. The cell‐cycle analysis was performed on a flow cytometer (Influx, BD, San Jose, CA, USA). Cellular apoptosis was analyzed using Apoptosis Analysis Kit (Beyotime, Shanghai, China) according to the manufacturer's protocol on a flow cytometer (Influx, BD, San Jose, CA, USA).