Pi rnase staining solution

The HepG2 cells were seeded in a 6-well plate (5 × 10⁵ cells/well) and allowed to adhere overnight at 37 °C and 5% CO₂. The cells were then incubated for 24 h with NO-CIETA 21a (2.4 μM) and 21b (8.1 μM), and DMSO (0.001%) as a control. The cells were then washed twice with ice-cold 1X PBS and collected after trypsinization [55 (link)]. Then, the cell pellet was washed twice with ice-cold 1X PBS and fixed overnight with ice-cold 70% ethanol at −20 °C. After that, the cells were washed once with ice-cold PBS and the second wash with ice-cold PBS-2% FBS. The cell pellet was re-suspended in 500 μL propidium iodide (PI)/RNase staining solution (BD Biosciences) and 0.1% triton x-100 for 30 min at room temperature in the dark and analyzed within 1 h by BD Accuri C6 flow cytometer (Becton-Dickinson, Mountain View, CA, USA) [56 (link)]. Data were analyzed by MFLT32 software and 10,000 events with slow flow rates were recorded for each sample.

Cells treated with ADV at varying concentrations were centrifuged at 200g for 5 min at room temperature. After aspirating the medium, cells were resuspended in cold PBS/1% BSA. The cells were then fixed in 70% ethanol while vortexing and stored at −20°C for 2 h. Before analysis, cells were centrifuged two times at 200g for 10 min at room temperature to remove the ethanol. Cells were rinsed twice with PBS/1% BSA and stained for 15 min with 1 μg/ml PI/RNase staining solution (BD Biosciences). The samples were measured by FACSCalibur (BD Biosciences). The fraction of cells at each cell cycle phase was quantified by fitting the experimentally obtained histogram with the Dean-Jett-Fox model in FlowJo software version 7.6.4 software (Treestar Inc.).

Cells were stained with the Annexin V/FITC Apoptosis Detection Kit I (BD Pharmingen, Franklin Lakes, NJ, USA) 48 h after receiving a 10 Gy radiation dose to look for signs of apoptosis. Using a Coulter-XL flow cytometer (Beckman Coulter Inc., Brea, CA, USA) and an EXPO32 ADC, we were able to capture and analyze pictures of apoptosis. The total apoptosis rate of tumor cells was calculated by adding the apoptosis rates observed in the early (lower-right) and late (upper-right). After 24 h, cells that had been irradiated with 10 Gy were collected and preserved in cold ethanol (1 mL PBS + 2 mL absolute ethanol) for studying the cell cycle. The cells were stained with a PI/RNase staining solution (BD Pharmingen; Franklin Lakes, NJ, USA). After that, we analyzed the cells cycles distributions (G1 and G2 phase) using flow cytometry (Coulter-XL; Beckman Coulter Inc., Brea, CA, USA) and Mod Fit 3.0 (Verity Software House, Inc., USA).

Cell apoptosis of hAMSCs under SC and SF conditions at the indicated time points was accomplished by utilizing the Annexin V and 7-AAD based FCM assay as we previously described [5 (link)]. In detail, hAMSCs under SC and SF conditions were collected and dyed with the commercial FITC Annexin V Apoptosis Detection Kit with 7-AAD (BioLegend, USA) according to the manufacturers’ instructions and detected by FCM assay (BD Biosciences, USA).
As to cell cycle analysis, hAMSCs under SC and SF conditions (preconditioned in SF medium for 48 h) were fixed with 70% cold ethanol (Tianjin, China) after washing with cold 1 × PBS (Gibco, USA). Then, the cells were incubated with the commercial PI/RNase staining solution (BD Pharmingen, USA) at 4 °C for 30 min and detected by using the FACS Canto II (BD Biosciences, USA).

Knockdown of CTEN gene expression was performed by siRNA-mediated gene silencing. Six hours after transfection cells were treated with 2 mM thymidine (Sigma-Aldrich) for 12 h. For allowing cells exit from S phase, the thymidine medium was replaced with complete growth medium. Next, cells were treated with 400 μM L-mimosine (Sigma-Aldrich) for 14 h. In order to release cells from chemical blockade, the L-mimosine medium was replaced with complete growth medium (time 0). At the indicated time, cells were suspended in chilled 70% ethanol and fixed at −20 °C overnight. For analyzing cell cycle phase distribution by DNA content, ethanol was removed and PI/RNase staining solution (BD Pharmingen, San Diego, CA, USA) was added to suspend cells. Cells were incubated at room temperature for 15 min and then analyzed by flow cytometry BD FACSCanto II.