Facscalibur cytometry

Approximately 1 × 10⁵ cells were plated on six‐well dishes and treated as indicated. Floating and adherent cells were fixed in cold ethanol (4 °C; 70% in PBS) overnight. RNaseA (100 μg·mL⁻¹) was added to the cells at 37 °C for 30 min, followed by propidium iodide staining (50 μg·mL⁻¹; 30 min) in the dark and flow cytometric analysis using FACSCalibur cytometry (Becton Dickinson, Franklin Lakes, NJ, USA). Approximately 10,000 events were calculated for each sample and the distribution of cell cycle was analyzed using cell quest (Becton Dickinson).

Cells were seeded into 6-well plates at 5 × 10⁵ cells/well. After overnight incubation, they were treated with 8 μM DIM for 48 h and medium with same concentrations of DMSO was used as control. After incubation, cells were harvested, then double stained with Annexin V-FITC/PI using an apoptosis analysis kit (KeyGEN BioTECH, CHN), and subjected to flow cytometry analysis for detection of apoptosis. 10,000 cells per sample were analyzed by a BD FACSCalibur Cytometry (BD Biosciences) to quantify apoptotic cells (Annexin V-FITC positive cells).

MCF-7 cells were seeded into 6-well plates at 5 × 10⁵ cells/well. After overnight incubation, they were pretreated with 20 μM DIM for 2 h and then exposed to 10 Gy of γ-irradiation. Cells were harvested at 48 h after irradiation before being double stained with annexin V-FITC/PI using an apoptosis analysis kit (KeyGEN BioTECH, Nanjing, China) and subjected to flow cytometry analysis for detection of apoptosis. 10,000 cells per sample were analyzed by a BD FACSCalibur Cytometry (BD Biosciences, Franklin Lakes, New Jersey, USA) to quantify apoptotic cells (annexin V-FITC positive cells).

RA-FLS were transfected with the indicated oligos or plasmids in six-well plates. For cell cycle analysis, the cells were collected after 48 h of transfection. The cells were washed with PBS and then fixed with 70% ice-cold ethanol overnight at − 20 °C, washed with PBS, resuspended with 400 μl PBS, and then incubated with 100 μg/ml RNaseA (Kaiji, China) for 30 min at 37 °C and with 50 μg/ml propidium iodide (PI) (Kaiji, China) for another 30 min at 4 °C. After incubation, the cells were subjected to DNA content analysis using BD FACS Calibur cytometry and the results were analyzed with the ModFitLT software. For apoptosis analysis, the complete medium was changed to RPMI 1640 medium supplemented with 1% FBS to induce cell apoptosis, after transfection for 24 h. Cell apoptosis was evaluated by Annexin V-FITC and PI (BD Biosciences, 556547) staining according to the manufacturer’s protocol, followed by flow cytometry analysis. Briefly, cells were collected and washed with ice-cold PBS and resuspended in 100 μl binding buffer. Then, 5 μl of Annexin V-FITC and 5 μl of PI were added to the cells, incubated for 15 min at room temperature in the dark, and an additional 400 μl of binding buffer was added to the reaction prior to analysis. The results were analyzed with the Summit v4.3 software.

Cell cycle was performed by flow cytometry analysis. After collecting cells and rinsing with cold PBS, and a total of 1 × 10⁶ cells were fixed with 70% ice-cold ethanol for 24 h at 4 °C. Cells were resuspended with cold PBS followed by incubation with 50 μg/ml propidium iodide and 0.1 mg/ml RNase A at 37 °C for 15 min. The DNA content of RASFs was acquired with BD FACS Calibur cytometry and analyzed by ModFitLT software.

Cells were pretreated with DIM for 2 h and then irradiated with γ-irradiation. After incubation for 48 h after irradiation, cells were collected, washed in PBS, and fixed in ice-cold 70% ethanol. The cells were then recentrifuged and incubated with PBS containing 200 μg/mL RNase A and 5 μg/mL propidium iodide (PI), and cell cycle distribution was analyzed by BD FACSCalibur Cytometry. Data was analyzed with Cellquest software (BD Biosciences, Franklin Lakes, New Jersey, USA).