Mod t software

Cell Counting Kit-8 (CCK-8; Beyotime, Shanghai, China) was obtained from Beyotime and be used to detect the viability of two cell line according to the manufactories' protocol. FAC (100 μM), DFO ((100μM)) and PBS were added to different groups respectively. Then the 96-well plate was assessed at 450 nm 24 hours, 48 hours and 72 hours after incubation. For the cell cycle assay, a total of 1 x 10 6 cells nearly 80% con uent from 6-well plate were collected and xed in 70% ethanol for 24 hours at 4 ℃ like previously reported 27 . Cells were then washed in Phosphate Buffer Saline (PBS) and stained with propidium iodide (PI) before being analyzed by ow cytometry (BD Biosciences). G1, S and G2/M phases were calculated by Mod t Software (Verity Software House, Inc.).

TMEM176A unexpressed and stably expressed H23 and H1299 cells were starved for 12 hours to synchronize, and cells were re-stimulated with 10% FBS for 24 hours. Cells were xed with 70% ethanol and treated using the Cell Cycle Detection Kit (KeyGen Biotech, Nanjing, China). Cells were then detected using a FACS Caliber flow cytometer (BD Biosciences, CA, USA). The cell cycle was analyzed also for H727 cells with or without knocking down TMEM176A. Cell phase distribution was analyzed using the Mod t software (Verity Software House, ME, USA).
To increase the sensitivity of apoptosis detection, TMEM176A unexpressed and stably re-expressed H23 and H1299 cells were treated with Doxorubicin at 0.8µg/ml and 0.6µg/ml for 24 hours respectively (35) . Apoptosis was also analyzed in H727 cell with or without knockdown of TMEMA176. The cells were prepared using the FITC Annexin V Apoptosis Detection Kit I (BD Biosciences, Franklin Lakes, NJ, USA) following the manufacturer's instructions and then sorted by FACS Calibur (BD Biosciences, Franklin Lakes, NJ, USA). Each experiment was repeated three times.

Propidium iodide (PI) single staining tested cell cycle distribution: Cells were centrifuged at 1000 rpm, resuspended with 300 µL phosphate-buffered saline (PBS), xed with 70% ice-ethanol and centrifuged at 1000 rpm once again. Resuspended in 400 µL PBS, cells were added with 50 µL Rnase A (2.5 mg/mL) to attain 250 µg/mL and incubated for half an hour. Then, the cell suspension was added with 50 µL PI (1 mg/mL) to reach 100 µg/mL and reacted for 30 min without light exposure. Since that, the cellular DNA distribution was tested within 1 h by ow cytometry and the data were evaluated by Mod t software (Verity Software House, Maine, USA).
AnnexinV-uorescein isothiocyanate (FITC)/PI double staining tested cell apoptosis: Cells were successively treated with ethylene diamine tetraacetic acid-free trypsin, centrifuged at 2000 rpm twice and suspended in 500 µL Binding Buffer. Subsequently, cells were stained by 5 µL Annexin -FITC (BD Company, New Jersey, USA) and 5 µL PI, and detected on a ow cytometer (Beckman Coulter, CA, USA) within 1 h.