Annexin 5 fitc pi apoptosis and cell cycle kit

In cell cycle assay, A total of 1 × 10⁶ cells were seeded in each six-well plate and further incubated for 12 hrs to reach 50% confluent. Then the cells were transfected with 3xflag-FEN1-WT or 3xflag-FEN1 mutants by using TG-transfection (Gene Technologies). Cells were treated with 1 mM HU ((Hydroxyurea) or ddH₂O 24 hrs later and harvested after 12 hrs. For UV treatment, cells were exposed to UV at 120 J/m² and collected after 2 hrs post-incubation. Cell cycle assay and apoptosis assays were conducted using Annexin V FITC/PI apoptosis and cell cycle kit (MultiSciences70-APCC101-100), samples were run on Beckman CoulterFC500 MPL and the cell cycle data were analyzed using MultiCycle AV DNA Analysis software.

HCFs were collected after treatments and fixed in 75% ethyl alcohol on condition of −20°C overnight. After three times of washing and centrifuging (in 600 rpm, 5 min), fixed cells were stained by the Annexin V FITC/PI apoptosis and cell cycle kit (Multi Science, China) for 30 min, then, HCFs were analyzed by a flow cytometer (BD Biosciences, United States). Images were processed by FLOWJO software (BD Biosciences, United States), and the percentage of G0/G1, S, and G2/M were calculated to analyze cell cycle.