Figure 5.

NAC partially reverses the SPR knockdown induced apoptosis and proliferation inhibition. (A) The cells were transfected with siSPR#3 for 4 h and replaced fresh DMEM containing 10% FBS for 24 h, then 5 mM NAC was added and cultured for another 24 h. The cells were harvested, and the intracellular ROS level was measured using a ROS assay kit. (B) The cells were treated as mentioned above in (A), then the cells were stained with PI/Annexin V, and the apoptotic cells were analyzed using flow cytometry. (C) The cells were treated as mentioned above in (A), then the cells were harvested and the caspase-3, cleaved-caspase-3, caspase-9, cleaved-caspase-9, PARP, and cleaved-PARP expressions were analyzed using western blot. (D) The cells were transfected with siSPR#3 for 4 h and replaced with fresh DMEM containing 10% FBS for 48 h to 80% confluence, then 1000 cells were seeded into 6-well plates, and the medium was replaced with fresh medium with or without 5 mM NAC every 2 days for 14 days. Then the cells were stained with crystal violet solution. (E) The cells were treated as mentioned above in (D) and the number of cells was counted every day for 3 days. The data are represented as the mean ± SD in triplicate. **P < 0.01; ***P < 0.001; ****P < 0.0001.