Figure 5.

Downregulation of cancer-derived IgG-lowered cellular ROS level. (a) HeLa cells were treated with IGHG1 siRNA or scrambled siRNA for 72 h. Cells were then incubated with fluorescent probe DHE at a final concentration of 10 μM in high-glucose DMEM without FBS for 20 min at 37 °C, washed three times with serum-free medium and analyzed using a flow cytometry. For each analysis, 10 000 events were recorded. Similarly treated cells were observed with a confocal microscope. The images are shown in the right panel. Scale bar, 50 μm. (b) Similar experiments were performed for HEp-2 and PC3 cells. (c) HeLa cells were transfected with IGHG1 siRNA or scrambled siRNA for 72 h. Cells were then incubated with fluorescent probe DCFH-DA at a final concentration of 10 μM in high-glucose DMEM without FBS for 20 min at 37 °C, washed three times with serum-free medium and analyzed with a flow cytometry. For each analysis, 10 000 events were recorded. Similarly treated cells were observed with a confocal microscope. The images are shown in the right panel. Scale bar, 50 μm. (d) Intracellular ROS (H₂O₂) level was monitored with flow cytometry with fluorescent probe DCFH-DA in HEp-2 and PC3 cells. HeLa cells were treated with IGHG1 siRNA or scrambled siRNA for 72 h. Total lysates (e) and culture medium (f) were used to detect cellular H₂O₂ level respectively. Similar experiments were performed using HeLa cells stably expressing IGHG1 shRNA or control shRNA (a, c, e, and f). Data shown are the mean±S.D. of three independent experiments (*P<0.05; **P<0.01)