Figure 3.

Association of ROS generation with anoikis resistance in suspended A549 cells. (A) A549 cells were grown in attached or suspended conditions for 24 h. For inhibitor treatments, A549 cells grown in suspension were treated with either 30 μM of NAC, a ROS scavenger, or 5 μM of DPI, a pan-NOX inhibitor. The ROS levels were measured by flow cytometry using a ROS-sensitive dye, CM-H₂DCFDA. (B) A549 cells grown in suspension were treated with the NOX inhibitors DPI or apocynin or 24 h, and cell viability was measured using an MTS assay. (C) Total cell lysates from A549 cells treated as in B were subjected to immunoblotting analysis using the indicated antibodies. (D–G) A549 cells grown in suspension were treated with plumbagin for 24 h prior to capturing images of the cell aggregates with a Zeiss inverted microscope (D), measuring ROS levels by flow cytometry (E), analyzing the levels of cell death by staining with green-fluorescent Calcein AM and red-fluorescent ethidium homodimer-1 (green=live cells; red=dead cells) (F), and performing immunoblotting analysis using indicated antibodies (G). The ratio of EGFR or phosphorylated EGFR to actin in the control cells was set at 1. Scale bar: 100 μm. The data are presented as averages of triplicate measurements with the error bars representing standard deviations (*P<0.05). These experiments were performed three independent times with comparable results.