Figure 4.

Effect of NOX4 levels on viability and growth of A549 cells grown in suspension. (A) Mock or NOX4-overexpressing A549 cells were grown in attached or suspended conditions for 24 h. NOX4 mRNA levels were determined by RT–PCR using GAPDH as a loading control. Nox4 protein levels were determined by immunoblotting using the indicated antibodies. (B) Mock or NOX4-overexpressing A549 cells were transfected with non-specific siRNA (si-control) or with siRNA specific for NOX4 (si-NOX4) for 24 h, and the levels of NOX4 mRNA were determined by RT–PCR. Nox4 protein levels were determined by immunoblotting using the indicated antibodies. (C) Mock or NOX4-overexpressing A549 cells were transfected with si-control or si-NOX4 and were grown in attached or suspended conditions for 24 h. The cells were imaged using a Zeiss inverted microscope. (D–F) A549 cells were transfected with si-control or si-NOX4 and were grown in suspended conditions for 24 h. The cells were subjected to viability analysis (D), immunoblotting analysis (E), and cell counting at the indicated times (F). Scale bar: 100 μm. The data are presented as averages of triplicate measurements with the error bars representing standard deviations (*P<0.05). Similar results were observed in three independent experiments.