Figure 5.

IREB2 knockdown and AQP3 overproduction promote endogenous DNA damage. (a) Knocking down IREB2, but not NEXN by siRNA causes increased γH2AX level in MRC-5V2 cell line. Upper: scheme for a siRNA DNA damage assay. Lower left: representative flow cytometric histogram showing increased γH2AX in cells with IREB2 knockdown compared to cells that were transfected with nontargeting siRNA. Lower right, a summary of at least three independent experiments for both IREB2 and NEXN, n > 20. Mean fluorescence intensity of each knockdown experiment was normalized to its corresponding nontargeting siRNA control. Error bar: SEM. (b) AQP3 overproduction increases endogenous γH2AX levels in MRC-5V2. Upper: full-length sequence-verified AQP3 fused with N-terminal GFP fusion (and GFP-Tubulin as a control) were transiently overproduced in MRC-5V2 cell line and the DNA damage levels for both nongreen and green cells were quantified by flow cytometry (details see Methods). Lower left: representative flow cytometric histograms of GFP-AQP3, GFP-Tubulin, and mock transfection. Lower right: summary, mean ± SEM, n ≥ 8.