Figure 4.

DNA damage assay at GABRA4 and NR2F1. A–F, GABRA4 and NR2F1 are lung cancer–associated DNA damageome proteins. A, Endogenous DNA damage assay scheme. B, GABRA4 and NR2F1 overproduction promotes DNA damage, respectively, quantified by H2AX levels using flow cytometry. DNA damage levels are compared and normalized to tubulin overproducing cells. Bar: median. n ≥ 7. Two-sample two-sided t test assuming equal variances. ^*, P = 0.0273; ^***, P = 0.0003. 3–5, gating strategy and representative flow cytometric density plots. C, mock transfection. D, Tubulin overproduction. E, NF2F1 overproduction. F, Histograms showing that NF2F1 overproduced cells increase high-DNA damage subpopulations compared with Tubulin in GFP+ cells. G–I, Benzo[a]pyrene (BaP) potentiates GABRA4-induced DNA damage. G, Endogenous and exogenous agent DNA damage assay scheme. H, BaP exposure sensitizes GABRA4-induced DNA damage. A total of 8 μmol/L BaP exposure for 72 hours induces additional DNA damage in GABRA4 overproduced but not tubulin overproduced cells. n ≥ 7. Two-sample two-sided t test assuming equal variances. ns, not significant; P = 0.7047; ^*, P = 0.0137; ^**, P = 0.0034. I, Model: GABRA4 overproduction increases endogenous DNA damage and then potentially overloads DNA repair pathways. The addition of an exogenous agent (BaP, e.g.) causes more DNA damage that cannot be repaired and lead to DNA damage catastrophe.