Figure 7.
Cells expressing phospho-deficient DHX9 are hypersensitive to DNA damage. (A) Cells expressing phospho-deficient DHX9 showed defects in cell survival. HeLa cells devoid of endogenous DHX9 by siRNA were transiently expressed with SFB-tagged DHX9 (WT, S321A, S321D and AA) or the corresponding empty vector. Viable cell numbers were counted and expressed as the ratio of cell survival relative to DHX9WT. Data are presented as the mean ± SD of three independent experiments. *** P < 0.001. (B) Cells expressing phospho-deficient DHX9 led to increased apoptotic cells. HeLa inducible cell lines were transfected with control or DHX9 siRNA, and the expression of SFB-DHX9 (WT, S321A and AA) was induced with doxycycline. Cells treated with DMSO or 1 μM cisplatin for 24 h were collected for Annexin V analysis using Flow cytometry. Data are presented as the mean ± SEM of three independent experiments. * P < 0.05; ** P < 0.01. (C, D) Ablating S321 phosphorylation sensitized cells to DNA damage agents. HeLa inducible cell lines were transfected with DHX9 siRNA to deplete endogenous DHX9. The expression of SFB-DHX9 (WT, S321A and S321D) was induced with doxycycline. The cell lines were incubated with CPT (C) or HU (D) at the indicated concentrations for 24 h, and the cell viability was determined using Cell TiterGlo 2.0. Values of cell viability are relative to vehicle-treated cells and presented as the mean ± SD. All statistical significance was determined by Student's t-test (two-sided). *** P < 0.001.