Figure 2.

Y14 Depletion Causes DNA Damage, Induces R-Loops, and Perturbs DDR

(A) The comet assay of siRNA-transfected HeLa cells. Immunofluorescence shows SYBR Green staining of DNA. (A, C, and E) Immunoblotting shows Y14 and α-tubulin.

(B) siRNA-transfected HeLa cells as in (A) were either not treated (−) or treated with 2 Gy IR followed by 16-h recovery. Box and whiskers plot shows the comet tail moment; mean ± SD were from ∼300 cells in three independent experiments.

(C) Immunoblotting of siC- or siY14-transfected HeLa cell lysates was performed using antibodies against the indicated proteins.

(D) siRNA-transfected HeLa cells were treated with 10 Gy IR followed by recovery for 30 min. Immunofluorescence was performed using anti-53BP1 and anti-Y14. Arrowheads, Y14-depleted cells. Scale bar, 10 μm. The number (mean ± SD) of 53BP1 foci per cell is shown in the bar graph; ∼200 cells were counted in three independent experiments.

(E) siRNA-transfected HeLa cells were subjected to immunofluorescence using antibody S9.6; the images were merged with DAPI staining (scale bar, 10 μm). Box and whiskers plot shows quantification of nuclear (left) and mock or actinomycin D-treated nucleolus-excluding nucleoplasmic (right) S9.6 signals. For each transfection, ∼100 cells were scored from three independent experiments.