Figure 2.

TonEBP is recruited to DNA damage sites and induces m6A RNA methylation. (A) U2OS cells were subjected to laser microirradiation followed by double staining for TonEBP and γH2AX. Representative images are shown (scale bar: 2 μm). (B) Top: cells were transfected with plasmids expressing GFP-tagged Yc1 (upper panel) or ΔRHD (lower panel). After 24 h, cells were laser microirradiated at the positions marked by a red dashed line (left). Green fluorescence images were taken 0–60 s after irradiation. Bottom: the fluorescence intensity in the microirradiated area at each time point was determined from 20 cells. Mean ± SD, P < 0.01 from 10 to 60 s. (C) Immunoblot analysis of the knockdown efficiency of siRNA in U2OS cells. Heat shock cognate 71 kDa protein (HSC70) was used for loading control. (D) Cells were transfected with scrambled siRNA (scr) or TonEBP-targeting siRNA (TonEBP) for 24 h and then subjected to laser microirradiation. After 2 min, immunostaining was performed for m6A and γH2AX. Left: representative images of a nucleus in each condition. Right: the laser stripe intensity was determined for 40 cells in each condition. Mean ± SD, *P < 0.01. (E) siRNA-transfected cells were subjected to laser microirradiation followed by incubation for 2 min and immunostained for Pol κ and γH2AX. Left: representative images. Right: percentage of Pol κ laser stripe-positive cells from 10 cells. Mean ± SD, n = 4. *P < 0.01. (F) Cells were immunostained for full-length TonEBP and METTL3; a representative set of images are shown. (G) Cells were transfected with two plasmids expressing GFP-TonEBP_Yc1 and mCherry-METTL3 for 24 h. Green and red fluorescence images were obtained 2 min after laser microirradiation. (H) siRNA-transfected cells were transfected a second time with a plasmid expressing GFP-METTL3, and green fluorescence images were taken 2 min after laser microirradiation. (I) siRNA-transfected cells were transfected a second time with a plasmid expressing GFP-Yc1, and green fluorescence images were taken after laser microirradiation. (J) Cells were transfected with various combinations of scrambled-siRNA (−), TonEBP-targeting siRNA, and METTL3-targeting siRNA for 24 h as indicated. Cells were subjected to 0–100 J/m² of UV irradiation, and the cell survival percentage after 24 h was measured. Mean ± SD, n = 3; *P < 0.01; NS, P > 0.05. (K) Cells were transfected as (J) and treated with 0–10 μM CPT for 24 h, and then the cell survival percentage after 24 h was measured.