Figure 3.

Synemin functions in non-homologous end joining. GFP-based reporter assays for (A) HR and (B) NHEJ. Cal33 cells stably transfected with DRGFP or pimEJ5GFP recombinant plasmids were depleted of synemin. The number of GFP-positive cells was analyzed by FACS, provided in Supplementary Figure S5A; (C) NHEJ activity in mCherry–Synemin-overexpressing Cal33-pEJ5GFP cells. Analysis performed by FACS as indicated under (A,B); (D,E) Immunoblots and fold changes from synemin-depleted and 6-Gy irradiated SAS cells showing total and/or phosphorylated forms of DNA-PKcs, ATM. β-actin served as loading control; (F,G) Immunoblot and fold change of DNA-PKcs from whole cell lysates of 6-Gy X-ray irradiated and mock-treated SAS mCherry–Synemin transfectants. β-actin served as loading control; (H–J) Kinetics of DNA-PKcs S2056, 53BP1 and γH2AX foci upon synemin knockdown at different time-points post 1-Gy X-rays in SAS cells; (K) Representative immunofluorescence images of residual DNA-PKcs S2056 foci of synemin knockdown and control cell cultures 1 h after 1-Gy X-rays (bar, 10 µm). Data are presented as mean ± SD (n = 3; two-sided t-test; ** p < 0.01, *** p < 0.001; n.s., not significant (p ≥ 0.05)).