Fig. 6.

Interactions between POLD2 and Alt-NHEJ factors in translocation formation. (A and B) Translocation frequency in CRITR assay (30) using 293T cells transfected with siCTRL or siRNA targeting POLD2 (siPOLD2 #1, D2), POLQ (Q), LIG3 or combinations, and 72 h later transfected with the same siRNAs and Cas9/gRNA. Cells were collected after 48 h for flow cytometry (A) and RT-qPCR for the indicated transcripts (B). Data are presented as mean ± SE of n = 3 independent experiments. (C) PLA with antibodies against POLθ and the indicated targets at 2 h following 10 Gy IR or mock treatment. Representative PLA panels and quantification of PLA foci. (D) PLA with antibodies against POLβ and the indicated targets as in C. Greater than or equal to 90 cells were imaged per condition per replicate and data are presented as mean ± SE of n ≥ 2 independent experiments. P values calculated using a one-way ANOVA with Tukey’s correction. *P < 0.05, **P < 0.01, ****P < 0.0001.