Extended Data Fig. 1: Repair of spontaneous breaks by TMEJ in HR-deficient cells is delayed until mitosis.
From: POLθ-mediated end joining is restricted by RAD52 and BRCA2 until the onset of mitosis
a, Spontaneous γH2AX foci in G1 and G2 HeLa cells. Cells were transfected with siRNAs, labelled with EdU for 1 h and foci were immediately analysed after labelling in EdU− cells (microscopic dot blot on the left indicates the evaluated cell populations) (n = 4 independent experiments, except siRAD51 + siPOLθ: n = 3 independent experiments). b, Spontaneous γH2AX foci in HeLa cells treated with a second POLθ siRNA (siPOLθ_6). (i) Cells were labelled as in Fig. 1a and EdU−BrdU+ cells were analysed (n = 3 independent experiments, except siCTRL/siBRCA2 at 12 h: n = 4 independent experiments). (ii) Cells were labelled and analysed as in (a) (n = 3 independent experiments, except siCTRL/siBRCA2/siRAD51: n = 4 independent experiments). The data without siPOLθ_6 in (i) are also part of Fig. 2a,b whilst the data without siPOLθ_6 in (ii) are also shown in panel (a) of the present figure. c, Spontaneous γH2AX foci in fibroblasts. The same analysis as in (b) was performed with 82-6 hTERT (WT) cells (for (i): n = 4 independent experiments, for (ii): n = 3 independent experiments). d, Spontaneous chromatid breaks in G2 and mitotic HeLa cells. Cells were transfected with siRNAs and analysed by premature chromosome condensation (PCC) for G2 cells or a 2-h colcemid treatment for mitotic cells. Chromatid breaks were quantified per spread and normalized to 70 chromosomes (n = 3 independent experiments). e, Lagging chromosomes in ana/telophase HeLa cells. Cells were transfected with siRNAs and ana/telophases were identified by their morphology seen in the DAPI and phospho-histone H3(S10) (pH3) stainings (n = 4 independent experiments). f, Micronuclei in G1 HeLa cells. Cells were transfected with siRNAs, labelled as in Fig. 1a and EdU−BrdU+ cells were analysed in G1 (n = 4 independent experiments). All data show the mean ± s.e.m. Individual experiments, each derived from 40 (S/G2 foci, G2 spreads and lagging chromosomes), 60 (M spreads), 100 cells (G1 foci) or 200 cells (micronuclei), are shown as dots. *: P < 0.05, **: P < 0.01; ***: P < 0.001, ns: non-significant (One-way ANOVA). The exact P values are provided as source data. Source data are available online.
