Fig. 2: Lamin-A but not Lamin-B1 suppresses DSBs mobility in BRCA1-deficient cells treated with PARPi. | Nature Communications

Fig. 2: Lamin-A but not Lamin-B1 suppresses DSBs mobility in BRCA1-deficient cells treated with PARPi.

From: Nuclear deformability increases PARPi sensitivity in BRCA1-deficient cells by increasing microtubule-dependent DNA break mobility

Fig. 2

a Representative images of Immunofluorescence (IF) with anti- Lamin-A/C or Lamin-B1 antibodies of p53-deleted Brca1F/F MEFs. Cells were fixed before immunostaining. Arrowheads indicate nuclear invaginations. Scale bar: 10 μm. b Immunoblot for 53BP1, BRCA1 and Lamin-A (LMNA) in p53-deleted Brca1F/F Lmna+/+ or Brca1F/F Lmna−/− litter mates MEFs without any treatment or 72 h after Brca1 deletion with Hit&Run Cre, as indicated. Ponceau S staining is shown as loading control. Immunoblot for 53BP1 was processed in parallel using the same samples. c Immunoblot for 53BP1, BRCA1, Lamin-B1 (LMNB1) in p53-deleted Brca1F/F MEFs transduced with the empty vector control (vec) or two independent shRNAs against (mRNA) LmnB1 (B1) before and 72 h after Cre-mediated deletion of Brca1. Immunoblot for actin is shown as loading control. Immunoblots for 53BP1 and actin were processed in parallel using the same samples as the other two blots. d Representative IF images with anti-Lamin-B1 antibodies of Brca1F/F Lmna+/+ or Brca1F/F Lmna−/− MEFs, or with anti-Lamin-A antibodies of Brca1F/F MEFs transduced with vec or the shRNA against LmnB1 as in described in (b, c). Arrowheads indicate nuclear invaginations or blebs, respectively. e, f Quantification of normal nuclei and nuclei with blebs or invaginations/deformations in MEFs deleted of Lmna (e) or depleted of Lamin-B1 (f) as indicated and shown in (d) for n = 3 independent experiments with mean ± SEM. For each experiment, 50 cells were analyzed for condition. g Examples of 10 min traces of mCherry-BP1-2 foci in Brca1F/F Lmna+/+ or Brca1F/F Lmna−/− MEFs 72 h after Brca1 deletion, 6 h after PARPi addition in the absence or presence of taxol. Scale bar: 1 μm. h, i MSD of mCherry-BP1-2 foci in the indicated MEFs as described in (g), with SD. Total foci analyzed in (h): 1442 for Lmna+/+, 1487 for Lmna+/+ + taxol, 711 for Lmna−/− and 1374 for Lmna−/− + taxol from 42, 37, 21, 37 nuclei, respectively, from n = 3 independent experiments. Total foci analyzed in (i): 998 for vec, 1250 for shB1.1, 959 for shB1.2, from 30, 36, and 27 nuclei, respectively, from n = 3 independent experiments. Statistical analysis by two-way Anova for multiple comparison for (e, f). (****) p < 0.0001, P ≧ 0.05 is not significant. Source data are provided as a Source Data file. See also Supplementary Figs. 2 and 3.

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