Fig. 4: Sphingolipids affects DSBs mobility, DSB misrepair and survival after PARP inhibition in BRCA1-deficient MEFs. | Nature Communications

Fig. 4: Sphingolipids affects DSBs mobility, DSB misrepair and survival after PARP inhibition in BRCA1-deficient MEFs.

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

Fig. 4

a Immunoblot for BRCA1, LMNA/C and SPT1 in P53-deleted Brca1F/F MEFs transduced with the empty vector control (vec) or an shRNA against (mRNA) Spt1 (shSpt1) before or 72 h after Hit&Run Cre-mediated deletion of Brca1. b IF for Lamin-B1 of representative nuclei of Brca1F/F MEFs transduced with vec or shSpt1. c Quantification of nuclear deformations blebs or invagination/deformations as shown in (b). Data are from 150 nuclei from n = 3 independent experiments (50 cells/experiment) with mean ± SEM. Statistical analysis by 2way Anova for multiple comparison: (***) P < 0.001 (0.0005 for EV and 0.0003 for shSpt1); P ≧ 0.05 is not significant. d Representative 10 min traces of mCherry-BP1-2 foci in the MEFs transduced with the empty vector or shSpt1 72 h after Cre-mediated deletion of Brca1 and 6 h treatment with PARPi. e MSD of mCherry-BP1-2 foci as shown in (d) for n = 3 independent experiments, with SD (total foci analyzed: 763 from 24 nuclei for vec and 1201 foci from 32 nuclei for shSpt1). f Representative metaphases showing aberrant mis-rejoined chromosomes in MEFs with or without shSpt1. DNA is visualized with DAPI. Arrows show chromosomes in aberrant structures. g Quantification of mis-rejoined chromosomes as shown in (f). Each dot represents a metaphase. Bars shows the median for n = 3 independent experiment, with 10 metaphases each (total = 30 metaphases per condition). Statistical analysis by ordinary one-way ANOVA for multiple comparisons: (***) P < 0.001 (0.0002); (****) P < 0.0001; P ≧ 0.05 is not significant. h Representative survival assay of Brca1F/F MEFs transduced with vec or shSPT1, before or after Cre-mediated deletion of Brca1. PARPi was added for 24 h before wash. Colonies are stained with Methylene blue a week later. i Quantification of colony formation as shown in (h), normalized on MEFs growing without PARPi before Brca1 deletion. Data represents average and SEM for n = 3 independent experiments. All scale bars, 10 μm. Source data are provided as a Source Data file. See also Supplementary Fig. 4.

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