Extended Data Fig. 6: Stalled replication fork degradation due to FANCD2- but not BRCA2-deficiency is linked with induction of innate immune response.
From: Stalled replication fork protection limits cGAS–STING and P-body-dependent innate immune signalling
(a) pSTAT1 levels in BRCA2 knockdown cells at different times after treatment with HU (4 mM). (b) Knockdown of FANCD2 but not BRCA2 leads to cytosolic ssDNA accumulation by immunofluorescence staining with ssDNA antibody. Scale bars, 10 μm. (c) BRCA2 knockdown does not lead to HU-induced cytosolic ssDNA accumulation by quantification of Mean fluorescence intensity (MFI) with mean ± SD. One way Anova was used for statistics. (d) Picogreen staining of BRCA2 knockdown does not show HU-induced cytosolic DNA accumulation. Scale bars, 10 μm. Data shown represent three independent experiments in a, b, d, e. (e) FANCD2 knockdown leads to increased pSTAT1 in MEFs. (f) BRCA2 knockdown does not induce an increase of P-bodies. Number of P-bodies per cell was quantified and shown with mean ± SD. One way Anova was used for statistics. (g) Mirin restores fork protection in FANCD2 depleted cells. FANCD2-deficient PD20 cells expressing empty vector or FANCD2 gene were used and treated as illustrated. IdU/CIdU ratio was quantified with mean ± SD. One-way Anova was used for statistics. (h) Knockdown of DNA2 decreases P-bodies number in FANCD2-deficient cells. Number of P-bodies per cell was quantified and shown with mean ± SD. One-way Anova was used for statistics. (i) Cytosolic rDNA fragment accumulation in FANCD2-deficient cells detected by FISH with biotin-labelled rDNA 18S probe. Scale bars, 10 μm. Mean fluorescence intensity was quantified with mean ± SD. One-way Anova was used for statistics. ‘n’ refers the number of cells analysed across three independent experiments in c, f-i.
