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. Author manuscript; available in PMC: 2023 Jul 11.
Published in final edited form as: Nat Cell Biol. 2022 Jul 11;24(7):1154–1164. doi: 10.1038/s41556-022-00950-8

Figure 5. Stalled replication fork degradation due to FANCD2-deficiency depends on DNA2 and is linked with induction of innate immune response.

Figure 5.

(a) FANCD2 knockdown leads to cytosolic ssDNA accumulation. Mean fluorescence intensity (MFI) of cytosolic ssDNA per cell was quantified and shown with mean ± SD.

(b) FANCD2 knockdown leads to increased pSTAT1. U2OS cells were treated with control or FANCD2 siRNAs before treatment with 4 mM HU.

(c) FANCD2 knockdown leads to increased number of P bodies in response to HU. The number of P-bodies per cell was quantified with mean ± SD.

(d) Increased pSTAT1 and pTBK1 level in FANCD2 knockdown cells is dependent on cGAS. Cells were treated HU (4 mM).

(e) FANCD2 deficiency in replication fork end protection is rescued by DNA2 knockdown. FANCD2-deficient PD20 cells expressing empty vector or FANCD2 gene were used and pulselabeled with CIdU followed by IdU and treated with HU as illustrated. IdU/CIdU ratio was quantified with mean ± SD.

(f) Knockdown of DNA2 or treatment with mirin reduces pSTAT1 or pTBK1 level in FANCD2 depleted cells. Cells were transfected with indicated siRNAs before treatment with HU.

(g) Innate immune genes IL6 and CXCL10 expression is induced in FANCD2-deficient cells and the induction is reduced by DNA2 depletion or mirin treatment. Cells were untreated or treated with HU (4 mM, 16 h). Levels of IL6 and CXCL10 mRNAs were detected by real-time qPCR and relative fold change was quantified (n=2 independent experiments of mean from triplicates of each experiment).

(h) Inhibition of DNA2 or MRE11 decreases cytosolic ssDNA in FANCD2-deficient cells. Cells were untreated or treated with HU (4 mM, 4h). Immunofluorescence was carried out with ssDNA antibody. Mean fluorescence intensity (MFI) of cytosolic ssDNA per cell was quantified mean ± SD.

(i) Cytosolic rDNA fragment accumulation in FANCD2-deficient cells detected by FISH with biotin-labelled rDNA 28S probe. Scale bars, 10 μm. Cytosolic FISH intensity (MFI, mean fluorescence intensity) was quantified with mean ± SD.

One-way Anova was used for statistics in a-i. ****p<0.0001