Fig. 4: PARPi induced DNA replication stress in GRB2 depleted cells cause genomic instability, cytoplasmic DNA accumulation resulting in cGAS/STING activation and chemoattractant secretions. | Nature Communications

Fig. 4: PARPi induced DNA replication stress in GRB2 depleted cells cause genomic instability, cytoplasmic DNA accumulation resulting in cGAS/STING activation and chemoattractant secretions.

From: GRB2 stabilizes RAD51 at reversed replication forks suppressing genomic instability and innate immunity against cancer

Fig. 4

a Representative PicoGreen and DAPI staining after 10 μM Olaparib treatment for 48 h and the resulting cytoplasmic micronuclei formation in HAP-1 cells. Scale bar 10 μm (b) Quantitation of cytoplasmic micronuclei form three independent experiments with ±SD. c A comparison and time-course of Olaparib (10 μM) induced TBK-1 and IRF-3 phosphorylation between control and GRB2-KO HeLa cells. d GRB2 re-expression in GRB2-KO HeLa cells suppressed Olaparib (10 μM, 48 h) induced TBK-1 and IRF-3 phosphorylation. e Representative immunofluorescence images showing olaparib (10 μM, 48 h) treatment induces nuclear accumulation of phosphorylated IRF-3 (pIRF-3) in GRB2-KO HeLa cells. Scale bar 10 μm. f The increased TBK-1 and IRF-3 phosphorylation observed in GRB2-KO HeLa cells are independent of RAS (PD184352; left panels) or Akt (MK2206; right panels) signaling. g The increased TBK-1 and IRF-3 phosphorylation in GRB2-KO HeLa cells are MRE11 dependent, n = 3. h STING knockdown is sufficient to abrogate Olaparib (10 μM) induced TBK-1 and IRF-3 phosphorylation in GRB2-KO HeLa cells. n = 3. i The increased TBK-1 and IRF-3 phosphorylation in GRB2-KO HeLa cells are STING dependent, n = 3. i–k qRT-PCR showing Olaparib induce increased level of INF-B, CCL5 and CXCL-10 mRNA in HeLa cells with ±SD, n = 3. l Quantitation of cytokines array results from two independent experiments showing upregulated inflammatory cytokine released in culture medium with olaparib treatment, n = 2. m Recruitment of cytotoxic CD8 + T-lymphocytes (CTL) isolated from PBMC to GRB2-KO HeLa cells in response to Olaparib treatment, n = 3, ±SD are shown. n siRNA mediated STING knockdown in GRB2-KO HeLa cells abrogate PBMC isolated CD8 + CTL recruitment, n = 3, errors are in ±SD. The significance was analyzed by two-sided Student’s t test. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001; NS not significant. For (b–n), source data are provided as a Source Data file.

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