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. Author manuscript; available in PMC: 2020 Jun 1.
Published in final edited form as: Cancer Discov. 2019 Apr 23;9(6):722–737. doi: 10.1158/2159-8290.CD-18-1218

Figure 2. Olaparib activates the cGAS/STING pathway in cells derived from the K14-Cre-Brca1f/fTp53f/f GEMM.

Figure 2.

The murine isogenic cell line pair KB1P-G3−/+BRCA1 derived from the GEMM was treated with DMSO (0 μM Olaparib) or the indicated doses of olaparib. (A) At 72h post-treatment, cells were stained with picogreen, fixed and subjected to immunofluorescence for cGAS. (Left panel). Representative images of picogreen- (green) and cGAS (red)-stained micronuclei are shown (60x magnification). Scale bar, 8 μm. (Right panel). The number of cGAS-colocalized micronuclei was quantified and expressed as % of total nuclei. Error bars represent standard error of the mean (SEM) of 2 independent experiments. Statistical analysis was performed using two-way ANOVA with Tukey’s post-hoc test. (B) At 72h post-treatment, cells were subjected to immunoblotting for phosphorylated (pTBK1Ser172) and total TBK1 expression with vinculin as a loading control. Markers indicate molecular weight (MW) (kDa). Numbers below the blots represent normalized pTBK1 protein levels quantified by densitometric analysis. (C) At 24h post-treatment, cells were fixed and subjected to immunofluorescence for γ-H2AX (pH2AX Ser139) and pIRF3Ser396. (Left panel) Representative images of DAPI- (blue), γ-H2AX- (green) and pIRF3 (red)- stained cells are shown (20x magnification); scale bar, 8 μm. (Right panels). pIRF3 corrected integrated density/cell was expressed as fold change versus KB1P-G3 DMSO. Statistical analysis was performed using Kruskal-Wallis test with Dunn’s post-hoc test. Error bars represent SEM of 3 independent experiments. The number of cells displaying >5 γ-H2AX foci was quantified, and statistical analysis was performed using two-way ANOVA with Tukey’s post-hoc test. (D) At 72h post-treatment, RNA was extracted and qPCR was performed. IFNβ, CCL5 and CXCL10 mRNA levels were normalized to GAPDH internal control. Error bars represent SEM of 3–4 independent experiments. Statistical analysis was performed using Kruskal-Wallis test with Dunn’s post-hoc test.