Extended Data Fig. 2: Loss of cGAS or STING in BT549 TNBC cells results in increased sensitivity to Mps1 or Wee1 inhibition.

a) Immunoblots of wild type and cGAS^KO BT549 cell lysates stained with anti-cGAS and anti-ß-actin antibodies. ß-Actin is used as a loading control. b) cGAMP levels quantified in cell lysates (intracellular) or in tissue culture media (extracellular) isolated from cGAS^KO BT549 cells incubated with DMSO, 250 nM reversine, or 500 nM AZD1775 for 48 hours. cGAMP levels were normalised to harvested cell numbers. c) Representative images of 250 nM reversine- or DMSO-treated BT549 cGAS^KO cells stained with DAPI (DNA) and an anti-STING antibody. Scale bar equals 5 µm. d) Quantification of cells with peri-nuclear or non-peri-nuclear STING localization. The number of quantified cells – n – is indicated in the figure. e) Immunoblots of cell lysates from wild type, cGAS^KO, STING^KO, and cGAS; STING^DKO BT549 cells treated with DMSO or 250 nM reversine stained with anti-p-IRF3 (Serine 396), anti-IRF3, anti-p-STAT1 (Tyrosine 701), anti-STAT1, anti-p-STAT3 (Tyrosine 705), anti-STAT3, or anti-ß-Actin. ß-Actin is used as a loading control. f) DMSO-normalised cell count of wild type or cGAS^KO BT549 cells treated with 500 nM AZD1775 for 48 hours. g) Percentage of apoptotic cells quantified by Annexin V staining of wild type or cGAS^KO BT549 cells treated with 500 nM AZD1775 for 48 hours. h) DMSO-normalised cell count of wild type BT549 cells treated with a combination of 2.5 μM RU.521 and 250 nM reversine for 72 hours. i) DMSO-normalised cell count of wild type BT549 cells treated with a combination of 2.5 μM RU.521 and 500 nM AZD1775 for 48 hours. j) Quantification of mitotic abnormalities observed in cGAS^KO BT549 cells expressing Histone H2B-mCherry treated with DMSO, 250 nM reversine or 500 nM AZD1775 assessed by live cell imaging, the number of assessed mitotic cells – n –is indicated in the figure. k) Immunoblots of wild type, STING^KO, or cGAS; STING^DKO BT549 cell lysates stained using anti-STING, anti-cGAS and anti-β Actin antibodies. ß-Actin is used as a loading control. l) Control DMSO-normalised cell counts of wild type, cGAS^KO, STING^KO, or cGAS; STING^DKO BT549 cells treated with 500 nM AZD1775 for 48 hours. Error bars in (b, f–i, l) represent the s.e.m, n=3 biological replicates (b, g–i, l), n=6 biological replicates (f), significance was tested with a two-sided t-test.

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