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Fig. 8 | BMC Cancer

Fig. 8

From: Linking hypoxia, DNA damage and proliferation in multicellular tumor spheroids

Fig. 8

1% O2 is sufficient to induce DNA damage in A673 monolayers and inhibition of ATR promotes H2AX phosphorylation. a The effect of maintaining monolayers of A673 cells in 1% O2 for 12 h was monitored. Immunofluorescence indicates the formation of distinct γ-H2AX foci (red) in A673 cells. b Western blot shows elevation of γ-H2AX levels in A673 cells maintained in 1% O2 for 12 h. c Alkaline COMET assay shows induction of COMET tails (a direct measure of DNA damage) when A673 cells were maintained in 1% O2 for 12 h. Tail moments were quantitated using OpenCOMET [43] and are plotted as the mean and standard deviation. d Bar graph showing the percentage of γ-H2AX-positive A673 cells when monolayers were maintained for 12 h in either 20% O2 or 1% O2 in vehicle, 1% O2 in KU55933 (ATM inhibitor), or 1% O2 in VE-821 (ATR inhibitor). Mean and standard deviation from two independent experiments are shown. e Immunofluorescence for γ-H2AX (red) in A673 monolayers maintained for 12 h in either 20% O2 or 1% O2 in vehicle, 1% O2 in KU55933 (ATM inhibitor), or 1% O2 in VE-821 (ATR inhibitor). ATR inhibition under hypoxia results in cells with either intense pan-nuclear γ-H2AX or distinct foci, while only distinct γ-H2AX foci are seen under hypoxia in the presence of either vehicle or ATM inhibitor. One way ANOVA with Tukey’s multi-comparison post-test. ***P < .001, **P < .01, *P < .05, ns P > .05

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