Fig. 6: Inhibiting the ATR pathway re-sensitises JEG3R cells to MTX.

A JEG3 and JEG3R cells were subjected to a kinome-wide siRNA screen in the presence and absence of MTX used at the IC₅₀ of the corresponding cell line. Changes in cell survival were monitored by Crystal Violet staining. Table: LFC; Log2 fold changes in cell number in response to MTX following silencing of the indicated target as compared to non-targeted siRNA-transfected cells, SI sensitivity index to MTX calculated as in [87], TI toxicity index as fold changes in cell number following silencing of the indicated target in the absence of MTX. B JEG3 and JEG3R cells and their corresponding ATR CRISPR clones (#1 and 2) were treated with a dose range of MTX for 72 h before being subjected to Crystal Violet staining. C–F JEG3 or JEG3R cells were treated with the indicated concentrations of ATR (VX-970), CHK1 (LY-2603618) or WEE1 (MK-1775) inhibitors (shown as ATRi, CHKi and WEEi, respectively). C–E Cell lysates were subjected to Western blotting for the indicated targets. Detection of Vinculin was used as a loading control. Results representative of three biological replicates. F Cells were additionally treated with/without IC₅₀ of MTX. Cell survival changes were revealed using Crystal Violet staining. Data are fold changes of mean ± SEM of representative experiments from biological triplicates with n = 4. G ZIP analysis for synergistic interaction between the indicated inhibitors and MTX. Upper panels; Dose-Response matrices. Lower panel, ZIP score contour line plots. Tables show the average and maximum synergy scores. Statistics: (F) Student t-test. ***p < 0.005, **p < 0.01, *p < 0.05, ns not significant.