Fig. 2

A 5FU resistant cells stably expressing MINK1sgRNA (#1 and #2) and NTsgRNA were treated with 5FU for 48 h and cell viability was determined by MTT assay (n = 3 and 2-way ANOVA, ****P < 0.0001). B Indicated cells were treated with 5FU for 48 h, after which cell death was determined by annexin V/7AAD assay using flow cytometer. Bar diagrams indicate the percentage of cell death (early and late apoptotic) with respective treated groups (Mean ± SEM, n = 3, Two-way ANOVA, ****P < 0.0001). C Left panel: Indicated cells were treated with 10 μM of 5FU for 48 h, after which immunostaining was performed for γ-H2AX. Right panel: The number of foci from panel C are indicated as bar diagram. Two-way ANOVA, ****P < 0.0001. D Indicated cells were treated with 5FU for 48 h and immunoblotting (n = 3) was performed with indicated antibodies. E PDC2 MINK1WT cells were implanted in right upper flank of athymic male nude mice and PDC2 MINK1KO cells were implanted in left upper flank, after which they were treated with 5FU at indicated concentration. At the end of the experiment mice were euthanized, tumors were isolated and photographed (n = 5). F Tumor growth was measured in indicated time points using digital slide caliper and plotted as a graph (mean ± SEM, n = 5, Two-way ANOVA, *P < 0.05). G Bar diagram indicates the tumor weight measured at the end of the experiment (mean ± SEM, n = 5, Two-way ANOVA, ****P < 0.0001). H After completion of treatment, tumors were isolated and paraffin-embedded sections were prepared as described in materials and methods to perform immunohistochemistry with indicated antibodies. Scale bars: 50 μm. I Lateral view of fluorescent transgenic [Tg(fli1:EGFP)] zebrafish embryos at Day 0 and Day 5 injected with Dil-Red stained PDC2 control and MINK1 KO cells with and without treatment of 5FU. J The tumor growth and migration were assessed by an increase in fluorescence intensity on the 5th day compared to the day of injection. The quantitation of fluorescence intensity (I) was performed using ImageJ software and represented as fold change of fluorescence intensity where day 0 reading was taken as baseline (mean ± SEM, n = 6, Two-way ANOVA, ****P < 0.0001). K Zoomed image of distal part of embryo (5days post injection) to monitor migration of tumor cells.