Fig. 5

A In vitro MINK1 kinase assay was performed using three compounds potentially binding to MINK1 (based on IUPHAR database). All compounds (10 µM) were incubated with recombinant human MINK1 along with substrate MBP and ATP and further subjected to ADP-Glo™ Kinase Assay as described in materials and methods section (n = 3), one-way ANOVA, *P < 0.05. B Determination of EC50 value for kinase activity of top two MINK1 inhibitors selected from panel (A) (n = 3), one-way ANOVA, ****P < 0.0001. C, D The indicated cells were treated with indicated concentration of MINK1 inhibitors and cell viability was determined by MTT assay. Selection of highest dose of Lestaurtinib and Pexmetinib that does not affect cell viability when treated alone (viability > 80%) in 5FU resistant OSCC lines (n = 3). E 5FU resistant cells were treated with indicated doses of MINK1 inhibitors (50 nM Lestaurtinib, 500 nM Pexmetinib) in combination with increasing concentrations of 5FU for 48 h, after which cell viability was determined by MTT assay (n = 3), 2-way ANOVA, ****P < 0.0001. F 5FU resistant OSCC lines and PDC2 cells were treated with indicated doses of MINK1 inhibitors (50 nM Lestaurtinib, 500 nM Pexmetinib) in combination with increasing concentrations of 5FU for 48 h, after which cell death (early and late apoptotic) was determined by annexin V/7AAD assay using flow cytometer. Bar diagrams indicate the percentage of cell death with respective treated groups (Mean ± SEM, n = 3), Two-way ANOVA, ****P < 0.0001. G Left panel: Indicated 5FU resistant OSCC lines and PDC2 cells were treated with 5 μM of 5FU and/or 50 nM of Lestaurtinib for 48 h, after which immunostaining was performed for γ-H2AX as described in materials and methods. Right panel: The number of foci from panel (G) are indicated as bar diagram. Two-way ANOVA, ****P < 0.0001. H Indicated 5FU resistant OSCC lines and PDC2 cells were treated with Lestaurtinib for 48 h, after which immunoblotting (n = 3) was performed with indicated antibodies. I Effect on 5FU IC50 upon Lestaurtinib treatment in cells with or without MINK1 knockout in indicated 5FU resistant OSCC lines and PDC2 cells (n = 3), *P < 0.05 by 2-way ANOVA.