Fig. 3

A Using a lentiviral approach, 5FU resistant OSCC lines and PDC2 were stably transfected with ShRNA which targets 3′UTR of MINK1 mRNA (MINK1 UTRKD). For ectopic overexpression, pLenti CMV/TO Puro DEST MINK1 and control vector were transiently transfected to indicated MINK1 UTRKD cells and immunoblotting (n = 3) was performed with indicated antibodies. B MINK1 was ectopically overexpressed in 5FUR cells stably expressing MINK1ShRNA targeting 3′UTR and treated with 5FU at indicated concentration for 48 h, after which cell viability was determined by MTT assay (n = 3), 2-way ANOVA, ****P < 0.0001. C Cells were treated as indicated in (B) panel and 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), 2-way ANOVA, ****P < 0.0001. D Left panel: MINK1 was overexpressed in 5FUR cells stably expressing MINK1ShRNA targeting 3′UTR and treated with 5FU 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 (D) are indicated as bar diagram. Two-way ANOVA, ****P < 0.0001. E MINK1 was overexpressed in chemoresistant cells stably expressing MINK1ShRNA targeting 3′ UTR, followed by 5FU treatment for 48 h, and immunoblotting (n = 3) was performed with indicated antibodies. F 5FU sensitive cells were stably transfected with either pSilencer™ 4.1-CMV puro-MINK1WT or pSilencer™ 4.1-CMV puro-MINK1 K54R (kinase dead) followed by puromycin selection (up to 5 µg/ml). Two clones (C1 and C2) from each line were selected and immunoblotting was performed with indicated antibodies. G 5FU sensitive OSCC lines stably expressing MINK1 WT or MINK1 K54R (two clones C1 and C2 from each line) were treated with 5FU at indicated concentrations for 48 h, after which cell viability was determined by MTT assay (n = 3), 2-way ANOVA, ****P < 0.0001.