A Scatter plot showing the correlation between the expressions of BTK mRNA and NANOG, CD133, TIM-3, and KLF4. B Cell viability assay revealed higher cisplatin resistance, indicated by sphere and ionizing radiation (IR)-resistant SAS and TW2.6 cells compared with the parental cells. C The protein BTK expression in SAS, TW2.6 and HSC-3 parental, sphere, and IR cell lines as shown by Western blotting. D E Effect of BTK-knockdown on BTK, TIM-3, and CD133 expressions at both mRNA and protein levels. F Immunofluorescence staining showing the effect of shBTK on the BTK expression, TIM-3, CD133, and KLF4 proteins in spheres formed by SAS cells. Blue stain = DAPI, nuclear staining. GAPDH is loading control; WT, wild-type. All assays were performed at least twice in triplicate and expressed as mean ± SD. *p < 0.05, **p < 0.01.