Fig. 1. Broad-spectrum kinome profiling identification of CDK6 upregulation in lenvatinib-resistant HCC cells with enhanced self-renewal ability.

a, b A limiting dilution assay showed that lenvatinib-resistant PLC/PRF/5 and Huh7 cells had enhanced self-renewal ability as evidenced by the increase in the stem cell frequency compared to the corresponding mock controls (LenR: lenvatinib-resistant) (n = 2 independent experiments, one-sided extreme limiting dilution analysis). c Long-term colony formation assay of mock and LenR PLC/PRF/5 and Huh7 cells. Cells were grown in the absence or presence of lenvatinib at the indicated concentrations, and they were then fixed and stained. The corresponding IC₅₀ values were calculated and compared (n = 3 independent experiments). d The XO44 probe indicated upregulation of a kinase of approximately 36 kDa in lenvatinib-resistant PLC/PRF/5 and Huh7 cells compared to mock cells (PLC/PRF/5 n = 4 and Huh7 n = 3 independent experiments). e Venn diagram showing five upregulated proteins common to the two lenvatinib-resistant HCC cell lines with a 2-fold cutoff after mass spectrometry analysis (n = 1). CDK6 was found to be the most upregulated gene in lenvatinib-resistant PLC/PRF/5 while the second in Huh7. f Phylogenic kinase mapping illustrates the kinase profiles in LenR HCC cells. g Quantitation of CDK6 expression by the parallel reaction monitoring (PRM) approach (n = 2 independent experiments). h Western blot analysis showed that CDK6 protein was upregulated in lenvatinib-resistant PLC/PRF/5 and Huh7 cells. i Heatmap showing the changes in the phosphorylation levels of kinases in lenvatinib-resistant PLC/PRF/5 cell line (n = 3 independent experiments). j IHC staining showed the upregulation of CDK6 in lenvatinib-resistant HCC patients (n = 3 patients) when compared to sensitive group (n = 4 patients) (two-tailed t test). The protein expression was quantified using ImageJ. Data was presented as mean ± standard deviation. Source data are provided as a Source Data file.