Figure 5.

The mutational status and expression of tuberous sclerosis complex (TSC)1/2 in a panel of human hepatocellular carcinoma (HCC) cell lines and their sensitivity to mammalian target of rapamycin inhibitor treatment are shown. (A) RNA sequencing revealed that PLC HCC cells carried a TSC2 mutation. (B) The TSC2 mutation identified by RNA sequencing in PLC was confirmed by Sanger sequencing at genomic level. (C) Protein expression levels of TSC1 and TSC2 in a panel of HCC cell lines were determined by western blotting with the use TSC1-specific and TSC2-specific antibodies. PLC, H2P and H2M cells showed relatively low TSC1 as well as TSC2 protein expression. H2M was derived from H2P. (D) Sanger sequencing revealed a stop-gain mutation in TSC1 and a missense mutation in TSC2 in H2P cells. The stop-gain mutation may account for the loss of TSC1 protein expression in H2P cells. (E) Schematic diagram showing the TSC1 and TSC2 mutants identified in PLC and H2P cells. (F) Eight HCC cell lines with defined mutational status of TSC1/2 were subjected to rapamycin treatment at the indicated concentrations for 4 days, followed by fixation and crystal violet staining for visualisation.