Fig. 4

HGS knockdown impairs cell viability specifically in hepatoblastoma cells with high β-catenin signaling. a HuH6^shCTNNB1 cells were cultured in the presence or absence of 2 μg/ml doxycycline for 96 h, seeded in 12-well plate and transfected with scramble or siRNA targeting specifically HGS and cultured for 72 h. Untransfected cells (siRNAimax lipofectamine alone) were also included as a control. Cell density was estimated 72 h after cell transfection by crystal violet staining. b Cells were cultured and transfected as in A and were stained with DiOC6₍₃₎ and PI. Proportion of apoptotic cells was determined by flow cytometric experiment. Note that apoptotic cells showed low labelling for both DiOC6₍₃₎ and PI. Means of three independent experiments are shown. c Cells were treated as in A and cleaved caspase3, HGS and β − actin protein levels were examined by immunoblotting. d HuH7 were transfected with scramble or siRNA targeting specifically HGS and cultured for 72 h. Untransfected cells (siRNAimax lipofectamine alone) were also included as a control. Proportion of apoptotic cells was determined by flow cytometric experiment after DiOC6₍₃₎/IP staining. e HuH7 cells were transfected as in D and cleaved caspase3, HGS and β − actin protein levels were examined by immunoblotting. Protein extract from HuH7 treated with 2 μg/ml doxorubicin for 16 h was added as positive control for cleaved caspase3 expression