Additional file 2: Figure S1. of A kinome siRNA screen identifies HGS as a potential target for liver cancers with oncogenic mutations in CTNNB1

HuH6shCTNNB1 growth rate in response to Dox treatment is dependent of cell density. A-HuH6shCTNNB1 cells were cultivated in the presence or absence of 2 μg/ml Dox for 3 days. Cells were then seeded at 3 × 103 cells per well in 12–well plates and cultivated in the presence or absence of 2 μg/ml Dox for 7 days. Cell density was estimated at the indicated times by crystal violet assay and quantified as indicated in Materials and Methods. A representative picture of four independent experiments is shown. Results are expressed as the mean ± S.D. Plain and dashed lines show the growth of HuH6shCTNNB1 cells without and with Dox, respectively. B-HuH6shCTNNB1 cells were cultivated in the presence or absence of 2 μg/ml Dox for 3 days. Cells were then seeded at 30 × 103 cells per well in a 12–well plate and cultivated in the presence or absence of Dox for 7 days. Cell density was estimated at the indicated times by crystal violet assay. A representative picture of four independent experiments is shown. Results are expressed as the mean ± S.D. Plain and dashed lines show the growth of HuH6shCTNNB1 cells without or with Dox, respectively. C- HuH6shCTNNB1 cells were cultured in the presence or absence of 2 μg/ml Dox for 3 days and cells were seeded at low density (0.02 × 106 cells/well in 6-well plates) and cultured for another 24 h. Proportion of cells in G0/G1, S or G2/M phases was determined by assessment of DNA content with PI staining and flow cytometry. Black and grey rows represent untreated HuH6shCTNNB1 and Dox-treated HuH6shCTNNB1 cells, respectively D- HuH6shCTNNB1 cells were cultured in the presence or absence of 2 μg/ml Dox for 3 days and cells were seeded at medium density (0. 2 × 106 cells/well in 6-well plates) and cultured for another 24 h. Proportion of cells in G0/G1, S or G2/M phases was determined by assessment of DNA content with PI staining and flow cytometry. Black and grey rows represent untreated HuH6shCTNNB1 and Dox-treated HuH6shCTNNB1 cells, respectively E- Assessment of apoptosis by flow cytometry with DiOC6(3) and IP staining. Plain black (untreated) and plain grey (2 μg/ml Dox) columns show apoptosis in HuH6shCTNNB1 seeded at medium density (0.2 × 106 cells/well), whereas dashed black (untreated) and dashed grey (2 μg/ml Dox) columns show apoptosis in HuH6shCTNNB1 seeded at low density (0.02 × 106 cells/well). Means ± S.D. of three independent experiments are presented. (PDF 172 kb