Fig. 3
siRNA screen based on immunofluorescence imaging. a Identification of kinases required for HuH6 viability. A kinome siRNA library was used to transfect HuH6 cells: 687 genes were individually targeted (four siRNAs per gene) and three independent experiments were performed. Cells were immunolabeled 72 h after transfection with anti-cleaved caspase 3 (apoptosis marker) and anti-Ki67 (cell proliferation markers) antibodies and nuclei were labeled with DAPI (cell count) before the acquisition of images and readout of phenotypes. Three phenotypes were analyzed: (i) cell numbers through DAPI staining, (ii) Apoptosis and (iii) G0-phase arrest with Ki67. All siRNAs with median Z-scores >2 or < −2 were considered significant hits, and genes with at least two (out of four) ‘hit’ siRNAs were selected as candidate genes. Following this screen, 52 genes were identified as necessary for HuH6 survival or proliferation. The 52 outliers are listed in Table S2, additional Excel file b Counter-screen: Investigation of synthetic lethality relationship between candidate genes and stabilized β-catenin. HuH6 and HuH6shCTNNB1 were cultured in the presence or absence of 2 μg/ml doxycycline 72 h, and transfected with a siRNA library including the 52 outliers identified in the primary screen (four siRNAs per gene). Each screen was repeated three times to obtain biological replicates. Transfection was carried out and phenotypes were assessed as described in A. Outliers were called if |robust Z-score| > 2 for at least two phenotypes and two out of four siRNAs, only in cells with high β-catenin signaling. Following the counter-screen, five genes (listed in Table 2) were identified as having a potential lethal synthetic relationship with mutant β-catenin