Fig. 2

Dependency on EGLN1 is partially mediated by HIF1α stabilization. a Western blot analysis showing CRISPR/Cas9-mediated knockout (KO) of EGLN1 in NCI-H23 cells. A non-targeting sgRNA (NT) has been used as a negative control. β-actin is the loading control. b Competition assay showing reduced proliferation in NCI-H23 cells KO for EGLN1. Cells infected with a sgRNA targeting ATP2A2 gene are the positive control. For each time point the ratio between GFP-positive (infected) and GFP-negative (uninfected) cells has been calculated and normalized on T0. Statistical significance has been calculated comparing the normalized ratio for each sample with NT. Data are mean ± SEM; *p < 0.05; N = 3. c Sensitivity curve of NCI-H23 cells to EGLN1 inhibitor molidustat. Data are mean ± SEM; N = 3. d Heatmap showing lung cancer cell lines ordered by dependency on EGLN1 gene. e Heatmap showing sensitivity to EGLN1 inhibitor molidustat in a panel of lung cancer cell lines. f and g Correlation analysis between EGLN1 and HIF1A (f) or VHL (g) CERES scores in lung AD cell lines. Cyan dots represent KRAS-mutated cell lines, black dots represent KRAS-WT cell lines. h Western blot analysis for HIF1α or EGLN1 expression on NCI-H23/Cas9 cells infected with sgRNAs for HIF1A, EGLN1 or both. β-actin is the loading control. i Western blot analysis with anti-HIF1α antibodies, performed on NCI-H23 cells treated with molidustat for 72 h at the indicated concentrations. β-actin is used as loading control. j Competition assay performed on NCI-H23 cells. Cells infected with a non-targeting sgRNA (NT) are the negative control. NT, EGLN1 or HIF1A sgRNAs carried by a GFP-containing plasmid have been used to infect the cells. For double KO, EGLN1 sgRNA-GFP containing plasmid was used to infect cells which were already KO for HIF1A. For each time point the ratio between GFP-positive (infected) and GFP-negative (uninfected) cells has been calculated and normalized on T0. Statistical significance has been calculated comparing the normalized ratio for EGLN1 KO with double KO. Data are mean ± SEM; *p < 0.05; N = 3. k Schematic representation of the proposed mechanism