Figure 3. EGLN1 deletion inhibits proliferation in EGLN1-dependent cell lines.

A. Effects of EGLN1 deletion on HIF1A levels. Whole cell lysates of ES2-Cas9 with the indicated sgRNAs. Equal amounts of protein were analyzed by immunoblotting with GAPDH included as a marker of equal loading. Deletion of EGLN1 leads to increased HIF1A phosphorylation. *sgRNAs used for subsequent experiments.

B. EGLN1 deletion reduces proliferation in EGLN1-dependent cells. Cells were serially passaged every 3–4 days. Results are representative of 3 independent experiments and data is graphed as mean ± standard deviation of 3 replicates. *p<0.05

C. EGLN1 deletion does not affect proliferation in EGLN1 independent cells. Cells were serially passaged every 3–4 days. Results are representative of 3 independent experiments and data is graphed as mean ± standard deviation of 3 replicates.

D. VHL deletion reduces proliferation in EGLN1-dependent cells. Results are representative of 2 independent experiments and data is graphed as mean ± standard deviation of 3 replicates. *p<0.05

E. Luciferase competition assay. Renilla-expressing and Firefly+Cas9-expressing cells are mixed at 1:1 and then sgRNAs are expressed. Ratio of Renilla/Firefly luciferase was quantified, and cells were serially passaged every 3–4 days. EGLN1 KO cells (red) were compared to negative controls (blue) and positive controls (black). EGLN1 deleted cells dependent on EGLN1 are outcompeted by EGLN1 WT cells over time in a proliferation assay, whereas EGLN1 deleted cells insensitive to EGLN1 are not. Results are representative of 2 independent experiments and data is graphed as mean ± standard deviation of 3 technical replicates. Empty Vector – Empty Vector with no sgRNA, Chr2 Intragenic – sgRNA targeting intergenic region of chromosome 2, LacZ-1 – sgRNA targeting LacZ gene, PolR2D-1 – sgRNA targeting RNA Polymerase 2 Subunit D, PolR1C-1 – sgRNA targeting RNA Polymerase 1 Subunit C.