Figure 1. A genome‐wide CRISPR‐Cas9 screen identifies SLC1A3 as a contributor to L‐asparaginase (ASNase) resistance in PC3 cells.

1. IncuCyte cell proliferation curves of PC3 cells treated with the indicated concentrations of ASNase.
2. IncuCyte cell proliferation curves for ASNS knockout (sgASNS) and control (sgNon‐targeting) PC3 cells in the absence and presence of ASNase.
3. Flow chart for a genome‐wide CRISPR‐Cas9 functional screen in PC3 cells.
4. Volcano plots for the MAGeCK pipeline analysis of the sgRNA abundance from the screen. Green dots indicate positive controls and red dots indicate candidates with a fold discovery rate (FDR) < 0.003.
5. IncuCyte cell proliferation curves of SLC1A3 knockout (sgSLC1A3) and control (sgNon‐targeting) PC3 cells in the absence and presence of ASNase treatment. #3 and #4 represent two different sgRNAs targeting SLC1A3.
6. Radioactive labeled aspartate and glutamate uptake measurement in control (sgNon‐targeting) and SLC1A3 knockout (sgSLC1A3) PC3 cells. #3 and #4 represent two different sgRNAs targeting SLC1A3. Radioactive labeled leucine uptake was used as a control. Data were normalized to the reads of control PC3 cells.
7. Endogenous levels of aspartate, asparagine, glutamate, and glutamine in control (sgNon‐targeting) and SLC1A3 knockout (sgSLC1A3) PC3 cells with or without ASNase for 3 days. Median peak intensity was used for the read normalization.
8. IncuCyte cell proliferation curves of SLC1A3 knockout (sgSLC1A3#3) PC3 cells treated with ASNase and supplemented with either esterified aspartate (Asp, 6 mM) or esterified glutamate (Glu, 6 mM), and esterified leucine (Leu, 6 mM) as a control.

Data information: For IncuCyte proliferation assays, images were taken every 4 h and the cell confluence was calculated by averaging three mapped images per well. All results were calculated from three replicates and presented as mean ± SD, unless otherwise stated. The P‐value was calculated by two‐tailed unpaired t‐test by Prism7. **P < 0.01, ***P < 0.001.