Extended Data Fig. 6. The XPR1 dependency is not affected by phosphate levels in the tissue culture.

medium a) The concentration of phosphate in the growth medium of DepMap cell lines does not determine the extent of XPR1 dependency. Concentrations of phosphate were estimated from manufacturer formulations (see methods) and the pearson correlation between growth medium phosphate and XPR1 dependency is indicated.

b) Experimental procedure for manipulating tissue culture medium and assessing its effect on XPR1 dependency. The same parental cancer cell line was engineered to express firefly luciferase and Cas9, or Renilla luciferase alone. After a one-week adaptation to lowered phosphate, the two variants were mixed together and infected with sgRNA-encoding lentivirus. After selection for lentivirus-infected cell lines, the initial representation of Cas9:parental cells was determined by measuring the ratio of Firefly:Renilla luciferase using a DualGlo assay (Promega). One week after infection (Day 16 of the protocol), the extent to which genetic perturbation was detrimental to cell viability was determined using the DualGlo assay.

c) The XPR1 dependency is maintained in a low phosphate medium condition. SNGM and ES2 were profiled in the assay outlined in panel b. Note that the CERES score - displayed below the plot - represents the viability defect of the cell line 21 days after knock-out of XPR1 and growth in the indicated growth medium. N=5 technical replicates representative of N=2 experiments.

d) The viability of cells (as measured by total protein content) was measured in parallel with total phosphate as in Figure 4d. N=3 technical replicates representative of N=4 independent experiments per cell line.