Extended Data Fig. 7: Transcriptional profiling reveals a phosphate-related homeostatic response after XPR1 inactivation.
a) Experimental workflow to determine the transcriptional profile of XPR1 inactivation across many different cancer cell lines. See methods for full details; N = 1 transduction event for panels b-g. b) The total number of cells per cell line de-multiplexed from the 10X scRNAseq library. c) The total number of unique transcripts measured for each cell, as measured by unique molecular identifiers (UMIs). Box plots represent the 1st – 3rd quartiles, with whiskers representing the minimum and the maximum. The exact N for each sample in c is indicated in panel b. d) UMAP projection of the 2,501 cells from the indicated cell lines (determined by SNP profiles) and perturbations (indicated by cell-surface antibody ‘hash-tag’ labeling). Arrows indicate the degree to which the average transcriptional profile changes between the control sgRNA and the sgXPR1 infection condition. e) Summary of transcriptional effects across cell lines after inactivation of XPR1. Middle, the log-fold change of the top 500 differentially expressed genes after regressing out the effect of cell cycle. Left, summary annotations for each cell line include XPR1 dependency (XPR1 CERES), the overall transcriptional change (average log2 fold-change), and the degree of cell cycle arrest observed in the single-cell data (ΔG0/G1). The pearson correlation of these values is shown above the heatmap. Right, the pearson correlation of the top 500 differentially expressed genes between each cell line. f) Differentially expressed genes - after correcting for cell cycle - in the less sensitive cell lines (COV413a, JHOS4, OVCAR4, HEC6, and JHUEM1). Significance was assessed by the limma-voom pipeline using a two-tailed statistical test (see methods). g) Same as in f, but for the highly correlated cell lines RMG1, IGROV1, and OVISE. h) Four days after induction of shXPR1_2 (IGROV1) or shXPR1_4 (OVISE) using doxycycline, the amount of secreted FGF23 was measured in the conditioned medium using ELISA. N = 2 technical replicates representative of N = 3 independent experiments. i) 72 hours after treatment with the XPR1 inhibitor XRBD, the indicated proteins were detected using immunoblot. N = 1 technical replicate representative of N = 2 independent experiments. j) Top, western blot analysis of SLC34A2 and XPR1 in the SLC34A2-high yet XPR1-insensitive lung cancer cell lines, five days after infection with lentivirus expressing the indicated sgRNA. Bottom, viability of the indicated cancer cell lines was assessed using Cell Titer Glo after a five day XRBD treatment to inhibit XPR1. Points represent the mean of N = 3 technical replicates; error bars represent standard error of the mean. Data are representative of N = 2 independent experiments.
