Extended Data Fig. 3: Slc33a1 dependency is abrogated in Nrf2-deficient cell lines and dependency is recapitulated across large human cancer cell line CRISPR-screens.

a, Slc33a1 expression quantified by qPCR in cells transduced with sgRNA-resistant cDNA (*) overexpression constructs. Bars represent mRNA expression normalized to Actb then to PGK Ctrl samples, and the error bars represent mean ± s.d. from the mean across n = 4 independent experiments. P values were determined by unpaired two-tailed Student’s t-test. b, Fluorescence competition assay plot displays %RFP + cells marking sgCtrl or sgSlc33a1 infected cells over time. This experiment was performed once with n = 4 biologically independent samples obtained from two independent cell lines per genotype that were tested with two independent sgRNAs. P values were determined by two-way ANOVA with Tukey’s post-hoc multiple comparisons test. c, NRF2 western blot of KPK2 and a mixed population of pUSEPR-sgNrf2 KPK cells (KPKN2). Histone H3 was used as a loading control. Gel has been cropped to remove non-relevant cell lines and nonspecific NRF2 bands. d, Fluorescence competition assay plot displays day 2 normalized %RFP + cells marking sgCtrl or sgSlc33a1 infected cells over time. Data are representative of n = 3 independent culture wells per cell line per sgRNA. e, Colony formation assay of the indicated cell lines infected with sgCtrl or sgSlc33a1. Data from n = 3 independent culture wells per cell line per sgRNA. f, Quantified colony formation assay via the integrated pixel density assessed by ImageJ software. Related to (e). Data are representative of n = 3 independent culture wells per cell line per sgRNA. g, CERES normalized scores obtained across all cell lines. Dotted red line represents the median CERES scores of all core-essential genes defined by the DepMap consortium (19Q2, n = 563 cell lines). h, CERES normalized scores obtained across KEAP1/NRF2-mutant or wild-type cancer cell lines (n = 563 total cells). Values above comparison denote the fold change relative to the average CERES score of the wild-type group. P values were determined by unpaired two-tailed Student’s t-test. i, Co-dependencies arising from the DepMap dataset cluster NRF2 with SLC33A1, SUCO, TAPT1, DNAJBL11 and ADPGK. Each node depicts a gene. Connecting lines indicate a direct co-dependency shared between each node (ref. ²⁸). j,k, Waterfall plot of the rank ordered Pearson correlation coefficient between (j) SLC33A1 CERES scores versus or (k) ATF4 CERES scores versus all genes screened in the DepMap consortium (19Q2, n = 563 total cells). l,m, Fluorescence competition assays of sgRNA infected cells. Plot displays day 2 normalized %GFP + cells marking (l) sgCtrl or sgSuco edited or (m) sgCtrl or sgTapt1 edited cells. Data are representative of n = 3 independent culture wells per cell line per sgRNA and representative of three independent experiments with similar results. All error bars depicted represent mean ± s.d. unless otherwise stated. Data from a single experiment are shown in c, d, e, and f are representative of two independent experiments with similar results. Data for experiments a, b, d, l and m are available as source data (Source_Data_Extended_Data_1; Source_Data_Extended_Data_2).