Extended Data Fig. 2: In vitro validation of Slc33a1 as a Keap1-mutant-specific dependency.
From: Keap1 mutation renders lung adenocarcinomas dependent on Slc33a1
a, Schematic of lentiviral vectors utilized for fluorescence competition assays. b, Experimental validation pipeline for gene essentiality by fluorescence competition assays. c, Fluorescence competition assays of KP or KPK isogenic pairs targeted with sgCtrl or two independent sgRNAs targeting Arf4. Plot displays day 2 normalized %GFP + (pUSPmNG) cells at day 10 post infection with sgCtrl or sgArf4. Data are representative of n = 3 independent culture wells per cell line per sgRNA. d, Quantified colony formation assay via the integrated pixel density assessed by ImageJ software. Related to Fig. 1f. Data are representative of n = 3 independent culture wells/cell line/sgRNA. e, Schematic of LVt-TSTOP lentiviral vector utilized for doxycycline inducible miR30a-based shRNA expression. TRE, tetracycline-response element (TRE3GS); rtTA, reverse tetracycline-controlled transactivator (Tet-On 3 G); P2A; 2 A self-cleaving peptide; Puro, Puromycin-resistance cassette. f, Colony formation assay of KP1 and KPK1 cells transduced with LVt-TSTOP vectors expressing shRenilla or shSlc33a1. Data are representative of n = 3 independent culture wells per cell line per shRNA. g, Quantified colony formation assay via the integrated pixel density assessed by ImageJ software. Related to (f). Data are representative of n = 3 independent culture wells per cell line per shRNA. h, Slc33a1 expression quantified by qPCR in cells treated with and without doxycycline (10 ng/mL) for 48 hours. Bars represent mRNA expression normalized to Actb then to -dox conditions, and the error bars represent mean ± s.d. from the mean across n = 3 or 4 independent experiments. P values were determined by unpaired two-tailed Student’s t-test. i, Slc33a1 expression quantified by qPCR 48 hours post-transduction with sgRNA-CRISPRi constructs. Bars represent mRNA expression normalized to Actb then to sgCtrl 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. j, Left: Representative microscopy images of cells transduced with sgRNAs mediating transcriptional repression of the Slc33a1 promoter (Scale bar = 100 μm). Right: Colony formation assay of dCas9-KRAB expressing cells in the presence of the indicated promoter-targeting sgRNAs. Data are representative of n = 3 independent culture wells per cell line per sgRNA. k, Quantified colony formation assay via the integrated pixel density assessed by ImageJ software. Related to (j). Data are representative of n = 3 independent culture wells per cell line per sgRNA. All error bars depicted represent mean ± s.d.. Data from a single experiment are shown in c, d, f, g, j, and k are representative of two independent experiments with similar results. Data for experiments c, h, and i are available as source data (Source_Data_Extended_Data_1; Source_Data_Extended_Data_2).
