Extended Data Fig. 1: Validation and quality control of inducible LKB1 restoration model and genome-scale CRISPR/Cas9 screen.
From: LKB1 inactivation modulates chromatin accessibility to drive metastatic progression
a. Schematic of restorable Lkb1TR/TR alleles. SA = splice acceptor, SD = splice donor, FRT = flippase recognition target. b. Schematic of the derivation of LKB1-restorable cell lines. c. Expression of LKB1 by immunoblot over a time-course of 4-OHT treatment, represented in hours (h) and days (d). HSP90 is a sample processing control. 25% and 10% of input after six days of 4-OHT treatment is shown for a visual comparison. d. Expression of LKB1 by immunoblot in LR1 and LR2 cells treated with vehicle or 4-OHT compared to a KPT cell line and a KPT;Lkb1−/− cell line. HSP90 is a sample processing control. e. RNA-sequencing reads mapping to the Lkb1 locus following six days of 4-OHT or vehicle treatment. f. Subcutaneous growth assay following injection of cell lines into recipient NSG mice. Tamoxifen or vehicle treatment was initiated on day 0. Mean tumor volume as measured by calipers of six tumors per condition +/− SEM is shown. g. Intravenous (i.v.) transplant assays. Left: Representative lung histology. Right: Change in % tumor area in LKB1-restored cells. Mean area of four mice per condition +/− SEM is shown. **p = 0.001, ***p = 0.0001, n.s. = not significant with a two-sided t-test. Scale bars represent 5 mm. h. Cumulative population doublings recorded over 12 days of 4-OHT treatment. Each cell line and condition was cultured and analyzed in triplicate. Mean +/− SEM is shown. **p = 0.0002 for LR1, **p = 0.0001 for LR2. i. Left: Representative image of clonogenic growth in LR1 cells. Right: % normalized area of cell growth. Each treatment group was cultured and analyzed in triplicate. Mean +/− SEM is shown. *p < 0.01, **p < 0.001, n.s. = not significant with a two-sided t-test. Scale bars represent 10 mm. p = 0.0001 for LR1 and p = 0.0059 for LR2. j. Heatmap of Pearson correlation matrix of log-normalized counts across all samples in the genome-scale CRISPR/Cas9 screen. k. Log2 fold enrichment of negative control sgRNAs and sgRNAs targeting Lkb1 at day 12 versus day 0.
