Fig. 1: Parallel in vivo and in vitro CRISPR screens in A549 and in vivo specific function of MEN1 in lung cancer.
a, Schematic representation of the CRISPR screen experiment design. A549 cells with stable Cas9 expression were transduced with Epi-Drug sgRNA library and selected with puromycin for 3 days (D0). Parallel in vitro and in vivo screens were performed for 3 weeks (D21). Samples were collected for PCR amplification and NGS. b, Dropout (genes with sgRNA reduced in D21; blue dots) and enriched (genes with sgRNA increased in D21; red dots) genes in LUAD A549 xenograft compared with A549 2D cultured cells. The P values of positive and negative selections and log2(fold change) were defined and calculated using MAGeCK. MEN1 is the top ranked enriched gene. c, Xenograft tumor growth curve in immunodeficient mice inoculated with control (sgCtrl) or MEN1 knockout (sgMEN1-1, sgMEN1-2) A549 cells. Each data point represents mean ± s.e.m. tumor volumes (n = 5 in sgCtrl, sgMEN1-1 and sgMEN1-2 groups). Two-way ANOVA was used for the growth curves. *P < 0.05, **P < 0.01. d, Colony formation of A549 cells with (sgMEN1) and without (sgCtrl) knockout of MEN1. Cells were seeded in six-well plates in duplicate and allowed to grow for 8 days before staining with crystal violet. Scale bars, 7 mm. e,f, KEGG analysis of differential genes in MEN1 knockout versus control A549 cells in vitro (e) and in vivo (f). The x axis represents the number of genes. Wald tests defined in DEseq2 were used to calculate P values. g, RT–qPCR showing the expression of representative cytokine-related genes in MEN1 knockout A549 cells relative to control. Housekeeping gene TBP was used as a control. Mean ± s.e.m. of three biological replicates is shown (unpaired two-tailed Student’s t-test). h, KEGG analysis of differential genes in MEN1-low versus MEN1-high patients from the TCGA LUAD cohort. Wald tests defined in DEseq2 were used to calculate P values. i, Boxplot showing the abundance of representative cytokine genes in MEN1-high versus MEN1-low patient tumors in the TCGA LUAD cohort. Twenty patients with the highest and the lowest MEN1 expression were assigned to each group. Horizontal lines in the box represent the upper quartile, median and lower quartile from top to bottom. Vertical extending lines mark the 5th to 95th percentile (unpaired two-tailed Student’s t-test). *P < 0.05, **P < 0.01. IgA, immunoglobulin A; IL, interleukin; Padj, adjusted P value; Rep, replicate. a, Created with BioRender.com.
