Fig. 1: A genome-wide CRISPR knockout screen identifies genes essential for maintaining the contractile phenotype of VSMCs.
a Schematic diagram showing the generation of an EGFP reporter-expressing MOVAS cell line. P2A, a self-cleaving peptide. b Representative flow cytometry and quantification of EGFP expression in the reporter MOVAS cell line in response to 48 h of TGF-β treatment (blue line), serum starvation (red line), or PDGF-BB treatment (green line). WT, wild-type; TGF-β, transforming growth factor-β; PDGF-BB, platelet-derived growth factor BB. n = 3 independent experiments. c Schematic diagram of the genome-scale CRISPR knockout screen. d Flow cytometry analysis of EGFP reporter knock-in MOVAS cells that were transduced with or without the pooled lentivirus encoding a genome-wide sgRNA library with two rounds of cell sorting. Wild-type (WT) MOVAS cells were utilized as a negative control for EGFP fluorescence. e Identification of the top candidate genes using the MAGeCK algorithm. Pink circles represent the top candidate genes that have been previously reported to regulate the VSMC phenotype. MAGeCK, Model-based Analysis of Genome-wide CRISPR/Cas9 Knockout. RRA score, robust rank aggregation score. f RT‒qPCR analysis of Acta2, Cnn1, Tagln, and Myh11 gene expression in the A7r5 rat VSMC cell line transfected with the scrambled siRNA (20 nM) or siRNA targeting Lemd3 (20 nM) for 24 h. The data are presented as the relative fold changes to siRNAscramble (n = 5 independent experiments). Error bars indicate s.e.m. Statistical analysis was performed using ordinary one-way ANOVA with Tukey’s multiple comparisons test for panel b and two-sided unpaired Student’s t test for (f).
