Fig. 3

The effect of CRISPRi knockdown of p110γ on the migration of malignant B cell lines and CLL cells. a Confirmation of p110γ CRISPRi knockdown specificity. JVM3 cells were transfected with either dCas9-GFP expression vector alone (Control) or co-transfected with dCas9-GFP plus vectors expressing p110γ-targeting guide RNAs (at a 1:3 ratio). After 24 h, p110 isoform expression within sorted GFP+ transfectants was assessed by qPCR. b p110γ knockdown significantly impaired the migration of JVM3 and Ramos cells. Cells were transfected as above and then subjected to a transwell migration assay. Data represent average fold change in migration of GFP+ cells (n = 3). c p110γ knockdown significantly reduced the migration of primary CLL cells regardless of their IgVH mutation status. CLL cells were transfected with either dCas-GFP expression vector or dCas9-GFP plus vectors expressing p110γ-targeting guide RNAs then assessed 48 h later using a transwell migration assay. Percent of cells migrating toward SDF1α was determined by flow cytometry counting of live GFP-expressing cells present in the upper and lower chambers after 3 h