Figure 2 | Scientific Reports

Figure 2

From: Dual-sgRNA CRISPR/Cas9 knockout of PD-L1 in human U87 glioblastoma tumor cells inhibits proliferation, invasion, and tumor-associated macrophage polarization

Figure 2

Transfection of CRISPR/Cas9 plasmids and genomic editing efficiency in human U87 cells. (A) Transfection of GFP tagged Cas9-g82 and Cas9-g165 in U87 cells was evaluated by fluorescent microscopy. GFP expression indicates successful transfection of Cas9-g82/GFP and Cas9-g165/GFP in U87 cells at day 2. (B) Optimization of Cas9-g82/165 transfection. Cas9-g82/165 was transfected at different concentrations with either 12.5 µM or 25 µM of the HDR template, and Cas9 expression was evaluated by western blot analysis. Cas9 is fused with an N-terminal FLAG tag, and actin was used to evaluate protein levels. While concentrations above 1.5 ug/mL lead to Cas9 transfection, low toxicity was observed with a concentration of 1.5 ug/mL. (C) The T7E1 assay was performed to determine the efficiency of PD-L1 gene-editing at the genomic level. Multiple bands for a single sample indicate successful gene editing by Cas9. A ladder (M; lane 1), and a positive control (T7E1 ctl; lane 2) were used to confirm the size of the uncut product (Ctl) and the efficacy of the assay, respectively. Gel electrophoresis indicates all CRISPR/Cas9 plasmids were able to edit PD-L1 at the genomic DNA level, with an increased editing efficiency observed with Cas9-g82/165 + HDR. Scale bar = 50 um.

Back to article page