Figure 3.

PD-L1 knockout by CRISPR/Cas9 plasmids in U87 cells. (A) Western blot analysis of PD-L1 protein levels. U87 cells transfected with the CRISPR/Cas9 plasmids were analyzed and quantified to determine PD-L1 protein knockout efficiency of both (B) deglycosylated and (C) glycosylated PD-L1. The strongest PD-L1 knockout was detected in Cas9-g82/165 + HDR treated U87 cells. (D and E) PD-L1 knockout by Cas9-g82/165 + HDR was further confirmed by western blot analysis in human breast cancer MDA MB 231 cells. (F) PD-L1 localization in U87 cells. U87 cells were stained for PD-L1, and PD-L1 expression (green) was examined on the membrane and cytoplasm using the structural protein vimentin (red), and the nucleus using DAPI (blue). Images suggests PD-L1 is expressed throughout the cell, with high expression seen in the nucleus. (G) Immunofluorescence labeling of PD-L1 in U87 cells treated with or without Cas9-g82/165 + HDR. (H) Quantification of the mean fluorescence intensity of individual cells suggests that PD-L1 was significantly reduced in Cas9-g82/165 + HDR treated U87 cells. Control cell # = 266, Cas9-g82/165 + HDR treated cell # = 253. (I) Western blot analysis of nuclear and membrane/cytoplasm fractions of U87 cells confirms PD-L1 expression in the cytoplasm and nucleus. Histone H3 was used to identify the nuclear fractions. Cells treated with Cas9-g82/165 + HDR showed a significant decrease in PD-L1 in both the membrane and nuclear fractions. Quantification of (J) glycosylated and (K) deglycosylated PD-L1 in the nuclear and membrane fractions of U87 cells treated with or without Cas9-g82/165 + HDR. (D–E) and (I–K) represent a single experiment. All other experiments were performed in triplicate and results were normalized for direct comparisons. Data represents the mean ± SEM. Statistical significance was evaluated with either a one-way ANOVA followed by Tukey’s post-hoc analysis (B, C), or a t-test (E and H). *p < 0.05, **p < 0.01. Scale bar = 50 µm.