Figure 3.

PCM1-depleted GBM cell lines generated using CRISPR/Cas9. A CRISPR/Cas9 plasmid co-expressing a GFP reporter for Cas9 and gRNA directed against human PCM1 was used to transfect L0 and S3 GBM cells and generate cell clones depleted of PCM1. GFP-positive clones were FAC-sorted and expanded for screening by WB and immunostaining. (A) WB of L0 lysates shows that, compared with clone D5, clone F9–derived cells displayed an absence of a band for PCM1. β-Actin was used as a loading control. (B) Immunostaining of L0 clones shows the presence of PCM1-positive clusters (green) in clone D5 but not clone F9–derived cells (right panels). (C, D) Similarly, S3 cells generated from clone B9 lacked detectable PCM1 by WB (C) and ICC (D) compared with clone F11.