MOESM16 of Glioma cell proliferation is enhanced in the presence of tumor-derived cilia vesicles
2018-11-06T05:00:00Z (GMT) by
Additional file 16. CRISPR/Cas9 depletion of IFT88 and effect on ciliogenesis in L0 GBM cells. A CRISPR/Cas9 plasmid (pU6-gRNA-CMV-Cas9:2a:GFP; Sigma-Aldrich) co-expressing a GFP reporter for Cas9 and gRNA directed against human IFT88 (Target ID: HS0000334248; IFT88 gRNA target sequence: GCCATTAAATTCTACCGAA) was used to transfect parental L0 cells and generate cell clones depleted of IFT88. L0 cells were grown on 10 cm2 plates and transfected (Lipofectamine 2000) at 60% to 70% confluence with 0.5 μg/ml of the CRISPR/Cas9-encoding plasmid DNA. Twenty-four to 48 h after transfection, GFP+ cells were sorted as individual clones into 96-well plates containing 250 μl of DMEM/F12 medium supplemented with hEGF and bFGF using a BD FACS Aria II Cell Sorter. GFP+ clones were FAC-sorted and expanded for screening by western blot (WB) and immunostaining for acetylated alpha-tubulin+ cilia. (A) WB of L0 cell lysate showing that, compared to parental L0 (control) cells, clone C9-derived cells displayed an absence of a band for IFT88. β-Actin was used as the loading control. (B) Percentage of aaTub+ cilia in control and C9 clones. ***p < 0.001 (Student’s t test).