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. Author manuscript; available in PMC: 2014 Feb 1.
Published in final edited form as: Cancer Discov. 2012 Nov 15;3(2):198–211. doi: 10.1158/2159-8290.CD-12-0353

Figure 2. BUB1B validates as a candidate GBM-lethal gene in vitro.

Figure 2

(A) BTIC-specific effects of BUB1B knockdown, visualized using shRNA-GFP+ BTICs and NSCs 6 days after post-transduction with pGIPz-shRNA virus. Knockdown of KIF11/EG5, which encodes a microtubule motor protein critical for bi-polar spindle formation during mitosis, was used as a positive control for both RNAi pathway activity and cell proliferation.

(B–C) Examination of BUB1B knockdown by western blot and RT-qPCR analysis in BTIC-G166 and NSC-CB660 cells.

(D) Comparison of the effects of BUB1B knockdown on in vitro expansion of multiple BTIC and NSC lines and normal human astrocytes (NHAs). **indicates student’s t-test p <.01. See methods for a description of how BTIC isolates were developmentally subtyped.

(E) Limiting dilution assays (LDA) for in vitro tumor sphere formation. BTIC-0131 cells and also un-passaged primary GBM tumor cells (448T) were transduced with indicated LV-GFP-shRNAs, diluted and assayed for sphere formation after 14 days. Linear regression analysis was used to evaluate significance.