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. Author manuscript; available in PMC: 2014 Apr 11.
Published in final edited form as: Nat Commun. 2013;4:2956. doi: 10.1038/ncomms3956

Figure 8. GRG6 overexpression phenocopies the effects of FOXG1 and TLE silencing in BTICs.

Figure 8

(a) Model of antagonistic contributions of TLE and GRG6 to FOXG1 transcription repression activity. FOXG1 mediates transcriptional repression of target genes (e.g., p21Cip1) when in a complex with TLE. The transcription repression activity of FOXG1:TLE can be antagonized by GRG6, which can compete with TLE for FOXG1 binding but does not have transcriptional corepressor activity. (b) FFPE sections from normal brain (n=13) or GBM (n=15) tissues were subjected to immunohistochemistry with anti-GRG6 antibody, followed by counterstaining with hematoxylin. A representative image is shown for each group. Scale bar: 50 μm. (c) Quantification of the percent of GRG6-positive nuclei in normal brain or GBM specimens (mean±SEM; GBM, P=6.8×10−8; n=13; normal brain, n=10; t-test). (d) Western blot analysis of endogenous GRG6 expression in normal brain and GBM specimens, as indicated. β-ACTIN is shown as loading control. Molecular size markers are indicated in kDa. (e) Western blot analysis of BT048 cells using anti-FLAG epitope or anti-GRG6 antibodies following transduction with FLAG-GRG6-expressing lentivirus or empty vector (EV) control lentivirus. Exogenous GRG6 exhibits retarded mobility compared to endogenous (Endog) GRG6 due to the FLAG epitope. One representative result is shown (n≥3). (f) Quantification of the sphere-forming ability of BT025 and BT048 cells passaged for two generations after transduction with empty vector or GRG6 lentivirus (mean±SEM; BT025, primary spheres, P=1.1×10−3, secondary spheres, P=1.5×10−3; BT048, primary spheres, P=1.7×10−4, secondary spheres, P=6.2×10−3; n≥5; t-test). (g) Western blot analysis of p21Cip1 (p21) expression in BT048 cells after transduction with empty vector or GRG6 lentivirus. (h) Quantification of the percent of BT048 cells that incorporated BrdU after transduction with GRG6 lentivirus compared to empty vector lentivirus (mean±SEM; P=3.1×10−4; n=7; t-test). (i) ChIP analysis of p21Cip1 promoter occupancy in BT048 cells transduced with either empty vector (lanes 2–5) or GRG6 (lanes 7–10) lentivirus. Experiments were performed using rabbit anti-FOXG1 (lanes 3 and 8), rabbit anti-TLE1 (lanes 4 and 9), or control (Ctrl) rabbit (lanes 5 and 10) antibodies, followed by PCR with primers specific for the p21 promoter. Input genomic DNA (Input; lanes 2 and 7) was subjected to PCR with the same primers. DNA standards (Stands, lane 1) are shown and their size indicated in base pairs. One representative ChIP experiment is shown (n≥3).