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. 2012 May 9;14(6):745–760. doi: 10.1093/neuonc/nos088

Fig. 5.

Fig. 5.

Effect of pCU on run-on transcription. (A–B) Simultaneous visualization of transcription (green) and γH2AX (red) in pCU-treated U87 and 4910 non-GICs and GICs with and without radiation. Cells were transfected with pCU alone or pCU plus radiation and incubated with BrUTP at the final concentration of 5 mM. Cells were fixed and stained with anti-BrdU antibody and anti-phospho H2AX antibodies for 1 h and counterstained with species-specific Alexa Fluor-conjugated secondary antibodies for 1 h. Before mounting, cells were treated with DAPI and analyzed under a confocal microscope (Olympus BX61 Fluoview). Overlay of images was done using SPOT Advanced software (Windows version 4.0.8). (C–D) Expression of survivin mRNA. Total RNA was extracted from both non-GICs and GICs, and mRNA expression of survivin was determined by RT-PCR. GAPDH was used as a loading control. (E–F) Expression of survivin protein. Cell lysates from pCU-treated U87 and 4910 non-GICs and GICs with and without radiation were analyzed for expression of survivin by Western blotting. (G–H) Expression of survivin mRNA after transfection with full-length H2AX (FLH). Total RNA was extracted from both non-GICs and GICs, and mRNA expression of H2AX and survivin was determined by RT-PCR. (I–J) Lysates from FLH-treated cells with or without radiation were analyzed for γH2AX, H2AX and survivin proteins by Western blotting.