Fig. 3.

Simultaneous downregulation of uPAR and cathepsin B with radiation-enhanced accumulation of cells in the sub-G0/G1 phase. (A–B) U87 and 4910 non-GICs and GICs were transfected with pSV and pCU with or without radiation as described in Materials and Methods. uPAR and cathepsin B expression levels were determined by Western blotting. (C–D) Expression of uPAR and cathepsin B at the mRNA level. Total RNA was extracted from both non-GICs and GICs, and mRNA expression levels of uPAR and cathepsin B were determined by RT-PCR. (E–F) Distribution of cells in different phases of cell cycle. Non-GICs and GICs transfected with pSV and pCU with or without radiation were trypsinized and stained with propidium iodide as per standard protocols. Changes in cell-cycle phases were determined by measuring cellular DNA content using a flow cytometer. Histograms represent the percent of cells in sub G0-G1, G0-G1, S, and G2-M phases. (G–H) Cells were stained for apoptosis using TdT-mediated dUTP nick end-labeling (TUNEL) assay. Data shown are representative of 3 experiments. (I–J) Quantification of apoptotic cells expressed as percent of DAPI-stained cells. Bars represent the mean ± SD of 3 experiments (**P < 0.001).