Figure 5 (IMAGE)
Caption
BIRC5 mRNA Expression in NET Cell Line. (A). Radiation increases BIRC5 gene expression of NCI-H720 cells. Cells growing as clusters in suspension in triplicate wells were subjected to one dose of 15 Gy X ray radiation. Control cells (0 Gy) were not subjected to radiation. After 2 days, BIRC5 gene expression normalized to that of housekeeping ACTB gene was quantified by reverse transcription-PCR. Mean and range (n = 3) of relative BIRC5 expression are depicted. (B) siRNA-mediated BIRC5 knock-down in NCI-H720 cells. Single-cell suspensions of NCI-H720 were transfected with a non-specific siRNA (Neg. ctrl.) or with one of two siRNAs against BIRC5 (BIRC5#1 and #2) at a concentration of 8 nM. Whole cell lysates were prepared from the transfectants after 2 days and subjected to immunoblotting to detect BIRC5 and housekeeping calnexin proteins. Different portions of the same blot were used to detect the two proteins. Relative band intensities as measured by image densitometry are listed. (C) BIRC5 knock-down reduces NCI-H720 proliferation. Cells were transfected with siRNAs as described for panel B. Average cross-sectional area of cell clusters in cultures of transfectant cells 6 and 9 days after siRNA transfection was determined by quantitative analysis of light microscopy images. Mean and range (n = 3) of fold-change in the average cross-sectional area of cell clusters during the 3 days are depicted. (D) BIRC5 knock-down does not enhance radiation sensitivity of NCI-H720 cells. Cells were transfected with siRNAs as described for panel B and transfectants were dissociated a day later into single-cell suspensions (0.2 million cells/ml) and immediately treated with a single dose of radiation (1.5, 3, or 6 Gy). Average cross-sectional area of cell clusters in cultures of transfectant cells 6 days after radiation was determined by quantitative analysis of light microscopy images. Mean and range (n = 4) of average cell cluster radius relative to non-irradiated (0 Gy) cells are shown.
Credit
Correspondence to - Renuka Iyer - Renuka.Iyer@RoswellPark.org
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