Fig. 2

Ras overexpression and Rb deletion in vitro, induce differential expression of MHC-I, Rae1α, Rae1αβγδε, Fas, FasL, Mult1, H60a, H60b and H60c. Murine astrocytes were transformed in vitro for the overexpression of Ras, the deletion of Rb or both. In addition, two cell lines were derived from tumors that develop in SCID mice after transplantation of in vitro transformed astrocytes (T653, and T731). Expression of cell surface molecules, as indicated, was assessed by flow cytometry after cell staining with specific antibodies, as described in material and methods. Mean fluorescence intensity numerical values were normalized and given a value of 1.0 for the parental cell (cRb ^loxP/loxP), and the fold change of expression for the transformed astrocytes was then calculated. The expression for (a) MHC-1, (b) Rae1, (c) Rae1αβγδε, (d) Fas, and (e) FasL is shown. Results represent the media +/− S.D. from three independent experiments. mRNA expression for Mult1, H60a, H60b and H60c (f) was assessed by Real Time PCR using specific primers and SYBR Green dye as described in material and methods. All expression levels of interested genes were normalized to the housekeeping gene β actin. Gene expression values were then calculated based on the ΔΔCt method. Results represent the media +/− S.D. from three triplicates. Statistical significance was set at p < 0.05