Fig. 5
DNA-damage response and induction of aneuploidy in Survivin-transduced glioma cells. a Western Blot analysis showing the activation of a DNA damage response and a stabilized p53 protein in U251-MG and SVGp12 cells that overexpress Survivin. The membranes were blotted with anti-p53 (53 kDa), anti-p21waf/cip (21 kDa), anti-p53s15 (53 kDa), anti-γH2AX (16 kDa), anti-ATM (350 KDa), anti-pATM S1981 (350 kDa) and anti-pCHK2 T68 (62 kDa) antibodies. The indicated values represent the relative band density (fold increase) obtained by densitometric analysis when compared to the empty vector-transduced control. Membranes were re-probed with α-actin (42 kDa) to confirm equal loading. b, c, d SKY-Analysis reveals chromosomal instability (increased numerical and structural chromosomal aberrations) in Survivin-overexpressing U251-MG cells compared to mock-control. b shows representative karyograms of mock-control (upper figure) and Survivin-overexpressing cells (lower figure) with white arrows indicating clonal aberrations already present in the parental cell line. Survivin-overexpressing U251-MG show additional non-clonal structural changes indicated by purple arrows and also increased ploidy. The number of such non-clonal structural aberrations per metaphase was significantly increased compared to mock-control (c). Additionally, when counting gains and losses of chromosomes per chromosome and metaphase (compared to the mean number of a particular chromosome in the parental cell line), we found a significant higher number of “off-mode” chromosome numbers (d). **p < 0.001, ***p < 0.0005. e Indirect immunofluorescence analysis of U251-MG cells transduced with empty vector (Control) or with Survivin-vector. Note the non-aligned chromosomes (white arrowheads) in Survivin-transduced cells either containing or lacking immunosignals for centromers (white arrows). All data were collected 72 h after transduction of cells