Figure 1

OPN mRNA and protein levels of either non-irradiated or irradiated MDA-MB-231 cells after siRNA transfection. A. Quantitative real-time PCR: OPN mRNA levels of untreated cells and cells treated with siRNA targeting OPN or nonsense siRNA. Representative values of OPN mRNA levels (72 h after transfection) treated with OPN-specific siRNAs were normalized to those treated with nonsense siRNA. The value of the OPN mRNA level of cells that were treated with nonsense siRNA at 0 Gy was arbitrarily established as 100%. Data represent the average values (± SD) of three independent experiments (* p < 0.05, ** p < 0.001). B./C. Western blot: Western blot analyses of OPN with OPN specific antibody 0-17 (IBL). B. MDA-MB-231 cells were transfected with siRNA Mix as well as OpnS or with nonsense siRNA (non) for 24 h. Thereafter, MDA-MB-231 cells were incubated with serum-free culture media for another 24 h and 48 h. The Western blot shows the extracellular OPN protein levels (50 kDa) of MDA-MB-231 cells 48 h and 72 h after transfection with OPN specific siRNA Mix and OpnS, with nonsense siRNA (non) and untreated MDA-MB-231 control cells (UT). The Western blot shows one representative result out of two independent experiments. C. Intracellular OPN protein levels (64 kDa) of MDA-MB-321 cells 24 h after transfection. Cells were either untreated (UT) or treated with OPN specific siRNA Mix and OpnS or with nonsense siRNA (non) with and without irradiation at 2 Gy. The Western blot shows one representative result out of three independent experiments. Actin served as an internal loading control.