Figure 3.

siRNA knock down blocks radiation-induced increase of the pro-apoptotic genes Pkcδ or Bax. A, Dose response curve of irradiated salivary gland monolayer cultures. Cells were counted 24 hours after the indicated dose of γ-ray radiation. 7.5Gy single dose irradiation resulted in a significant (P < 0.05), 40% reduction in cell survival, and was used in all subsequent experiments. Radiation normally increases Pkcδ (B) or Bax (C) gene expression. However, nanoparticle-mediated siRNA knock down of Pkcδ or Bax expression blocks the radiation-induced increase. Cells were irradiated 24 hours after siRNA/nanoparticle addition, and mRNA levels were assayed by qPCR after an additional 24 hours. Results are representative of three replicates and compared to the untreated controls. D–E, Total protein extracts were analyzed by Western blot. Densitometric analysis of control and sample band ratios was conducted. The untreated control level of each protein was designated as 100%. D, Radiation-induced Pkcδ protein expression showed a statistically significant decrease in the presence of siRNA (i + Pkcδ) compared to the scrambled siRNA (scr) negative control. E, Radiation-induced Bax protein level was also reduced in cells in the presence of siRNA/nanoparticle complexes (i + Bax). (*P < 0.05, **P<0.001, n=3) Statistical significance was calculated using one-way analysis of variance with Tukey’s multiple comparison tests.