Expression of PrPC, Oct-4 and nestin in differentiating ES cells after siRNA-mediated knockdown of PrPC. (A) PrPC displayed immunoreactive bands at 35, 32 and 27 kDa. Reactivity was also detected for Oct-4, β-actin and nestin at 43, 42, 120 kDa, respectively. (B) Computerized quantification of migration bands indicated that PrPC expression was significantly reduced (P < 0.05) on Days 16 and 20 compared to scrambled control. Oct-4 levels were reduced at Day 8 with no differences between treatments. (C) PrPC mRNA levels were reduced in siRNA-PrPC ES cells at Days 16 and 20 compared to controls. (D) siRNA-PrPC ES cells showed lower levels of nestin levels on Days 16 and 20 compared to scrambled control. (E) PrPC and nestin immunoreactive signals were reduced in siRNA-PrPC ES cells compared to control colonies at day 20 of differentiation. Merged PrPC, nestin and DAPI for (e) scrambled control and (f) siRNA ES cells. Superscript (*) indicate significant (P < 0.05) difference. Bar scale pictures: (Up-left) 500 µm; (Up-right) 200 µm; (Down-left) 200 µm; (Down-right) 200 µm. Bar scale pictures: 200 µm.