Figure 2.

(A) RT-PCR blot in which siRNA (10, 25, and 50 nM) was used to specifically silence IR-B in DU145 cells. Cells treated with a non-silencing (NS) siRNA acting as a negative control. After 24 h, sample RNA was extracted and RT-PCR was used to assess the expression of the IR-B isoform. This is representative of blots performed at least three times. (B) Representative RT-PCR blot shows the effect of 50 nM IR-B siRNA in DU145 cells after 24, 48, and 72 h of treatment. A non-silencing siRNA acted as a negative control. This is representative of blots performed at least three times. (C) Q-PCR ΔΔCT IR-B/IR fold expression (%) normalized to non-silencing samples. IR-B was silenced for 24, 48, and 72 h in DU145 cells. Error bars represent the SE of the mean from three experiments each repeated in duplicate (n = 3). (D) DU145 cells were seeded in six well plates (0.1 × 10⁶ cells per well) in the presence or absence of target siRNA to the IR-B or non-silencing (NS) siRNA for 24 h and then switched to serum-free media for a further 24 h followed by dosing with IGF-II or insulin for 48 h. Changes in cell proliferation were assessed by direct count of viable cells in a hemocytometer. Results shown are the mean of three independent experiments each repeated in triplicate. Data are represented as mean ± SEM. (E) (i) DU145 cells were seeded in 6 well plates (0.1 × 10⁶ cells per well) in the presence or absence of target siRNA to the IR-B or non-silencing (NS) siRNA for 24 h and then switched to serum-free media for a further 24 h followed by dosing with IGF-II or insulin for 30 min. Western blotting was performed to show protein abundance of p-AKT/AKT, p-MAPK/MAPK, and p-IGF-IR/IGF-IR (n = 3 experiments). (ii) shows a western blot and optical density measurements for levels of IR-β following IR-B silencing and (iii–v) shows the mean (n = 3) optical densitometry measurements from the western blots for p-AKT/AKT, p-MAPK/MAPK, and p-IGF-IR/IGF-IR respectively: a representative example of which is shown in (E) (i).