Figure 7.

Changes in IGFBP-2 gene expression in response to different treatments in either 5 mM or 25 mM glucose conditions. (A) LNCaP cells were set up as described in Fig. 5A and pre-treated with an AMPK inhibitor – Compound C (2 µM) for 1 h before dosing with 5 mM metformin for another 24 h. The mRNA was extracted and Q-PCR was performed. Values of IGFBP-2 gene expression were normalised to the housekeeping gene (18S) (n = 3). Western blot insert shows successful inhibition of metformin-induced AMPK phosphorylation by Compound C. (B) LNCaP cells were transfected with 75 nM siRNA for α1 and α2 subunits of AMPK or 25 mM non-silencing control, whereas seeding in six-well plates (0.3 × 10⁶ cells/well) with 5 mM glucose and cultured as described in Fig. 1A. After 24 h treatment with 5 mM metformin, mRNA was extracted and Q-PCR was performed. Values of IGFBP-2 gene expression were normalised to the house-keeping gene (18S) (n = 3). (C) Changes in % cell death of LNCaP cells silenced with 75 nM siRNA for AMPK or 25 nM non-silencing control, whereas seeding in six-well plates (0.3 × 10⁶ cells/well) with 5 mM glucose and cultured as described in Fig. 1A. After 24 h treatment with 60 nM Docetaxel for further 24 h, levels of cell death were assessed using trypan blue cell counting (n = 3). Western blot insert shows effective silencing of AMPK. (D) Changes in % cell death of LNCaP cells set up as described in Fig. 6C but silenced with 30 nM IGFBP-2 siRNA or 25 nM non-silencing control and treated with 5 mM metformin or/and with 60 nM Docetaxel for 24 h. Cell death was assessed by counting using trypan blue (n = 3). Western blot insert shows effective silencing of IGFBP-2.