Fig. 3.

Glaucarubinone and gemcitabine synergistically reduced the amounts of active PAK1 and PAK4 in vitro. The effects of glaucarubinone (Glau) or gemcitabine (Gem) alone, or in combination (GG), on the amounts of total PAK1 and active phospho-PAK1 (pPAK1) (A and C) or total PAK4 and active phospho-PAK4 (pPAK4) (B and D) were determined in MiaPaCa-2 (A and B) or PANC-1 (C and D) cells by Western blotting using appropriate antibodies as described in Section 2. The concentrations of glaucarubinone used approximated the IC₅₀ values calculated from the proliferation assays (Fig. 1), and were 50 nM and 300 nM for MiaPaCa-2 and PANC-1 cells, respectively. The concentrations of gemcitabine used approximated the IC₅₀ values determined in similar proliferation assays (data not shown), and were 50 nM for both MiaPaCa-2 and PANC-1 cells. Variations in protein loading were corrected by GAPDH expression, and the corrected pPAK1/PAK1 or pPAK4/PAK4 ratios from untreated control cells [28] were taken as 100%. The data represent mean ± SEM, summarised from three independent Western blots. *p < 0.05; **p < 0.01 compared to control. ^#p < 0.05 compared to Glau treatment.