Figure 4. Effect of nimbolide on the mRNA and protein expression of GSK-3β and β-catenin in the hamster buccal pouch.

(mean ± SD; n = 3). (A) Quantitative RT-PCR analysis of GSK-3β and β-catenin. The fold change in transcript expression for each gene was determined using the 2−ΔΔCt method. Data are the mean ± SD of three separate experiments. Statistical significance was determined by the Mann–Whitney test (p < 0.05). (B) Western blots showing expression levels of phosphorylated and unphosphorylated GSK-3β and β-catenin in control and experimental animals. Gapdh and histone H2B were used as loading control for cytosol and nuclear proteins respectively. (C) Background subtracted protein bands were quantified and normalized to Gapdh. Phosphorylated proteins are normalized by their unphosphorylated forms. Each bar represents the protein expression ± SD of three determinations. (D) Immunohistochemical analysis of p-GSK-3β^Ser9 and p-β-catenin ^{Ser33,Ser37,Thr41}. ^♣ significantly different from control (p < 0.05). *significantly different from DMBA-treated group (p < 0.05) The groups are indicated as C (Control), D (Hamsters painted with DMBA for 12 weeks), D + N (Hamsters painted with DMBA for 12 weeks followed by nimbolide administration from 12 to 16 weeks), D + N (Hamsters painted with DMBA for 12 weeks followed by wortmannin administration from 12 to 16 weeks).