Figure 4.
Lupeol suppresses expression of STAT3-regulated gene products involved in proliferation, survival and angiogenesis. (A) The HepG2 cells (2 × 106 per ml) were treated with 50 μM lupeol for indicated time intervals, after which whole-cell extracts were prepared and 30 μg portions of those extracts were resolved on 10% SDS–PAGE, membrane sliced according to molecular weight and probed against cyclin D1, Bcl-xL, survivin, VEGF and MMP-9 antibodies. The same blots were stripped and reprobed with β-actin antibody to verify equal protein loading. The results shown are representative of two independent experiments. (B) The HepG2 cells (3 × 105 per ml) were treated with 50 μM lupeol for the indicated time intervals, after which cells were harvested after treatment and RNA samples were extracted. Then, 1 μg portions of the respective RNA extracts was used for reverse transcription to generate corresponding cDNA. Real-time PCR was performed to measure the relative quantities of mRNA. Each RT product was targeted against Mcl-1 and survivin probes, with GAPDH as endogenous control for measurement of equal loading of RNA samples. Results were analysed using Sequence Detection Software version 1.3 provided by Applied Biosystems. Relative gene expression was obtained after normalisation with endogenous GAPDH and determination of the difference in threshold cycle (Ct) between treated and untreated cells using 2-ΔΔCt method. (C) Treatment with lupeol decreases binding of STAT3 to VEGF promoter. Chromatin immunoprecipitation (ChIP) assay was performed in HepG2 cells to examine binding of STAT3 on VEGF promoter. Cells were treated with lupeol for indicated time and crosslinked. The cell pellets were processed for ChIP as detailed under Supplementary Materials and Methods. Ratio of quantitative real-time PCR signal in STAT3 immunoprecipitate relative to IgG (negative control) immunoprecipitate is plotted. Mean±s.d. of three experiments (*P<0.05).