Evidence-Based Complementary and Alternative Medicine / 2013 / Article / Fig 4

Research Article

Ganoderma tsugae Extract Inhibits Growth of HER2-Overexpressing Cancer Cells via Modulation of HER2/PI3K/Akt Signaling Pathway

Figure 4

Effect of GTE on the gene expression and protein stability of HER2. (a) SKOV-3 cells were treated with GTE (0.25 or 0.5 mg/mL) or the vehicle for 24 h. The mRNA level of HER2 was measured by semiquantitative RT-PCR as described in Section 2. (b) SKOV-3 cells were transfected with a luciferase gene plasmid construct driven by HER2 promoter (pHER2-luc) for 6 h and then treated with various concentrations of GTE (0, 0.25, and 0.5 mg/mL) for 24 h. The activity of HER2 promoter was measured by a reporter gene assay, as described in Section 2. The relative light units (RLU) of luciferase activity were normalized against β-gal activity. (c) SKOV-3 cells were pretreated with 20 μg/mL of cycloheximide (CHX) for 30 min and then treated with GTE (0.5 mg/mL) or the vehicle for 12, 24, and 48 h. Stability of HER2 was determined by measuring the protein’s half-life. (d) SKOV-3 cells were treated with GTE (0.5 mg/mL) for 12, 24, and 48 h. To detect polyubiquitinated HER2 (HER2- ), HER2 was immunoprecipitated and subjected to Western blot analysis using an antibody to ubiquitin. The total protein levels of HER2 and actin in the whole-cell extracts were also detected by Western blotting. (e) SKOV-3 cells were pretreated with proteasome inhibitor (LLnL) or the vehicle for 30 min and then treated with GTE (0.5 mg/mL) for 24 h. The protein level of HER2 was measured by Western blotting. P, parental SKOV-3 cells; C, vehicle control. Data are presented as the mean ± SD of three independent experiments. and versus the vehicle-treated control group.
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