Screening Active Compounds from Garcinia Species Native to China Reveals Novel Compounds Targeting the STAT/JAK Signaling Pathway

Figure 4

Assessing the mechanism of apoptosis induction for the compounds. (a) mRNA expression level of proapoptotic genes after treatment. NCI-H1650 cells were treated with different concentrations of each compound for 24 hr. Cells were harvested for RNA extraction. Q-PCR was performed by the TaqMan method on total RNA using primers specific for the PUMA, NOXA, and CDIP genes. (b) Immunoblot demonstrating the effect of GB32, GB39, and GB40 treatment on ERK, AKT, and STAT3 in NCI-H1650 cells. Treatment compounds were incubated for 8 h, and then whole cell lysates were processed for western blot analysis. Immunoblots were probed with the indicated antibodies. GAPDH served as a loading control. (c) GB39 significantly inhibits JAK2 of JAK family in a dose-dependent manner. After compound and substrates were added, the enzyme reaction was initiated by adding JAK2 enzyme and incubated for 2 hours. The reaction was then stopped, and the signal was detected using a Caliper LabChip 2000 plate reader. (d) GB40 significantly inhibits JAK2 of JAK family in a dose-dependent manner. After compound and substrates were added, the enzyme reaction was initiated by adding JAK2 enzyme and incubating for 2 hours. The reaction was then stopped, and the signal was detected using a Caliper LabChip 2000 plate reader. (e) HepG2 cells were pretreated with GB39 and GB40 for 2 h before stimulation by IL-6 (10 ng/mL) for 15 min. Whole cell lysates were processed for western blot analysis, and immunoblots were probed with the indicated antibodies. GAPDH served as a loading control.

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