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Celastrol Stimulates Hypoxia-Inducible Factor-1 Activity in Tumor Cells by Initiating the ROS/Akt/p70S6K Signaling Pathway and Enhancing Hypoxia-Inducible Factor-1α Protein Synthesis

Figure 2

Celastrol increases the HIF-1α protein level by enhancing its translation.

2a. Celastrol did not affect HIF-1α transcription. After treatment with different concentrations of Celastrol for 6 h, total RNA was extracted, and the mRNA levels of HIF-1α and RPL13A were determined by RT-PCR. The relative expression of HIF-1α was normalized to that of RPL13A. The values are presented as the means ± SD of three independent experiments. 2b. Celastrol enhanced the expression of the HIF-1α P402A/P564A mutant. HeLa cells were seeded in 6-well plates and transiently transfected with either pcDNA-V5 empty vector (ctrl) or pcDNA3-P402A/P564A-HIF-1α-V5 (mtHIF-1). Twenty-four hours later, the cells were treated with 1–2 µM Celastrol for 6 h under normoxia. Mutant HIF-1α expression was analyzed by western blotting with the anti-V5 antibody. 2c. Celastrol did not affect HIF-1α ubiquitination. HepG2 cells were treated separately with 4 µM Celastrol, 10 µM MG132 or Celastrol plus MG132 for 4 h under normoxia, and the ub-HIF-1α protein level was determined by western blotting using the anti-HIF-1α antibody. 2d. The effect of the protein synthesis inhibitor CHX on Celastrol-induced HIF-1α accumulation. HepG2 cells were treated with or without 4 µM Celastrol for 6 h. Then, 10 µM CHX was added to the culture medium, and the cells were collected at the indicated times (left). HepG2 cells were cultured in medium containing 100 µM CoCl2 with or without 4 µM Celastrol for 4 h, then 10 µM CHX was added, and the cells were collected at the indicated times (right). The HIF-1α protein level was analyzed by western blot analysis. 2e. Celastrol induced AKT/p70S6K activation under normoxia and hypoxia. HepG2 cells were challenged with the indicated doses of Celastrol for 6 h under normoxia or hypoxia. The protein levels were analyzed by western blotting with the corresponding antibodies. 2f. Celastrol induced AKT/p70S6K activation under serum starvation. HepG2 cells were cultured in serum-free medium for 24 h. Then, 10% FBS, 4 µM Celastrol or both were added, and the cells were cultured for another 6 h. The protein expression was analyzed by western blotting with the corresponding antibodies. 2 g. Celastrol induced the accumulation of ROS. HepG2 cells were treated with 4 µM Celastrol for 12 h under normoxia. The levels of ROS were measured by DCFH-DA staining and subsequently assayed by flow cytometry. 2 h. The effect of Celastrol-induced HIF-1α accumulation depends on ROS-mediated AKT activation. HepG2 cells were pretreated with 5 mM NAC or 10 µM LY294002 for 1 h. Then, 4 µM Celastrol was added to the culture medium, and the cells were cultured for another 6 h. The protein expression was determined by western blotting with the corresponding antibodies.

Figure 2

doi: https://doi.org/10.1371/journal.pone.0112470.g002