Figure 5

AMPK-α1-knockdown THP-1 cells are highly sensitive to TNF-α-induced apoptosis. (a and b) TAK1^−/− MEF cells were transfected with mock or Myc-TAK1 vector. At 24 h after trasnfection, cells were treated without (a) or with (b) TNF-α (100 ng/ml) for 24 h, and then stained with BD Cycletest Plus-DNA reagent in accordance with the manufacturer’s protocol. The stained cells were analyzed with FACSCalibur system and apoptotic cell death was determined with Modfit LT 3.0 software. Error bars represent mean±S.D. of three independent experiments. (c and d) The wt THP-1 (c) and AMPK-α1^KD THP-1 cells (d) were cultured with culture medium containing 2% FBS for 24 h, and then treated with or without TNF-α (100 ng/ml) for different times, as indicated. Cells were harvested and stained with FITC Annexin V Apoptosis Detection Kit (BD Biosciences) in accordance with the manufacturer’s protocol. Samples were analyzed with FACSCalibur system and apoptotic cells were determined with CellQuest software. Error bars represent mean±S.D. of three independent experiments. (e–g) The wt THP-1 and AMPK-α1^KD THP-1 cells were cultured with culture medium containing 2% FBS for 24 h, and then treated with or without TNF-α (100 ng/ml) for different times, as indicated. Caspase-3 (e), caspase-8 (f), and caspase-9 (g) activities were measured using the CaspACE kit according to the manufacture’s instructions. Error bars represent mean±S.D. of three independent experiments. *P<0.01. (h) The genomic DNA extracts were prepared as described in Materials and Methods, run on 1.8% agarose gels, and visualized under UV illumination