Fig. 7.

Downregulation or inhibition (Inhi) of phospholipase D1 (PLD1) or PLD2 attenuates bleomycin-induced mitochondrial (mt)DNA damage in bronchial epithelial cells. A: bronchial airway epithelial (Beas2B) cells grown in 35-mm dishes (~90% confluence) were treated with MitoTempo (100 μM) for 3 h before exposure to bleomycin (10 mU/ml) for 24 h. B: Beas2B cells grown in 35-mm dishes (~60% confluence) were transfected with scrambled (sc)RNA or PLD2 small interfering (si)RNA (50 nM, 24 h) before bleomycin challenge as in A. C: Beas2B cells in 35-mm dishes (~90% confluence) were pretreated with 250 nM PLD1 inhibitor VU0155609, 500 nM PLD2 inhibitor VU0364739, or a mixture of 250 nM PLD1 + 500 nM PLD2 inhibitors for 3 h prior to bleomycin challenge (10 mU/ml) for 24 h. mtDNA damage was determined by Q-PCR-based measurement using isolated whole genomic DNA, both mitochondrial and nuclear DNA, as indicated in materials and methods. DNA damage was expressed as the ratio of lesion frequency to fragment. Values are means ± SD from 3 independent experiments in triplicate. *P < 0.05, **P < 0.005.