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Role of hypoxia induced PKA on mitochondrial respiratory capacity.

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posted on 2013-10-10, 06:34 authored by Satish Srinivasan, Joseph Spear, Karunakaran Chandran, Joy Joseph, Balaraman Kalyanaraman, Narayan G. Avadhani

Macrophages grown under normoxic or hypoxic conditions in the presence or absence of H89 or KH7 were used for measuring Oxygen consumption in a Seahorse XF extracellular flux analyzer. Cells were maintained under either hypoxia or normoxia in presence of inhibitors for 12h. Before measurement of respiration, the cells were washed with fresh medium without inhibitors and plated on XF24 plates and incubated for additional 3h. A) Respiration measurement in presence of H89 (1µM) and KH7 (10µM) is shown. DNP=2,3 dinitrophenol. Rot/AA=Rotenone and Antimycin. This is a representative profile of three replicates B) The maximum OCR of cells treated with H89 or KH7 under hypoxia. Maximum OCR was calculated by subtracting residual OCR after adding Rotenone and Antimycin from OCR measured after uncoupling with 100µM DNP. The values were normalized to 100µg of total cellular protein. C) Maximum OCR in cells treated with different concentration of H89. Maximum OCR calculation and normalization were as described for B). Protection against loss of respiratory capacity by H89 was significant at 300nM and 500nM as indicated, compared to untreated hypoxic control. The values were derived from three separate experiments.*, p<0.05;**, p<0.005.


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