Figure 2

Accumulation and elimination of 8-Cl-ATP and effects on ATP production. (A) 10 μM 8-Cl-Ado treatment, hatched bars, perturbs basal OCR as compared to untreated, solid bars, in MCF-7, gray bars, and BT-474 cells, black bars. Effects of altering ETC complexes with the agents, oligomycin, FCCP, antimycin, and rotenone on OCR in (B) MCF-7 and (C) BT-474 cells treated with, black circles, and without, gray circles, 10 μM 8-Cl-Ado. (D) 10 μM 8-Cl-Ado treatment, hatched bars, reduces basal glycolysis as demonstrated by decreased ECAR in MCF-7, gray bars, and BT-474 cells, black bars. (E) Time dependent depletion of the endogenous ATP pool, (F) the accumulation of 8-Cl-ATP, and (G) changes in the 8-Cl-ATP/ATP ratio in MCF-7, ○, and BT-474 cells, ●. Cells were treated with 10 μM 8-Cl-Ado for the indicated times and acid extracts were analyzed by HPLC to measure nucleotide levels. (H) The elimination of 8-Cl-ATP in MCF-7, ○, and BT-474 cells, ●. Cells were treated with 10 μM 8-Cl-Ado for 3 days, washed with PBS, and cultured in fresh medium. At the indicated times, acid extracts were analyzed as above.