Figure 6.

BI-1 reduces mitochondria bioenergetics. (A) Activity of mitochondrial dehydrogenases was measured in control ([−] doxy) and BI-1 overexpressing ([+] doxy) HeLa cells. Values were normalized for the number of viable cells (trypan blue exclusion). Background absorbance was read at 690 nm and subtracted from readings at 440 nm (A440nm − A690nm). The difference between samples was significant (P < 0.05, by unpaired t-test). (B) Endogenous oxygen consumption rates in control (no doxy) and BI-1-overexpressing (doxy) cells were monitored by an oxymeter before (NT) and after exposure to oligomycin (2 μM) and FCCP (1 μM). Results are expressed as nanomoles of O₂ per minute per 1 × 10⁶ cells. Results for untreated control versus BI-1-expressing cells were statistically significant (P = 0.0015). (C) RCR in control ([−] doxy) and BI-1 overexpressing ([+] doxy) cells was calculated by dividing O₂ consumption before and after addition of oligomycin. The efficiency of oxidative phosphorylation coupling was comparable in control and BI-1-overexpressing cells. (D) ATP levels were measured in control versus BI-1-overexpressing cells using a bioluminescence method. Data represent relative luminescence units (RLUs) per 10⁶ viable cells (mean ± SD; n = 3; P = 0.002. (E) Levels of extracellular lactate were measured in control ([−] doxy) and BI-1-overexpressing ([+] doxy) cells. Results are represented as nanomoles of lactate per well (mean ± SD; n = 3; P = 0.021. (F) Lysates from control versus BI-1-overexpressing cells were normalized for total protein content and subjected to immunoblot analysis to detect AMPK, phospho-AMPK, acetylCoA carboxylase (ACC), and phospho-acetylCoA carboxylase (P-ACC). Anti-HA antibody was used to detect expression of BI-1-HA protein. Equivalent sample loading was shown by monitoring tubulin levels.