PMC

Trypan Blue Exclusion Assay assessment of cell viability of WT (open bars) and 123.7 cells (black bars) after menadione (MEN) treatment. Data are expressed as mean live cell count ± SD (n=5), *p<05 MEN vs. vehicle treated cells in each cell line.

A. ATP levels. ATP levels in WT cells (open bars) and 123.7 cells (black bars) exposed to normoxia (RA) or hypoxia (SH) with or without N-acetyl cysteine (NAC). Data are expressed as means of relative luminescence units (RLU) ± SD (n=4-7), ** p<005 WT-RA vs 123.7-RA, * p<01 WT-RA vs. WT-SH. B. cAMP levels. cAMP levels in WT cells (open bars) and 123.7 cells (black bars) exposed to normoxia (RA) or hypoxia (SH) Data are expressed as means ± SD (n=5-6), p= NS. C. COX IV Expression. Western blot of COX IV immunoreactivity in WT cells and 123.7 cells exposed to normoxia (RA) or hypoxia (SH) with or without N-acetyl cysteine (NAC). Data are representative of 3 individual experiments (n=3).

A. Reactive Oxygen Species Production assessed by DCF Fluorescence of WT cells (open bars) and 123.7 cells (black bars) exposed to 24h normoxia (RA) or hypoxia (SH), with or without N-acetyl cysteine (NAC). Data are expressed as relative fluorescence unit means ± SD (n=5), *p<.05 RA vs. SH; ^#p<.05 SH vs. SH + NAC. B. Effect of Antioxidant treatment on Trypan Blue Exclusion Assay. Assessment of WT (open bars) and 123.7 cells (black bars) live cell count in presence or absence of N-acetyl cysteine (NAC). Data are expressed as means live cell counts ± SD (n=5), * p<.05 RA vs. SH; ^#p<.05 SH vs. SH + NAC.

MTT Assay assessment of WT (A) and 123.7 (B) cell viability exposed to hypoxia (SH) at 0.1% O₂ (open bars), or 5% O₂ (grey bars) or to normoxia (RA). Data are expressed as O.D.₅₄₀ means ± SD (n=5), * p<.01 SH vs. RA control at one time point, + p<01 SH 0.1% vs. SH 5% at one time point. Trypan Blue Exclusion Assay. (C) Assessment of WT (open bars) and 123.7 cells (black bars) live cells count after 24 h normoxia (RA) or 24h hypoxia at 0.1% O₂ (SH). Data are expressed as means of live cell counts ± SD (n=10), *p<.001 RA vs. SH 0.1% O₂.

A. PKA activity assay 123.7 cells transfected with the active catalytic subunit of Protein Kinase A (PKA cat-α) or control vector (Vect) and exposed to hypoxia (SH) or to normoxia (RA). Data are expressed as counts per minute (CPM) means ± SD (n=5), * p<.005 PKA cat α transfected cells vs. vector controls of the same oxygen condition. B. PKA cat α expression. Western blot of active catalytic subunit of Protein Kinase A (PKA cat-α) immunoreactivity in PKA cat-α-transfected 123.7 cells compared to vector transfected cells. (Vect) exposed to normoxia (RA) and hypoxia (SH). Data are representative of 7 individual experiments (n=7).

A. Hypoxia-induced ROS Levels. DCF fluorescence in WT cells (open bars) and 123.7 cells transfected with PKA catalytic subunit (cat α; black bars) or with empty vector control ((Vect; grey bars) and exposed to normoxia (RA) or to hypoxia (SH). Data are expressed as relative fluorescence units (RFU) ± SD (n=5), * p<005 as indicated or PKA cat α transfected cells vs. vector controls of the same oxygen condition, **p<001 RA 24h vs. SH 0.1% 24h. B. Hypoxia-induced Cell Death. Trypan Blue exclusion cell count representing live WT cells (open bars) and 123.7 cells transfected with PKA catalytic subunit (cat α; black bars) or with empty vector control ((Vect; grey bars) and exposed to normoxia (RA) or to hypoxia (SH) for 24h. Data are expressed as means live cell counts ± SD (n=10), * p<05 RA 24h vs. SH 0.1% 24h. C. COX IV expression Expression of cytochrome oxidase IV (COX IV) in 123.7 cells exposed to normoxia (RA) or to 24 h hypoxia (SH) and transfected with the active catalytic subunit of Protein Kinase A (PKA cat-α) or empty vector control (Vector). Data are representative of 5 individual experiments (n=5). D. ATP Levels in WT cells (open bars) and 123.7 cells transfected with PKA catalytic subunit (cat α; black bars) or with empty vector control ((Vect; grey bars) and exposed to normoxia (RA) or to hypoxia (SH) for 24h. Data are expressed as mean relative luminescence units (RLU) ± SD (n=5-7), * p<005 RA 24h vs. SH 0.1% 24h for each condition.