Figure 5. PTEN overexpression results in increased mitochondrial oxygen consumption, mitochondrial ATP production and mitochondrial number.

a, OCR (oxygen consumption rate) was measured (Seahorse XF24 analyzer) in primary wt and tg MEFs (n=3 per genotype) under basal conditions and after addition of oligomycin, FCCP and Rotenone. b, Mitochondrial ATP production in wt and tg MEFs after agonist stimulation as described in the methods section. Where indicated, cells were treated with 100 µM ATP. Data are expressed as a percentage of the initial value. wt: 162 ± 8%; tg: 251 ± 18%. n=15 from three independent experiments and p<0.05 (mean ± s.e.m). c, Mitochondrial Ca²⁺ homeostasis measurements after agonist stimulation as described in the methods section. Where indicated, cells were treated with 100 µM ATP. wt: [Ca²⁺]_m peak 155 ± 8 µM. tg: [Ca²⁺]_m peak 131 ± 5 µM. n=12 from three independent experiments (mean ± s.e.m). d, Analysis of mitochondrial membrane potential (ΔΨ_m) in wt and tg MEFs. Cells where loaded with TMRM as described in the methods section. Where indicated cells were treated with FCCP to collapse completely the ΔΨ_m. The traces are representative of single cell responses (wt n=28; tg n= 33). e, Analysis of total and single mitochondrial volume and mitochondrial numbers as described in the methods section (wt n=43; tg n= 40 from three independent experiments and p<0.05). Mitochondrial morphology in wt and tg MEFs as revealed by mitochondrial targeted GFP visualization. Mitochondrial fragmentation index was calculated as described in the methods section (wt n=43; tg n= 40 from three independent experiments). Ordinates for the graphs of Network volume, Average mitochondrial volume and Mitochondrial number are Voxel/Cell, Voxel/Object and N° Object/Cell respectively. Error bars in a denote s.d.; error bars in d, e denote s.e.m. See also Figure S4.