Fig 1.
Bioenergetic phenotyping using Seahorse technology.
A: Mitochondrial stress test where OCR is expressed per μg protein. Indices of mitochondrial function, basal respiration, ATP production, proton leak, maximal respiration, respiratory reserve and non-mitochondrial respiration, were compartmentalized by a sequential application of pharmacological inhibitors, oligomycin, FCCP and a combination of rotenone and antimycin (R+A). B: The proportion of OCR due to ATP synthase, proton leak and non-mitochondrial oxygen consumption was quantified by taking “resting” OCR as 100% (left panel). The proportion of above three indices plus reserve capacity is quantified by taking “highest” OCR as 100% (right panel, n = 8). Note that “resting” and “highest” OCR are the sum of basal/maximal OCR and non-mitochondrial OCR. C: Glycolysis stress test was carried out by a sequential application of glucose (Glu) pharmacological inhibitors of OXPHOS (Oligo) and glycolysis (2-DG) to dissect basal acidification, glycolysis, glycolytic capacity, glycolytic reserve and non-glycolytic acidification. Change in ECAR is expressed per μg protein. D: ECAR is shown either as percentage of three components (glycolysis, glycolytic reserve, and non-glycolytic acidification) by taking “highest” ECAR as 100% (left panel) or per μg protein including glycolytic capacity, a sum of glycolysis and glycolytic reserve (right panel, n = 8).
Fig 2.
ATP production rate from OXPHOS and glycolysis.
A: OCR (circle, left Y axis) and PPR (square, right Y axis) of mitochondrial stress test, both expressed as per 2.0x104 cells. R is rotenone and A is antimycin B: PPR of glycolysis stress test expressed as per 2.0x104 cells. C: OCR from OXPHOS (left bar, left Y axis) and PPR from glycolysis (right bar, right Y axis) expressed as per 2.0x104 cells (n = 8). D: ATP production rate from OXPHOS (left bar) and glycolysis (right bar) expressed as per 2.0x104 cells (n = 8, p = 0.03).
Fig 3.
Shift in bioenergetic phenotype in response to metabolic modulators.
The change in relative bioenergetic phenotype of HCASMCs after exposure to 10 mM glucose (in glucose-free medium) (A, n = 8), 5 mM 2-DG (B, n = 8), 1 μM rotenone (C, n = 8), 1 μM antimycin (D, n = 7), 1 μM oligomycin (E, n = 8) and 0.75 μM FCCP (F, n = 8). Shift in bioenergetic phenotype can be detected as relative positional change from basal value (filled circle) after drug application (open square) when ECAR is plotted on the X axis and OCR on the Y axis. Panel G and H summarize percent changes of OCR and ECAR against control level.
Fig 4.
Cellular bioenergetics of HCASMCs during culture.
Summary of mitochondrial stress test using cells at passage (P) 7, 10 and 13. The proportion of OCR due to ATP synthase, proton leak and non-mitochondrial oxygen consumption when “resting” OCR was taken as 100% (top panels). The proportion of above three indices plus reserve capacity when “highest” OCR was taken as 100% (bottom panels, n = 8). Note that results of P7 are previously shown in Fig 1B.
Fig 5.
Effects of metabolic inhibitors on cellular ATP.
Effect of metabolic inhibitors on cellular ATP was measured after 10 min in the presence of 10 mM glucose (A) or 0 mM glucose (B and C) (n = 4). Rote: 1 μM rotenone. Anti: 1 μM antimycin. Oligo: 6 μM oligomycin. CCCP: 1 μM CCCP. 2-DG: 5 mM 2-DG. Significant difference was assessed using ANOVA. Note that rotenone application in the presence of 10 mM glucose (panel A second from left) did not cause significant change, but all other treatments with metabolic inhibitors significantly changed luminescence reading against control values.
Fig 6.
Effect of metabolic inhibitors on mitochondrial membrane potential.
Left panel of each set shows change in fractional fluorescence of the cells treated with 1 μM rotenone (A), 1 μM antimycin (B), 6 μM oligomycin (C) and 1 μM CCCP (D). Right panel of each set shows mean ±SEM of fractional fluorescence before and after application of rotenone (A, p<0.001, n = 33), antimycin (B, p<0.001, n = 30), oligomycin (C, p<0.05, n = 16) and CCCP (D, p<0.001, n = 17). Statistical significance was evaluated using paired Student’s t-test.
Fig 7.
Effects of metabolic inhibitors on cellular ATP:ADP ratio.
Effect of metabolic inhibitors on cellular ATP:ADP ratio measured after 15 min in the absence and presence of, left to right, DMSO (NS), 5 mM 2-DG (p<0.01), 1 μM rotenone (p<0.01), 1 μM antimycin (p<0.05), 6 μM oligomycin (p<0.001) and 1 μM CCCP (p<0.05). Statistical analysis was performed using Student’s un-paired t test (n = 4).