TOXSCI 160110 Repository data

Data used to created Figures 5-9

Bioenergetic phenotyping using Seahorse technology.

<p>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).</p

Effect of metabolic inhibitors on mitochondrial membrane potential.

<p>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 <i>t</i>-test.</p

Effects of metabolic inhibitors on cellular ATP.

<p>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.</p

Shift in bioenergetic phenotype in response to metabolic modulators.

<p>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.</p

Effects of metabolic inhibitors on cellular ATP:ADP ratio.

<p>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 <i>t</i> test (n = 4).</p

ATP production rate from OXPHOS and glycolysis.

<p>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).</p

Cellular bioenergetics of HCASMCs during culture.

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0177951#pone.0177951.g001" target="_blank">Fig 1B</a>.</p