Fig. 6: Asparagine rescues respiration inhibition by supporting aspartate pools. | Nature Cancer

Fig. 6: Asparagine rescues respiration inhibition by supporting aspartate pools.

From: Differential integrated stress response and asparagine production drive symbiosis and therapy resistance of pancreatic adenocarcinoma cells

Fig. 6

a, Oxygen consumption of clonal line N treated with vehicle, oligomycin or phenformin, as measured by a Seahorse metabolic flux analyzer (n = 4 samples per condition). b–e, Relative abundance of TCA cycle metabolites (b), aspartate (c), nucleotides (d) and asparagine (e) present in clone N cells treated with asparagine, oligomycin, asparagine + oligomycin or vehicle 4 h after treatment (n = 3 samples per condition). f, Labeled aspartate pools of clone N prelabeled with universally labeled 13C-glutamine and switched to medium containing unlabeled glutamine and treated with asparagine, oligomycin, asparagine + oligomycin or vehicle for 4 h (n = 3 samples per condition). g,h, Relative aspartate (g) and nucleotide levels (h) of siNT- or siAsns-transfected clone N cells treated with 1 nM oligomycin for 4 h (n = 3 samples per condition). i, Aspartate levels of oligomycin-sensitive clone N and oligomycin-insensitive clone V relative to vehicle 4 h after treatment (n = 3 samples per condition). j, Ratio of NADH/NAD+ of clone N and clone V treated for 4 h with oligomycin or vehicle (n = 3 samples per condition). k, Labeled aspartate pools of sensitive clone N and insensitive clone V were treated for 4 h with oligomycin or vehicle and switched to medium containing universally labeled 13C-glutamine and treated with oligomycin or vehicle for an additional 4 h (n = 3 samples per condition); *P ≤ 0.05, **P ≤ 0.01 and ****P ≤ 0.0001 by one-way ANOVA with a Tukey post hoc test (a–f and j) or two-tailed Student’s t-test (g–i and k); vehicle versus oligomycin P < 0.0001, vehicle versus phenformin P < 0.0001 (a); pyruvate: vehicle versus oligomycin P = 0.0017, vehicle versus oligomycin + asparagine P = 0.0019, asparagine versus oligomycin P = 0.0021 and asparagine versus oligomycin + asparagine P = 0.0023; (iso)citrate: vehicle versus oligomycin P < 0.0001, vehicle versus oligomycin + asparagine P < 0.0001, asparagine versus oligomycin P < 0.0001, asparagine versus oligomycin + asparagine P < 0.0001; aconitate: vehicle versus oligomycin P < 0.0001, vehicle versus oligomycin + asparagine P < 0.0001, asparagine versus oligomycin P < 0.0001, asparagine versus oligomycin + asparagine P < 0.0001; α-ketoglutarate (AKG): vehicle versus oligomycin P < 0.0001, vehicle versus oligomycin + asparagine P = 0.0002, asparagine versus oligomycin P < 0.0001, asparagine versus oligomycin + asparagine P < 0.0001; succinate: vehicle versus oligomycin P < 0.0001, vehicle versus oligomycin + asparagine P < 0.0001, asparagine versus oligomycin P < 0.0001, asparagine versus oligomycin + asparagine P = 0.0004; malate: vehicle versus oligomycin P = 0.0208 (b); oligomycin versus oligomycin + asparagine P = 0.0072 (c); oligomycin versus oligomycin + asparagine: CTP P = 0.0295, AMP P = 0.0301, ADP P = 0.0082, ATP P = 0.0449, UMP P = 0.0348, UDP P = 0.0035, UTP P = 0.0429, inosine P = 0.0276, IDP P = 0.0101, guanosine P = 0.0015, GTP P = 0.0147 (d); vehicle versus asparagine P < 0.0001, oligomycin versus oligomycin + asparagine P < 0.0001 (e); P = 0.0106 (f); P = 0.0003 (g); siNT + oligomycin versus siAsns + oligomycin inosine P = 0.0007, IMP P = 0.0045, IDP P = 0.006, CMP P = 0.0058, cAMP P = 0.0022, AMP P = 0.0171, ADP P = 0.013, UMP P = 0.0017, dU P = 0.0308 (h); N versus V P < 0.0001 (i); N vehicle versus V vehicle P = 0.0002, N oligomycin versus V oligomycin P < 0.0001 (j); P = 0.027 (k).

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