fig1

Figure 1. Mitochondrial respiration in TGCTs resistant and sensitive cell lines. (A) The representative trace from the measurement of mitochondrial respiration following the SUIT-008 protocol^[16]. The blue line represents the concentration of O₂ in the chamber and the red line shows the consumption of O₂ as flow per cells (pmol/s/10⁶ cells). A total of 1 × 10⁶ cells was used in a 2 mL chamber of an O2k-respirometer and mitochondrial respiration of the TGCT cells was measured in mitochondrial respiration medium MiR05^[15] with 20 mM creatine at 37 °C under continuous stirring at 750 rpm. The trace is from the measurement of the 2102EP cell line. After stabilization at ce, cells were permeabilized by Dig and chemicals were consecutively added to the O2k chamber following SUIT-008 protocol: PM; ADP; cyt c; G; S; U; Rot; Gp; U; Ama. All substrates were titrated in kinetically saturating concentrations and uncoupler FCCP was titrated in optimum concentration to reach the maximum flux. The descriptions above and below the graph show: CI: Complex I pathway; CI&II: complex I and II pathways; CII: complex II pathway; CII&Gp: complex II and GpDH pathways; LEAK: non-phosphorylating resting state of respiration; OXPHOS: the phosphorylating state of respiration; ET: noncoupled state of respiration at optimum uncoupler concentration. The labels above the red line assign the respiration after the titration steps. (B-G) Parameters of mitochondrial respiration in cell lines measured following SUIT protocol 008^[16] evaluated as O₂ flux/CS (pmol/s/IU). The columns represent the mean ± SEM of the respiratory capacities after the titration steps shown in Figure 1A. For evaluation of CIV activity, artificial substrates TMPD and ascorbate were titrated after Ama supporting electron flow to CIV, then sodium azide (200 mM) was added to inhibit CIV. The activity of CIV was evaluated as a difference between O₂ fluxes before and after the addition of sodium azide^[16]. 1411HP, 1777NRpmet: intrinsically resistant TGCT cell lines; T-cam: T-cam Cis - seminomas TGCT cell lines; H12.1/H12.1ODM, 2102EP/ 2102EP Cis, NTERA-2/ NTERA-2 Cis: non-seminomatous TGCT cell line pairs (sensitive/resistant); Hs1.Tes: control cell line. ^*P ≤ 0.05, ^**P ≤ 0.01, ^***P ≤ 0.001 for resistant vs. sensitive variant of the cell line; ^#P ≤ 0.05, ^###P ≤ 0.001 for 1777NRpmet vs. 1411HP line. TGCT: Testicular germ cell tumors; ce: routine respiration; Dig: digitonin; PM: pyruvate + malate; ADP: adenosine diphosphate; cyt c: cytochrome c; G: glutamate; S: succinate; U: uncoupler; Rot: rotenone; Gp: glycerophosphate; U: uncoupler; Ama: antimycin A; FCCP: carbonyl cyanide-p-trifluoromethoxyphenylhydrazone; CIV: complex IV.