Antimycin

Mitochondria and homogenates were loaded into Seahorse XF96 microplate in 20 μl of MAS. The loaded plate was centrifuged at 2,000 g for 5 min at 4°C (no brake) and an additional 130 μl of MAS for mitochondria or MAS containing cytochrome c (10 μg/ml, final concentration), in the case of homogenates, was added to each well with the same consideration as in the fresh protocol. In the case of brain and lung homogenates, alamethicin (10 μg/ml, final) was also added to the MAS containing cytochrome c solution to allow complete membrane permeabilization to substrates. Substrate injection was as follows: pyruvate + malate (5 mM each), NADH (1 mM), or 5 mM succinate + rotenone (5 mM + 2 μM) were injected at port A; rotenone + antimycin A (2 μM + 4 μM) at port B; TMPD + ascorbic acid (0.5 mM + 1 mM) at port C; and azide (50 mM) at port D. These conditions allow for the determination of the respiratory capacity of mitochondria through Complex I, Complex II, and Complex IV.
When using RIFS in tissue lysates, we loaded the same protein amount independently of the substrate. For liver, we loaded 4 and 8 μg for mitochondrial and homogenate samples, respectively. For WAT, we loaded 6 and 15 μg for mitochondrial and homogenate samples, respectively. We loaded the following protein homogenates for BAT (3 μg), heart (2 μg,), kidney (4 μg), brain (4 μg), skeletal muscle (6 μg), soleus (6 μg), and lung (15 μg).
We used frozen liver mitochondria to test OXPHOS inhibitors specificity and ATP allosteric inhibition of COX. Inhibitors (metformin, phenformin, 3‐nitropropionic acid, and potassium cyanide) or ATP at the indicated concentration were added to MAS after centrifugation of the plate containing the mitochondria.
When using RIFS in zebra fish muscle homogenates, 30 μg of homogenates was loaded into Seahorse XFe24 microplate in 50 μl of MAS. When using RIFS in deyolked zebra fish embryos (pool of 300 embryos per condition, 30 μg of homogenate loaded per well). The loaded plate was centrifuged at 2,000 g for 5 min at 4°C (no brake), and an additional 475 μl of MAS containing cytochrome c (10 μg/ml, final concentration) was added to each well. Substrate injection was as follows: NADH (1 mM) or 5 mM succinate + rotenone (5 mM + 2 μM) were injected at port A; rotenone + antimycin A (2 μM + 4 μM) at port B; TMPD + ascorbic acid (0.5 mM + 1 mM) at port C; and azide (50 mM) at port D. These conditions allow for the determination of the respiratory capacity of mitochondria through Complex I, Complex II, and Complex IV. The experiment was performed at 28°C.
When using RIFS in human cell lines, fresh and frozen THP‐1 cells were seeded into a Seahorse XF96 plate at 80,000 cells/30 μl/well. The plate was centrifuged at 2,300 g for 5 min with no brake. After centrifugation, 150 μl of MAS (fresh cells) or MAS supplemented with cytochrome c (10 μM) and alamethicin 2.5 μg/ml (frozen) were added to each well. The cartridge was loaded with desire substrates, for Complex I determination a mix of PMP + pyr‐mal + FCCP (fresh) or NADH (frozen). Complex II substrates were PMP + succinate + rotenone + FCCP for both fresh and frozen cells. Then, rotenone (2 μM) or antimycin A (10 μM) to inhibit Complex I or Complex II, followed by ascorbate–TMPD for Complex IV activity and finally azide 20 mM was injected.
The isolated blood cells were thawed and suspended in XF‐DMEM media to seed 150,000 monocytes/well, 300,000 lymphocytes/well, and 10 × 10⁶ platelets/well in 30 μl media. The plate was centrifuged at 1,300 g for 1 min with no brake, then rotated by 180 degrees and centrifuged again. After centrifugation, 150 μl of MAS or MAS supplemented with cytochrome c (10 μM) and ALA 2.5 μg/ml (frozen) were added to each well. The cartridge was loaded with same concentrations of substrates and inhibitors used for THP‐1 cells.

The inhibitory effect of different concentrations of antimycin A₁ on the mycelial growth of R. solani was determined by the mycelial growth rate method (Yang et al., 2020 (link)). The specific methods were as follows. Antimycin A₁ was dissolved in a small amount of DMSO (dimethyl sulfoxide), prepared in a 1 mg/mL antimycin A₁ solution with sterilized distilled water in a sterile environment and mixed with PDA medium. PDA medium with final concentrations of 0.416 μg/mL, 0.83 μg/mL, 1.66 μg/mL, 3.33 μg/mL, 6.66 μg/mL, 13.33 μg/mL and 26.66 μg/mL with DMSO concentrations less than 0.25% was prepared in a sterile workbench. In the centre of the plate, R. solani mycelium cake (5 mm) was inserted, the mycelia were facing down, the petri dish was sealed with a sealing film, and a plate containing an equal amount of sterile water with a DMSO concentration less than 0.25% was used as a blank control. Each treatment was repeated 3 times, and the culture was carried out in an incubator at 25°C. In all groups, the colony diameter of each plate was measured by the cross-crossing method, the colony growth inhibition rate of antimycin A₁ against R. solani was calculated, and the average inhibitory rate was taken. The following formula was used to calculate: $\mathrm{Inhibition}\mathrm{rate}\%=\left[\frac{\left(\mathrm{C}-\mathrm{d}\right)-\left(\mathrm{S}-\mathrm{d}\right)}{\left(\mathrm{C}-\mathrm{d}\right)}\right]\times 100\%$
C and S are the average diameters of fungal colonies in the blank control group and the antimycin A₁ treatment group, respectively, and d is the diameter of the fungal cake.

bEnd.3 cells were seeded into plates and cultured with DMEM containing 10% FBS and 1% P/S for 24 hours. Next, the medium was treated with DMEM containing relative concentrations of antimycin A (Sigma Aldrich, Cat. a8674) and rotenone (Sigma Aldrich, Cat. r887) for 3 hours. After washing gently with PBS, the medium was replaced by DMEM containing 10% FBS and 1% P/S, and culturing was continued for further assays.