Cytochrome c''

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.

After treating A431 and A388 cells with BTZ for 48 h, cells were collected and resuspended in a hypotonic buffer. Mitochondrial and cytosolic protein fractions, as mentioned earlier, were isolated following the protocol outlined by Uddin et al. [74 (link)]. Subsequently, the cytosolic fraction of A431 and A388 cells was resolved using 12% SDS-PAGE and probed with antibodies against cytochrome c and HSP60. The presence of cytochrome c in the cytosolic fraction indicated apoptosis initiation [73 (link)].

Jurkat cells at 2 ×10⁶ cells/mL were incubated in the presence of DMSO or 50 μM BIP-V5 inhibitor^{54 (link)} (Yuanye Biotechnology Co., Ltd, 579492-81-2) for 24 h. Two groups of above treated cells were each divided into four groups and incubated in the presence of DMSO, venetoclax at 2.5 μM, cp4-TAT at 20 μM, or cp5-TAT at 15 μM in serum-free RPMI medium for 4 h. Then the medium was supplemented with FBS to 10% and incubated for another 8 h. After being treated by venetoclax or CPs for totally 12 h, cell were processed with the Mitochondrial Separation Kit (Beyotime, C3601) to extract mitochondria. Cytochrome C in mitochondria and BAX proteins in the whole cells were detected by Western blot.