Mito tempo

Cells (1 × 10⁴) were seeded into 96-well culture plates overnight at 37 °C. Then, the cells were pretreated for 1 h with EUK-134 (Cayman Chemical Company, Ann Arbor, MI, USA) or Mito-TEMPO (Enzo Life Sciences, Farmingdale, NY, USA) and then co-incubated with or without 200 µM DFO (Sigma) for 24 h. Following the incubation period, 100 μL DMEM including 10 μL Cell Counting Kit-8 solution (CCK-8 reagent, Dojindo, Kumamoto, Japan) was added to each well; the samples were incubated for 2 h at 37 °C. Cell viability was analyzed by measuring the absorbance at 450 nm with a microplate reader (Synergy 2 Multimode Microplate Reader, Bio Tek, Winooski, VT, USA).

The conditionally immortalized mouse podocytes or murine glomerular endothelial cells (mGECs) were maintained in RPMI-1640 with L-glutamine (Invitrogen) containing 10% FBS and 1% penicillin/streptomycin on collagen I coated flasks under either permissive (33 °C with 10 units/ml of INF-γ), or non-permissive condition (37 °C without INF-γ) in a humidified incubator (95% air, 5% CO2). mGECs were incubated with normal glucose (5 mM) + D-mannitol (25 mM), or with high glucose (HG; 30 mM D-Glucose), or with control or diabetic serum (2.5%v/v), without or with MitoTEMPO (100 nM, Enzo Life Sciences International, PA) for up to 72hs. Transwell co-cultures were performed in collagen-I coated 6x well plates and 0.4um 24 mm inserts (Costar).

Serum and amino acid removal was performed by washing mPTCs with Hank’s balanced salt solution (55021 C, Sigma-Aldrich) and placing them in nutrient-deprived medium. Where indicated, autophagy was inhibited by addition of either PIK3C3/Vps34 inhibitor SAR-405 (5 μM in cell culture medium for 16 h, unless otherwise specified^{59 (link)}) or Spautin-1 (25 μM in cell culture medium for 16 h^{60 (link)}). Lysosomal proteolysis was inhibited by addition of BfnA1 (250 nM for 4 h, unless otherwise stated). Where indicated, oxidative stress was triggered by treating mPTCs with either hydrogen peroxide (0.5 mM for 1 h) or with ROT (250 nM for 8 h). Where indicated, mPTCs were treated with either the mitochondria-targeted oxidant scavenger Mito-TEMPO (10 μM in culture medium for 12 h, Enzo Life Sciences) or with a selective Src-family kinase inhibitor SU6656 (5 μM in culture medium for 12 h, Sigma Aldrich). Afterwards, the cells were processed and analyzed as described below.

Intracellular Ca²⁺ cycling activity in isolated rabbit ventricular myocytes was monitored using a Leica SP5 confocal laser scanning system with a 60 × 1.4 NA oil-immersion objective in XT mode using Ca²⁺-sensitive indicators Fluo-3 (Molecular Probes, Carlsbad, CA, United States), respectively. Cells were loaded with Fluo-3 for 10 min, and after 20 min de-esterification, the dye was excited at 488 nm with an argon laser. Emission was collected at 500–600 nm. CMs were studied in Tyrode solution (see above). Myocytes were paced via field stimulation at 1 Hz using extracellular platinum electrodes. To assess the SR Ca²⁺ load and decay kinetics, 10 mM caffeine was applied at the end of the experiments.
For triggered activity, myocytes were paced at 1 Hz for 30 s, and the latency between the last stimulus in the pacing train and the first SCW was calculated. To assess the effect of mitochondria-derived ROS on CMs from aged rabbits, myocytes were pretreated with the mitochondria-specific ROS scavenger (2-(2,2,6,6-tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl) triphenylphosphonium chloride (mito-TEMPO, 25 μM, Enzo Life Sciences, Farmingdale, NY, United States) for 10 min. The intracellular Ca²⁺ cycling measurements were performed under β-adrenergic stimulation with 100 nM ISO for HL-1 CMs and 30 nM ISO for aged rabbit CMs.