Ab96600

Cells were seeded at a density of 2 × 10³ cells/mm² in a 24-well plate. After 24 h, cells were stained with Mitotracker CMX-ROS (Ex/Em: 579/599) which accumulates in active (healthy) mitochondria with intact membrane potentials. Mitotracker CMX-ROS (200 nM) solution was pre-warm before incubating with cells for 40 min at 37°C. Cells then were washed twice in PBS and fixed in cold methanol for 10 min. Blocking was performed in PBS 0.1% Triton X-100 with Fetal Serum Bovine (FBS) at final concentration of 5%. Cells were incubated with primary antibody against Citrate synthase (ab96600, Abcam) over night at 4°C. Bound antibody were detected with 488 Alexa-Fluor-conjugated donkey anti-rabbit IgG antibody (Life Technologies). After two additional washes in PBS 0.1% Triton X-100, cells were incubated with a solution of 300 nM DAPI (Thermo Fisher Scientific, Waltham, MA, USA) in PBS for 10 min. The cells were again washed twice with PBS and the microscopy slides were mounted for detection with Mowiol 4-88 Reagent (Millipore, Billerica MA, USA cod. 475,904). Mitochondrial images were acquired with the confocal microscope LSM980 with 63× objective (NA 1.4) using Airyscan detector. Laser wavelength: 405, 488, 561 nm. Quantification of cell area (μm²) and fluorescence intensity was performed using Zen 3.1 software.

Laemmli buffer containing 4% SDS, 10% β-mercaptoethanol (M6250; Sigma-Aldrich), 20% glycerol (GI345; Melford), 0.004% blue bromophenol (A2331,0025; AppliChem), and 0.125 M Tris-HCl was added to protein lysates, followed by boiling at 95°C for 10 min. Samples were separated on SDS-PAGE and transferred to a nitrocellulose (Macherey-Nagel) or a polyvinylidene difluoride (Merck) membrane. The membranes were blocked with 5% milk (A0830; PanReac AppliChem) and probed with the following antibodies: anti–β-Actin (ab8227; Abcam), anti–β-Tubulin (ab15568; Abcam), anti-CBS (14787-1-AP; Proteintech), anti-CSE (12217-1-AP; Proteintech), anti-MPST (HPA001240; Atlas Antibodies), anti-MTCO1 (ab14705; Abcam), anti-Citrate Synthase (ab96600; Abcam), anti-SOD2 (13141; Cell Signaling), anti-TOM40L (ab236421; Abcam), anti-TIM50 (ab109436; Abcam), anti-HIF1α (sc-13515; Santa Cruz Biotechnology), anti-H3 (ab176842; Abcam), anti-V5 (Merck), anti-HA (H6908; Sigma-Aldrich), and anti-Biotin (HRP conjugate; 5571; Cell Signaling). Immunoblots were next incubated with a secondary antibody (anti-rabbit [AP132P; Merck] or anti-mouse [7076; Cell Signaling]) and visualized using the Western HRP substrate (Merck). ImageJ software was used to quantify the expression levels of the proteins.

Tissues or cells were homogenized in lysis buffer, nutated at 4°C for 1 hour, and centrifuged at 4°C for 15 min at 12,000g, and the supernatant was transferred to a new tube. Western blotting was performed as previously described (10 (link)), and samples were analyzed for protein abundance of FoxO1 (no. 2880, Cell Signaling Technology), 4-HNE (ab48506, Abcam), DRP-1 (ab56788, Abcam), pDRP-1 (no. 3455, Cell Signaling Technology), Mfn-2 (ab56889, Abcam), Nrf2 [Developmental Studies Hybridoma Bank (DSHB)], FoxO3 (DSHB), PRDX4 (DSHB), citrate synthase (ab96600, Abcam), and catalase (ab1877, Abcam).