Cell fractionation kit standard

The IL-8, TGF-β1 and bFGF concentrations in culture medium were measured using an ELISA kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions. The culture supernatants with or without poly I:C and anti-IL-8 antibody at the same doses as described above were collected at 24 h after the scratch and centrifuged at 3000 rpm for 15 min. Cell-free supernatants were then harvested and stored at −20 °C (for IL-8) or −80 °C (for TGF-β1 and bFGF) until further assay. The concentrations in each well were measured at 450 nm using a microplate reader (Bio-Rad). We also measured the protein levels using an ELISA kit of E-cadherin (R&D Systems), Vimentin (Cell Signaling Technology, Danvers, MA, USA) and Snail (Cloud-Clone Corp, Houston, TX, USA) according to the manufacturer’s instructions. Whole cell lysates were extracted using cell lysis buffer (Cell Signaling Technology) or nucleic/cytosolic fractions were extracted using cell fractionation kit-standard (Abcam) according to the manufacturer’s instructions and stored at −20 °C prior to use. Protein concentrations were determined using the RC DC protein assay kit (Bio-Rad).

Mitochondrial and nuclear fractions were separated from cultured cardiac myocytes using Cell Fractionation Kit-Standard (ab109719, Abcam). Then, the fractions were subjected to Western blot using an anti-AIF mAb.

Protein was extracted from preconditioned cAT-MSC-derived exosomes and DH82 using the Pro-Prep protein extraction solution (Intron Biotechnology). Concentration of the protein samples was analyzed using the DC Protein Assay Kit (Bio-Rad, Hercules, CA, USA). Nuclear proteins were isolated using the Cell Fractionation Kit-Standard (Abcam, Cambridge, MA, USA). For western blot assays, 25 μg of proteins were loaded and separated by SDS-PAGE. Bands from SDS-PAGE were transferred to polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA), which were then blocked with 5% non-fat dry milk and Tris-buffered saline. Membranes were incubated with primary antibodies against HIF-1α (1:500; LifeSpan BioSciences, Seattle, WA, USA), COX-2 (1:500, Santa Cruz Biotechnology, Dallas, TX, USA), STAT3 (1:500, LifeSpan BioSciences), phosphorylated (Tyr705) STAT3 (1:500, LifeSpan BioSciences), lamin A (1:500, Santa Cruz Biotechnology) and β-actin (1:1000, Santa Cruz Biotechnology) at 4°C overnight. The membranes were subsequently incubated with the appropriate secondary antibody for 1 h. Using an enhanced chemiluminescence detection kit (Advansta, Menlo Park, CA, USA), immunoreactive bands were detected and normalized to the housekeeping protein (β-actin).

For subcellular fractionation, MCF7 cells were seeded in 25 cm² culture flasks at a cell density of 1 × 10⁶ cells/flask and incubated for 24 h at 37 °C, 99% (v/v) humidity, and 5% (v/v) CO₂. After 24 h incubation, the medium was replaced with 5 mL of fresh medium containing 60 μM Ru1 or Ru2 (a concentration high enough to be detected) and incubated for 6 h at 37 °C. After incubation, the supernatant was collected; cells were detached from the culture flask and pelleted at 500× g for 5 min. Then, the cytosolic, mitochondrial and nuclear fractions were sequentially isolated using the Cell Fractionation Kit Standard (Abcam, Cambridge, MA, USA) according to the manufacturer’s instructions. Afterward, aqua-regia (HNO₃ and HCl in a 1:3 proportion) was added to the four fractions in 1:2 proportion (aqua regia–sample volume) and samples were incubated overnight at room temperature. The four fractions were then analyzed using a Horiba Jobin Yvon inductively coupled plasma atomic emission spectrometer as a paid service (Analytical Laboratory, Department of Chemistry, FCT-UNL) to determine the amount of ruthenium present in each sample.

Cells were cultured in DMEM without glucose, glutamine, phenol red, sodium pyruvate and sodium bicarbonate (Sigma-Aldrich Co., LLC) supplemented with 20 mM glucose, 2 mM glutamine, 44 mM sodium bicarbonate and 10% FBS (HyClone Laboratories, Inc.). Mitochondrial and cytosolic fractionation was performed using a commercially available kit (Cell Fractionation Kit-Standard; Abcam). Briefly, 3.0 × 10⁶ cells were suspended in the attached Buffer A and treated with Detergent I. After incubation at room temperature for 7 min, the sample was centrifuged at 5,000×g and 4 °C for 1 min, after which the supernatant containing the cytosolic fraction was collected. The resulting cell pellet was resuspended in Buffer A and treated with Detergent II. After incubation at room temperature for 10 min, the sample was centrifuged at 5,000×g and 4 °C for 1 min, after which the supernatant containing the mitochondrial fraction was collected. Each mitochondrial and cytosolic fraction sample was aliquoted for western blotting and FH activity assay using Colorimetric Fumarase Activity Assay Kit (Abcam). For FH activity assay, after the sample had been mixed with the attached substrate, enzyme mix and developer solution, absorbance at 450 nm was measured using Versamax (Molecular Devices, LLC) in the kinetic mode at 37 °C for 120 min.

For fractionation analysis, 2 × 10⁶ cells were seeded on 10 cm Petri dish and 24 h later treated with ICI and E2, followed by fractionation by Cell Fractionation Kit Standard (ab109719, Abcam, UK) according to the manufacturer’s instructions. Proteins of obtained fractions were measured by Pierce™ BCA Protein Assay Kit (Thermo Fischer Scientific, Waltham, MA, USA) and prepared in SDS-PAGE sample buffer (100 mM Tris-HCl (pH 6,8), 2% SDS, 20% glycerol, 4% β-mercaptoethanol, 0.5% bromophenol blue dye) for Western blot analysis. Anti-GAPDH and anti-H3 antibodies were used as controls for purity of cytoplasmic and nuclear fractions, respectively [18 (link),19 (link)]. Total cellular proteins for Western blot analysis were isolated in Ripa buffer with protease inhibitors (Roche, Basel, Switzerland). SDS-PAGE and Western blot analysis were carried out as described previously in [8 (link)]. Primary and secondary antibodies used in this study are listed in Supplementary Table S1.