100 mm culture dishes

5.5 × 10⁵ cells of human pRPE cells (Lonza) were plated in 100 mm culture dishes (Falcon) in RtEGM BulletKit Medium (Lonza) and incubated at 37°C in a humidified 5% CO₂ atmosphere. The medium was replaced twice weekly, and cells were passaged with 0.25% trypsin/0.1% EDTA (Biological Industries, Israel) upon reaching 90% confluence. Experiments were performed at passages 3–4.

For cell lysate
preparation, HeLa-cyt-AEQ cells were grown in 100 mm culture dishes
(Falcon, Cat. 353003). At confluence, cells were washed twice in PBS
and scraped in 250 μL of a buffer containing (in mM): 150 Tris,
0.8 phenylmethylsulfonyl fluoride (PMSF), and 0.1 ethylenediaminetetraacetic
acid (EDTA), pH 7.2. After three cycles of freeze-thawing, cells were
centrifuged (12,000g, 5 min at 4 °C), the pellet
was discarded while the supernatant was aliquoted and stored at −80
°C.
For AEQ reconstitution, 100 μL aliquots of HeLa-cyt-AEQ
lysate were supplemented with 140 mM β-mercaptoethanol (Sigma,
Cat. M6250) and 5 μM native coelenterazine (GoldBio, St. Luis,
MO, Cat. CZ5) and allowed to reconstitute O.N. (15–24 h) on
ice. For Ca²⁺ measurements, serial dilutions were prepared
in 150 mM Tris, supplemented with 10 mM EDTA, pH 7.2.
For calcium
measurements, 500 μL of cell lysate was aliquoted
into the acquisition chamber. The latter was mounted on the top of
SiPM using optical grease to assure optimal matching of the refractive
index. After 30 s of acquisition, 1 mL of 150 mM Tris supplemented
with 50 mM CaCl₂ was injected in the acquisition chamber.
After AEQ discharge, the trace was recorded until return to baseline
(about 4 min).

Isolation and culture of hASCs was performed according to a previous work [16 (link)]. In brief, after washing with an equal volume of 0.1 M phosphate-buffered saline (PBS; pH 7.4), lipoaspirates were digested with 0.075% collagenase type I (Washington Biochemical Corp.) at 37 °C for 60 min. The digested samples were centrifuged at 1200 × g for 10 min to obtain a high-density stromal vascular fraction (SVF), and they were resuspended in low-glucose Dulbecco’s modified Eagle’s medium (LG-DMEM) containing 10% fetal bovine serum (FBS), 100 mg/mL streptomycin, and 100 U/mL penicillin (growth medium (GM)). They were plated at 4 × 10⁴ cells/cm² in 100-mm culture dishes (Falcon). When they reached 70–80% confluence, the cells were passaged and hASCs before the third passage were used in the following study.
Osteogenic differentiation of hASCs was conducted using cultures of GM supplemented with 0.01 μM 1,25-dihydroxyvitamin D3, 50 μM ascorbate-2-phosphate, and 10 mM β-glycerophosphate. For adipogenic differentiation, the cells were cultured in adipogenic medium (AM) supplemented with 0.5 mM isobutyl-methylxanthine (IBMX), 0.1 μM dexamethasone (Dex), 10 μM insulin, and 200 μM indomethacin in growth medium. The effect of SrRan on the osteogenic differentiation of hASCs was studied using Sr (0, 25,100, 250, 500, 1,000, 1500, and 2000 μM) in osteogenic medium (OM).

Expression levels of various antioxidant proteins were evaluated via western blotting. Briefly, 5 × 10⁶ cells in 100 mm culture dishes (BD Falcon) were incubated with the indicated concentrations of Tempol (0.5–4 mM) for 48 h. Cells were washed with PBS and four volumes of lysis buffer (Intron Biotechnology, Seongnam, Gyeonggi-do, Korea) were added. Total proteins (30 μg) were resolved in 12.5% SDS-PAGE gels and then transferred to Immobilon-P PVDF membranes (Millipore, Billerica, MA, USA) by electroblotting. Membranes were probed with anti-SOD1, anti-catalase, anti-Trx1, anti-TrxR1, and anti-β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology). Western blots were developed using an EZ-Western Lumi Pico ECL solution kit (DoGen, Seoul, Korea). All band intensities were analyzed using the ImageJ software program (FujiFilm, Tokyo, Japan).

All-trans retinoic acid atRA (atRA) and 13-cis retinoic acid (13cRA) (Sigma Aldrich) were dissolved in dimethyl sulfoxide (DMSO). The final concentration of DMSO in medium was 0.2%. Cells cultured in medium supplemented with DMSO but without retinoids served as controls. The retinoid compounds and the cell cultures were handled under dimmed yellow light.
For rtPCR and RNA gel blot studies, medium (6 ml) supplemented with retinoids (10⁻⁷ M) or control medium was added to subconfluent cell cultures in 100 mm culture dishes (Falcon, Becton Dickinson) for 24 hours.