F 12 dmem

Hydrogen peroxide (H₂O₂) (Millipore Sigma, St. Louis, MO, USA) treatments were performed, as described [28 (link)]. Briefly, the cells were challenged with 200 μM H₂O₂ for 60 min and then harvested immediately after treatment upon washing with (1X) phosphate buffer saline (PBS, ThermoFisher Invitrogen, Grand Island, NY, USA). For all experiments, the cells were seeded 15–18 h prior to the experiments. Immediately before treatment, the cells were washed once with DMEM/F-12 (ThermoFisher Invitrogen, Grand Island, NY, USA) without any supplements, and the conditioned medium was saved for later use for studies where recovery was performed.

Adenovirus expressing mitochondria-targeted catalase was obtained as a gift from Dr. Marcelo Bonini from the University of Illinois at Chicago. WT hTERT and _nuchTERT cells were plated in DMEM/F-12 (ThermoFisher Invitrogen, Grand Island, NY, USA) medium with 10% Fetal bovine serum and then allowed to attach to six-well plate dishes overnight at 37 °C before the desired amount of viral particles were added. The next day, cells were washed in warm PBS (2X) and 500 µL DMEM/F-12 serum free medium were added to each well. 1 µL mito-catalase adenovirus was subsequently added to each well and incubated for one-hour at 37 °C, swirling every 15 min. Fresh medium with serum was added the following day. On the following day, the cells were treated with and without 200 μM H₂O₂ for 60 min. The samples were then collected for western analysis.

The human RPE cell line (ARPE-19) was obtained from ATCC (Manassas, VA). The ARPE-19 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM)/F12 (Invitrogen-Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% heat inactivated fetal bovine serum (FBS, Invitrogen), 100 U/ml of penicillin (Sigma-Aldrich, St. Louis, MO, USA) and 100 µg/ml of streptomycin (Sigma-Aldrich). The cells were grown in a humidified incubator with 5% CO₂ at 37 °C. For some experiments, cells were pretreated with different concentrations of scutellarin (25, 50, 100 μM) or AG90 (40 μM) for 2 h, and then challenged with H₂O₂ (1 mM) for 24 h.

Following informed consent, skin biopsies were collected from three patients with MAK-associated RP and used for fibroblast isolation and iPSC generation as described previously [6 (link)–8 (link)]. Briefly, dermal fibroblast cells were reprogrammed using the CytoTune non-integrating Sendai virus reprogramming kit according to the manufacturers protocol (Invitrogen/Thermo Fisher Scientific, Waltham, MA; CytoTune-iPS Reprogramming Kit; Cat#: A16517) [9 (link)]. Fibroblasts were plated on 6-well tissue culture plates and infected at a multiplicity of infection (MOI) of 5. At 12–16 h following transduction cells were washed and fed with fresh fibroblast cell growth media [(DMEM/F12, 5% heat inactivated FBS (Invitrogen/Thermo Fisher Scientific) and 0.2% primocin (Invivogen, San Diego, CA)]. At 7 days post-infection, cells were passaged onto 6-well LN521-coated cell culture dishes at a density of 30,000 cells/well and fed every day with E8 pluripotency media (Invitrogen/Thermo Fisher Scientific). At 3 weeks post-viral transduction, iPSC colonies were picked, passaged, and clonally expanded on fresh LN521-coated cell culture dishes. During reprogramming and maintenance of pluripotency, cells were cultured at 5% CO₂, 10% O₂, and 37 °C.

Primary Sertoli cells were isolated and cultured as previously described [26 (link), 27 (link)] with slight modifications. Briefly, three-week-old testes without tunica albuginea were sequentially treated with 0.5 mg/ml collagenase (Wako, 034–22363), 1 mg/ml hyaluronidase (Sigma, H3506) plus 1 mg/ml collagenase, and 1 mg/ml hyaluronidase in Dulbecco's modified Eagle's medium (DMEM) containing DNase I. Small pieces of seminiferous tubules were removed via filtering through a 100μm-pore-size filter. The purity of the isolated cells was confirmed by immunocytochemical staining with an anti-vimentin antibody (Progen Biotechnik; GP53). Isolated Sertoli cells were cultured with F12-DMEM (Invitrogen, 10565–018) mixed with 10 μg/ml insulin (Nacalai Tesque, 19251–24), 5 μg/ml transferrin (Sigma, T1147), and 5 ng/ml epidermal growth factor (BD Bioscience, 40010) at 34°C. The culture medium was changed at days 2 and 4, and Sertoli cells were stimulated with 1 μM RA (Sigma) at day 5 for 24 hours. Then, qPCR analyses were performed.

The tissue culture samples were collected and prepared as described in a previous study [11 (link)]; the tissues were cut into small blocks, washed twice with F-12/DMEM (Invitrogen, USA), centrifuged (1500 rpm, 5 min), and then cultured in F-12/DMED (Invitrogen) supplemented with 10% FBS (Gibco; Australia) and 1% penicillin–streptomycin (Invitrogen). Tissue explants were then placed into a CO₂ incubator (37 °C, 5% CO₂, approximately 0.1 g explants) (Thermo Fisher Scientific, USA) and cultured with 2 mL culture medium. IL-6 (PEPROTECH, USA) was diluted in 0.1% BSA (Sigma–Aldrich, USA) and PBS (Sigma–Aldrich); Corylifol A (MedChemExpress, USA) was diluted with 10% DMSO (Sigma–Aldrich), 40% PEG300 (MedChemExpress), 5% Tween-80 (Sigma–Aldrich) and 45% saline (Sigma–Aldrich).

Rabbit corneal fibroblasts were isolated from the cornea of New Zealand white strain rabbits as described in the previous study [41 ]. The rabbit heads were bought from the slaughter farm in Ijok, Malaysia. The corneal stroma was digested using collagenase type I solution in order to isolate the corneal fibroblasts. The corneal fibroblasts were then cultured on a 24 × 24 mm coverslip in 35 mm tissue culture dishes (Orange Scientific, Braine-L’Alleud, Belgium) with F12:DMEM supplemented with 10% FBS, 1% Glutamax, 1% antibiotic-antimycotic (Invitrogen) at 37 °C with atmospheric O² and 5% CO² level. The cell culture medium contained sodium bicarbonate and HEPES solution which was used as buffer to maintain the media at pH 7.2-7.6. The 100% confluent cells were treated with 3 densities of axenic trophozoites, which were 10⁴, 10⁵ and 10⁶ trophozoites per dish/well for triplicates at 3 time intervals (3, 6 and 24 h). The cells were fixed using 4% paraformaldehye and stained with giemsa. The area of remaining cells after cytolysis was observed under a light microscope and measured using the VideoTest Morphology software. The percentage of cytopathic effect (CPE) was calculated based on the surface area of empty spaces in the culture.