This article does not contain any studies with human participants or animals performed by any of the authors.
Cells and Culturing
Human conjunctival epithelial (HCjE) and corneal epithelial (HCLE) cells were cultured following the protocol provided with the frozen cell stock and consistent with previous publications [33, 34]. Growth and maintenance were under standard cell culture conditions (37°C, 5% CO2 and 100% relative humidity) until confluent, following established protocols  (see Supplementary 1 for detailed methodology).
SkQ1 Stock and Working Solutions
The stock solution of SkQ1, Mitotech-6, batch no. 040414M, was kindly provided by Mitotech S.A. Pharmaceuticals. It was prepared in a solvent containing sterilized 50% ethanol-0.9% NaCl solution. The SkQ1 concentration was 100 mg/ml. Following the instructions, this stock was stored at − 20 °C until use; it can be stored for at least 1 year from the preparation date. The working solutions of SkQ1 were prepared as 2 × serial dilutions in the maintenance medium as described above. Both the stock solution and working dilutions were kept in light-proof containers before and after being applied to the cultures.
Cell Viability, Dosing and Safety Ranges
Cell Viability Determination with Varying Concentrations of SkQ1 and Diluent
To evaluate the proper dosing and safety range of SkQ1 on healthy HCjE and HCLE cell cultures, the SkQ1 stock solution from Mitotech SA was serially diluted in culture medium, ranging from 0.25 nM to 250,000 nM, 10 × range. As a control, the diluent (50% ethanol) was also serially diluted in culture medium, ranging from 0 to 0.75%, equivalent to the concentrations carrying over from the SkQ1 stock. Cellular morphology changes were observed and recorded by a NIKON D5000 digital camera installed on an Olympus CK2 Inverse Phase Contract Microscope and transferred to a linked computer that ran “Control My Nikon” v3.0 software for further analysis. Quantitative cell viability was determined by the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay protocols established in the laboratory . The MTT assay is based on the conversion of MTT into formazan crystals by living cells, which determines mitochondrial activity optically . The calculated cell viability values were plotted against the SkQ1 concentrations used (see Supplementary 2 for detailed MTT methodology).
Cell Viability Determination when SkQ1 Is Combined with Sensitizing Reagent IL-1β or TNF-α
To rule out potential interferences from the combination of SkQ1 and pre-sensitizing agents on HCjE cells, the HCjE cell cultures were pre-sensitized for 1 h with 10 ng/ml of TNF-α or IL-1β as previously reported  and then exposed to various concentrations of SkQ1 in combination with the following: (1) culture medium only; (2) IL-1β (10 ng/ml); (3) TNF-α (10 ng/ml). Each treatment had three repeats. Controls included medium alone, TNF-α alone, IL-1β alone and SkQ1 alone. Exposure times under the standard incubation were 36 h. Experiments were run on 96-well plates, and results were photographically recorded.
Role of SkQ1 in Inflammation of HCjE Conjunctival Epithelial Cells
SkQ1 Treatment of HCjE Inflammation
The HCjE cells were grown in 96-well plates until reaching nearly 95% confluency, washed twice with pre-warmed medium and then given pre-treatments with freshly made maintenance medium containing varying concentrations of SkQ1 as indicated for 1 h. Then, 1 µl of freshly made IL-1β or TNF-α working solution was added to the SkQ1 pre-treated cells at final concentrations of 10 ng/ml. The treatments continued for 24 h. The control wells received the maintenance medium only. To acquire statistically meaningful results, each treatment in one experiment was repeated at least twice, and the entire experiment was repeated at least three times.
At 24- or 48-h SkQ1 treatments, 10 μl of samples was taken from each well and immediately analyzed for the concentrations of an inflammatory biomarker, prostaglandin E2 (PGE2), released in the culture medium. A monoclonal enzyme immunoassay (EIA) kit (Cayman Chemical Co., Ann Arbor, MI) was used following the manufacturer’s instructions and the protocol established in the laboratory . The degrees of PGE2 production were then calculated and plotted using the formula described in the Methods section.
Role of SkQ1 in Corneal Wound Healing
A single, one-dimensional “streak” defect in the epithelial cell lawn was created in a well by a common scratch method utilizing 200-µl or 1-ml Eppendorf pipette tips, referring to published methods [15, 38, 39].
SkQ1 Treatment Groups
Immediately after a wound was created, the cultures were gently washed twice with the expired Keratinocyte Serum Free Medium (KSFM, Gibco) to remove remaining dead cell sheets or debris and then re-grown in the maintenance medium. The SkQ1-treated groups received 50 nM SkQ1 in the maintenance medium. The control groups received the medium only with the solvent (ethanol). Since in the preliminary experiments the medium containing ethanol at 0.000002% concentration, equivalent to the amount that would be carried over from a 50-nM SkQ1 working solution, was tested to be intoxicating to the cells, we omitted the addition of the vehicle solvent to the controls.
End Point Measurements of the Corneal Wound-healing Process
To monitor and compare the wound-healing processes in the SkQ1-treated groups with those of the control groups, three markers were made alongside each wound at the bottom outside with a fine-tip marker pen to label the loci to be photographed immediately after scratching. These markers were used for alignment purposes: each was photographed together with the individual wounding spot at nearly the same orientation for all the photographing time points at 0, 4, 8 and 12 h from scratching till the wound was completely closed (healing). Photos were taken by a NIKON D5000 digital camera installed on an Olympus CK2 Inverse Phase Contract Microscope. Image files were recorded into a linked computer via “Control My Nikon v3.0” software.
To evaluate whether SkQ1 has a role in HCLE cell proliferation, extensively diluted single HCLE cells were inoculated into a 24-well plate in the growth and maintenance medium supplemented with various concentrations of SkQ1 ranging from 0 to 400 nM. Cell growth was tracked photographically at day − 1, 2 and 6 (corresponding to 24, 48 and 144 h). A standard MTT assay was conducted, except the stained cells were dried and photographed prior the addition of acidic isopropanol to dissolve formazan and to measure the absorbance at A572 and 690 nm. Two independent experiments were conducted, each with two repeats (see Supplementary 2 for detailed MTT methodology).
Cell migration during the corneal epithelial healing process has been demonstrated to be p38 MAPK dependent in an animal model . To evaluate if SkQ1 has a role in HCLE cell migration during the wound-healing process, 10 µM of SB203580, an inhibitor of p38 kinase, was added to the maintenance medium together with 0, 100 and 400 nM of SkQ1 after scratching. Closure of the wounds was photographically recorded at 0 and 10 h post-scratch. Two such experiments were conducted independently; each had two repeating wells, and each well had three spots to be photographed.
Evaluation of Inflammation on HCjE
The degree to which PGE2 was produced by HCjE cells sensitized by IL-1β or TNF-α under various concentrations of SkQ1 was compared with the corresponding degree of PGE2 production from cells without sensitization. Data were normalized for interpretation of results. Results were reported as mean ± SD (σ). A Student’s t test was performed for statistical analysis (see Supplementary 3 for specific normalization calculations).
Evaluation of Wound Healing on HCLE
The areas of wounds at various time points were calculated with ImageJ Fiji software. The whole wounded areas were used for the calculation against a reference with known dimensions, and rates of wound healing (%) of a specifically marked location at a particular time point were calculated. Results were reported as mean ± SD (σ) (see Supplementary 3 for specific calculation).