Panel a

The relative levels of 40 different cytokines were determined using mouse cytokine antibody array (Panel A, R&D System), according to the manufacturer’s protocol. Retinas were homogenized in PBS with protease inhibitors (cOmplete, mini, EDTA-free cocktail tablets, Roche). Samples were centrifuged at × 10,000g for 5 min to remove cellular debris, and Triton X-100 was added to a final concentration of 1%. Protein concentrations were quantified using total protein assay (Pierce 660 nm Protein Assay Kit, Thermo Fisher Scientific). Pre-mixed sample/antibody cocktails were incubated with blocked membranes overnight at 4 °C on a rocking platform shaker. After 3 washes, membranes were incubated with 800CW streptavidin (IRDye, LI-COR) for 30 min and washed 3 times before being scanned on the LI-COR odyssey imaging system. Data were analyzed with image studio lite 4.0 software (LI-COR). The average fluorescent intensity of each cytokine was quantified for each retinal sample; the light-dependent changes in cytokine expression were determined by dividing the light-exposed values by those of the dark-reared samples processed in parallel from the same genotypes (WT and Arr1^−/−) and performed in triplicate.

Relative expression of proinflammatory and immunomodulatory mediators in BAL fluid of mice inoculated with a lethal vs. sublethal dose of PVM (day 7) and mice inoculated with a sublethal dose of PVM alone (day 7 vs. day 21) was evaluated using the Proteome Profiler Array, Panel A and the Mouse Angiogenesis Profiler Array (both from R&D Systems, Minneapolis, MN, USA) as previously described [33 (link)]. Each profiler membrane was probed with 500 µL BAL fluid (100 µL from each of 5 mice per group). Raw data were normalized to levels of each cytokine detected in BAL fluid samples from uninfected controls. Quantitative evaluation of proinflammatory cytokines in BAL fluid was carried out by ELISA (DuoSet, R&D Systems).

Protein
expression patterns were determined from pooled conditioned culture
medium from the four MSC donors. The conditioned medium was collected
and centrifuged at 5000g for 5 min to remove any
cells or cell debris. The supernatant was transferred to new tubes
and stored at −80 °C until use. Human cytokine proteome
profiler (Panel A, R&D Systems, Minneapolis, MN, USA) was used,
according to the manufacturer’s instructions. For the analysis
of arrays, blots were threshold-adjusted and analyzed using ImageJ.
The intensity for a specific cytokine was then computed by averaging
over duplicated spots.

Protein lysates were prepared from snap frozen xenografts, and proteins (400 μg) were incubated with the Rat Cytokine Array Kit, Panel A, and the Human Angiogenesis Array Kit (R&D Systems; Minneapolis, MN, USA) according to the manufacturer's instructions. Collection and quantification of pixel densities from membranes was performed using the Fuji Las3000 (Fujifilm; Tokyo, Japan) and Image Reader LAS-3000 (Version 2.2; Fujifilm). Arrays for all samples were performed in duplicate, and an average density was calculated for each corresponding protein probed on the arrays based on the two pixel densities determined for each experiment.