Sodium l ascorbate

HSs and PSs were produced according to the self-assembly method, partially modified using 6-well plates [22 ,23 (link)]. Briefly, primary fibroblasts at P5 were seeded at 1.5 × 10⁵ cells/well and cultured for 26 days in DMEM supplemented with 50 μg/mL of (+)-sodium l-ascorbate (Sigma, St. Louis, MO, USA) until they formed manipulable sheets. Then, two fibroblast sheets were detached and superimposed to form the dermal equivalent. Dermal equivalents were incubated at 37 °C with 8% CO₂ for two more days to allow the sheets to fuse and thus form the new layer. After this period, primary keratinocytes at P2 were seeded on the dermal equivalent at 1.2 × 10⁶ cells/dermal equivalent to form the epidermal layer and cultured for seven days in DMEMH supplemented with 50 μg/mL of (+)-sodium l-ascorbate (Sigma). Then, the skin substitutes were raised to the air-liquid interface and cultured with a medium lacking EGF to obtain a stratified epithelium representative of skin in vivo. On day 14 of culture at the air-liquid interface, HSs and PSs were treated or not with the compound stock solutions or with MTX diluted in culture medium three times per week for one week. After a total of 56 days of culture, skin substitute biopsies were taken and analyzed.

Cells were incubated with 1 mM EU or 10 µM EdU (Berry & Associates, Inc.) for 30 min. Coverslips were fixed with 4% (w/v) PFA/PBS, and cells were permeabilized with 0.5% (v/v) Triton X-100/PBS. The click chemistry reaction was assembled with 10 mM sodium L-ascorbate (Sigma), 0.1 mM Biotin-TEG-azide (Berry & Associates, Inc.), and 2 mM Cu(II)SO4 (Sigma) in PBS. This was incubated at RT in the dark for 30 min. Following PBS washes, EU slides were incubated with primary antibodies against UBF and Nop52 as previously described (van Sluis and McStay 2015 (link)). Sites of synthesis were visualized with 1:200 Streptavidin conjugated TexasRed (Rockland) or Alexa647 (Jackson ImmunoResearch) for EU and EdU, respectively, combined with secondary antibodies (Jackson ImmunoResearch), diluted in 1% BSA/PBS for 1 h at 37°C.

Hydroxylation assays were performed in triplicate under normoxic conditions using liquid chromatography electrospray MS (LC-ESI-MS) (Figure 3, Figure 4). Conditions: 1 μm TgPhyA or DdPhyA, 100 μm full-length DdSkp1 or full-length TgSkp1 substrate, 50 μm (NH₄)₂Fe(II)(SO₄)₂ (Sigma-Aldrich), 1 mm sodium l-ascorbate (Sigma-Aldrich), and 500 μm 2-oxoglutarate disodium salt (Sigma-Aldrich) in HEPES (100 mm), pH 7.6. Mixtures were incubated at 37 °C for 1 h, or at specific time points for time course assays, and quenched using an equal volume of 1% (v/v) aqueous formic acid (Sigma-Aldrich). Reaction mixtures were analyzed using a Xevo G2-S Q-TOF mass spectrometer equipped with an electrospray source (Waters®) coupled to a Waters® ACQUITY UPLC System, unless otherwise specified. Instrument control and data processing were performed using MassLynx V4.1 software. An Aeris^TM 3.6-μm WIDEPORE C4 200 Å (Phenomenex) 4.6 × 50 mm column was used for separation.