Growth supplement

Human lung microvascular endothelial cells (HMVEC-l, Lonza, Basel, Switzerland) in the fourth passage were grown to 80% confluence in T75 flasks (Greiner Bio-One, Kremsmünster, Austria) containing 10 mL endothelial basal medium and growth supplements (Lonza). Where required, recombinant TNFα (Sigma-Aldrich, St. Louis, MO, USA) was added to a final concentration of 50 ng/mL and incubated for 10 h at 37 °C and 5% pCO₂. TNFα incubation times and dosage have been optimized recently in our labs [69 ]. Where required, heparinase III (0.1 mU/mL, Iduron, Alderley, UK) and chondroitinase ABC (0.5 mU/mL, Sigma-Aldrich) were added to the culture medium after 30 min of incubation with TNFα. To rule out CXCL-8 signaling through CXCR1 and CXCR2 and binding to DARC/D6, 0.5 μg/mL of each anti-CXCR1, anti-CXCR2 and anti-DARC/D6 antibody (Santa Cruz, Dallas, TX, USA) were added to the medium. After incubation for 90 min, recombinant CXCL-8 (Antagonis Biotherapeutics GesmbH, Graz, Austria) was added to the medium at a final concentration of 50 nM. After incubation for 8 h, cells were washed with PBS twice, scraped into 2 mL PBS/EDTA and centrifuged in a 2 mL Eppendorf tube at 500× g. Residual cells in the plate were collected with 2 mL PBS/EDTA, added to the cell pellet and centrifuged again at 500× g. The supernatants were discarded and the cell pellets were stored at −80 °C until further use.

HDFa (American Tissue Culture Collection, Manassas, VA, USA) were cultured in fibroblast basal medium (Lonza, Verviers, Belgium) containing growth supplements (Lonza), such as 2% FBS, 0.1% recombinant human insulin, 0.1% gentamicin sulfate amphotericin B, and 0.1% recombinant human fibroblast growth factor-B. The cells at a density of 5 × 10⁴ cells/mL (200 μL) were seeded in a 96-well plate until 80–85% confluency. The culture supernatant was replaced with 200 μL of serum-free DMEM containing test samples. After 24 h incubation, the cytotoxicity of the test samples was measured using MTT assay, and ELISA was used to determine the level of the wrinkle-forming mediator, MMP-1 (R&D Systems), in the culture supernatant.

Fibroblasts explanted from healthy adult lungs (Lonza, Walkersville, MD, USA) were
used. Fibroblasts were seeded in 6-well plates and maintained in fibroblast basal
medium with growth supplements (Lonza, Walkersville, MD, USA) and 20% FBS in a
humidified atmosphere of 5% CO2 at 37°C. Sub-confluent low passage fibroblasts
were incubated with human recombinant SAA (Peprotech EC Ltd, London, UK) at indicated
concentrations for 24h. Levels of IL-6 were determined in culture supernatants by
ELISA (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s
instructions. RNA was isolated from confluent fibroblasts using RNeasy Plus Micro
Kits (Qiagen, Hilden, DE) and reverse transcribed using Reverse transcription System
(Promega, Madison, WI, USA). StellARray platforms were used to measure gene
expression (Bar Harbor BioTechnology, Trenton, ME, USA).

Human primary hepatocytes of a single donor were obtained from Lonza (Walkersville, MD, USA). All products related to the culture including growth supplements, thawing medium, maintenance medium and plating medium were also purchased from Lonza. The primary hepatocytes were revived, cultured on collagen I coated cell culture plates or dishes, and used as per supplier's instructions.

Primary normal human epidermal keratinocytes (NHEK) were purchased from Lonza and cultured in KGM-2 growth medium with growth supplements (Lonza) and antibiotics (Life Technologies). Primary mouse epidermal keratinocytes were isolated from the neonates (0–2 days old) of experimental (K14.Cre⁺. Foxo1^L/L) and control (K14.Cre⁻. Foxo1^L/L) mice. Briefly, full thickness mouse skin was obtained and incubated with 2.5 U/mL Dispase II (Roche Diagnostics) overnight at 4 °C. The dermis was separated from the epidermis by 0.1% trypsin and 0.02% EDTA in PBS for 15 minutes at 37 °C. Keratinocytes from the epidermis were cultured in KGM-2 growth medium containing antibiotics. Cell cultures were maintained in a 5% CO₂ humidified incubator at 37 °C. Keratinocytes were passaged in KGM-2 growth media with supplements including standard insulin (8.6 × 10⁻⁷M). For assays cells were transferred to KGM-2 media with supplements except for the amount of insulin was reduced 100 fold unless otherwise stated, i.e. low insulin represents 1% of the amount of insulin in standard keratinocyte growth media. In some experiments, no insulin was present as indicated below.

BEAS-2B human bronchial epithelial cells from American Type Culture Collection (Manassas, VA, USA) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 100 μg/mL streptomycin, and 100 units/mL penicillin at 37 °C in a 5% CO₂ atmosphere. Human umbilical vein endothelial cell (HUVEC)s, obtained from pooled donners and cryopreserved at passage 1, were purchased from Lonza (Walkersville, MD, USA) and cultured in EGM-2 endothelial basal medium with growth supplements (Lonza) and 2% fetal bovine serum.
Anti-IκB, anti-phospho IκB, anti-NF-κB p65, anti-GSK-3β, anti-phospho GSK-3β, anti-phospho β-catenin, and anti-phospho LRP6 (Ser 1490) primary and secondary antibodies were obtained from Cell Signaling Inc. (Beverly, MA, USA). Anti-LRP6 antibody was obtained from Abcam (Cambridge, MA, USA), and anti-β-catenin antibody was purchased from BD Transduction Laboratories Inc. (Lexington, KY, USA). Anti-Axin, anti-β-actin, and anti-TATA box binding protein (TBP) antibodies were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Recombinant human Dickkopf-1 (DKK-1) and recombinant human Wnt3a, Wnt5a, Wnt7a and Wnt10b proteins were from R&D systems (Minneapolis, MN, USA). LPS from Klebsiella pneumoniae was obtained from Sigma-Aldrich (St. Louis, MI, USA).

Fibroblast basal medium, growth supplements, and NHLFs were purchased from Lonza (Walkersville, MD). Human-derived transforming growth factor beta (TGFβ), as well as the TGFβ receptor blocker SB431542, was obtained from R&D Systems, Inc. (Minneapolis, MN). Poly-L-lysine was purchased from Sigma-Aldrich (St. Louis, MO). The anti-Collagen I antibody was purchased from Fitzgerald (Acton, MA), while the alpha tubulin antibody was purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX). Alexa-fluor 488-conjugated donkey anti-rabbit secondary antibody, alexa-fluor 488-conjugated alpha-smooth muscle actin (α-SMA) primary antibody, and the beta actin primary antibody were purchased from Abcam (Cambridge, MA). ProLong gold antifade with DAPI mountant was obtained from Molecular Probes (Thermo Fisher Scientific, Waltham, MA).