Mmp 1

Immunoassays were performed to quantify the protein levels of selected characteristic molecular candidates in patient sera. The selection of analytes was based on transcriptome profiling results and previous studies [17 (link),18 (link),19 (link)]. Custom-made multiplex immunoassays (ProcartaPlex, Thermo Fisher Scientific, Waltham, MA, USA) were used to investigate the following analytes: Caspase-3, CD40, IL-8, MMP1, PAI-1, and VEGFA. Serum levels of ITGB3 (Abbexa, Cambridge, UK), LGALS2 (Abbexa, Cambridge, UK), and MMP9 (BioLegend, San Diego, CA, USA) were measured using singleplex immunoassays according to the manufacturer’s protocol. All data were analyzed following the manufacturer’s instructions. Additionally, an independent patient cohort consisting of COVID-19 patients with or without aspergillosis was investigated by the following immunoassays according to the manufacturer’s instructions: IL-8 (BioLegend, San Diego, CA, US), Caspase-3 (ThermoFisher, Waltham, MA, USA), and MMP1 (ThermoFisher, Waltham, MA, USA).

The 2,2-diphenyl-1-picrylhydrazyl (DPPH) was obtained from Sigma Aldrich (St. Louis, MO, USA). Mushroom tyrosinase (Sigma Chemical Co., USA). Matrix metalloproteinase-1 ELISA Kit (Abcam, Cambridge, MA, USA), procollagen type-C peptide (PIP) EIA kit (Takara, Shiga, Japan), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), phosphate-buffered saline (PBS), and MMP-1 were purchased from Invitrogen (Carlsbad, CA, USA). Dulbecco’s modified Eagle’s medium (DMEM), Fetal bovine serum (FBS) and Ham’s F-12 nutrient mixture, L-Glutamine, and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer (DMEM/F-12) were purchased from Lonza (Walkersville, MD, USA). LPS was purchased from Sigma-Aldrich (St. Louis, MO, USA). TNF-a and IL-6 were from ELISA Kit (BioLegend, San Diego, CA, USA), and the RAW264.7 mouse machrophage cells were from the Korean Cell Line Bank (Seoul, Republic of Korea). CCD-986sk human fibroblast cells were from American Culture Collection (Manassas, VA, USA).

Frozen section slides were fixed in 4% paraformaldehyde with 0.1% Triton X‐100 for 10 min. After 30 min of treatment in 3% H₂O₂ to remove nonspecific signals, blocking was performed in 5% donkey serum (Abcam, Cambridge, UK) for 1 h after washing. Sections were incubated with collagen type I (1:100 dilution; Invitrogen), collagen type III (1:100 dilution; Invitrogen), elastin (1:100 dilution; Bioss Antibodies, Woburn, MA, USA), matrix metalloproteinase (MMP‐1; 1:100 dilution; Invitrogen), and tissue inhibitor of metalloproteinase (TIMP‐1; 1:100 dilution; Bioss Antibodies) antibodies at 4°C overnight, washed three times with PBS, and incubated with horseradish peroxidase (HRP)‐conjugated donkey anti‐rabbit antibody for 1 h. After washing with PBS, AEC+ high sensitivity substrate chromogen (DAKO, Glostrup, Denmark) was used as color developer for HRP. Hematoxylin (DAKO) was used for counterstaining.