Ec growth medium

Liver and lungs were dissected from female C57/BL6N mice at 6 to 9 weeks old and placed in ice-cold sterile PBS. Samples were moved to a sterile tissue culture hood, and single-cell suspensions were prepared using the Miltenyi Octo Dissociator as described above. ECs were enriched from the single-cell suspensions using CD31 MicroBeads (Miltenyi Biotec, 130-097-418), following the manufacturer’s instructions, under sterile conditions. Cells were cultured in collagen-coated (Sigma, C8919) plates with EC growth medium (Cell Applications, 211-500).

For qRT-PCR analyses, HAEC (Cell Applications, San Diego, CA, USA) between passages 4 and 10 were maintained on 0.1% bovine gelatin-coated plates (Midsci, St. Louis, MO, USA) at 37 °C and 5% CO₂. EC growth medium (Cell Applications, San Diego, CA, USA) was supplemented with 5% fetal bovine serum (FBS, Life technologies, Carlsbad, CA, USA) and 1% penicillin-streptomycin (P/S, Carlsbad, CA, USA) to promote cultivation. Confluent HAEC monolayers were exposed to UFP, Adam10 inhibitor, Iwr1 (10 µM, Sigma Aldrich, St. Louis, MO, USA), or lithium chloride (LiCl, 20 mM) respectively in neutralized M199 media (Life Technologies, Carlsbad, CA, USA). Total RNA was collected following 6 h of treatment. RNA purification and reverse transcription were performed as previously described [28 (link)]. qPCR master-mix (Applied Biological Materials Inc., Richmond, BC, Canada) was used for PCR amplification. mRNA levels were assessed and compared by the ΔΔCt method. mRNA expression was normalized to human actin expression. Sequences of primers are listed in Table 1.

Liver and lungs were dissected from female C57/BL6N mice at 6 to 9 weeks old and placed in ice-cold sterile PBS. Samples were moved to a sterile tissue culture hood, and single-cell suspensions were prepared using the Miltenyi Octo Dissociator as described above. ECs were enriched from the single-cell suspensions using CD31 MicroBeads (Miltenyi Biotec, 130-097-418), following the manufacturer’s instructions, under sterile conditions. Cells were cultured in collagen-coated (Sigma, C8919) plates with EC growth medium (Cell Applications, 211-500).