Sc514

Mouse primary chondrocytes were isolated from the femoral condyles and tibial plateaus of 5-day-old mice by digestion with 0.2% collagenase (Sigma, St. Louis, MO, USA; C6885) as described previously [15 (link)]. Chondrocytes (5–6 × 10⁵) were seeded into 6-well plates in Dulbecco’s Modified Eagle’s Medium containing 10% fetal bovine serum and antibiotics. The next day, the cells were serum-starved overnight, and then treated with each peptide for 24 h at the concentrations indicated in each figure legend. All peptides were synthesized by Anygen Inc. (Gwangju, Korea). The amino acid sequences of the four peptides were as follows: Cramp, GLLRKGGEKIGEKLKKIGQKIKNFFQKLVPQPEQ; LL-37, LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES; HP (2–20), AKKVFKRLEKLFSKIQNDK; HP-MA, AKKVFKRLGIGKFLHSAKKF. IL-1β (GenScript, Piscataway, NJ, USA; Z02922), TNF-α (EMD Millipore, Temecula, CA, USA; GF027), and SC514 (Tocris Bioscience; 3318) were also used for cell experiments at the concentrations and periods indicated in the figure legends.
For KD analysis of Cramp, the cells were treated with hyaluronidase for 2 h in serum-free medium, and then transfected with either 10 nM of control or Cramp siRNA (Ambion, Austin, TX, USA) using Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA, USA; 13778150). Chondrocytes were harvested at 36 h after treatment with IL-1β.

Primary human myometrial cells at passage ≤ P4 were split equally between 6 or 12 well-plates and grown to 80–90% confluency. At this point media was changed to serum-reduced (0.1% FCS) media for 18 to 24 h before discarding from each well. Five hundred microliters of fresh media (0.1% FCS) was produced containing: Pen-NBD, Pen(43-56)-NBD, Sc514 (Tocris bioscience, 3318); or controls (equivalent volume DMSO, Pen-NBD Mutant, NBD alone) at indicated concentrations. The inhibitor/control media was added to wells as a pre-incubation step. After 1 h, 10 ng/ml IL1β (Peprotech, 200-01B), or equivalent volume DMSO vehicle, was added to each well.
For western blotting experiments cells were washed with PBS before being lysed using sucrose cell lysis buffer (62.5 mM Tris-HCl pH6.8, 2% SDS, 10% saccharose) containing 20 μl/ml protease inhibitor (Sigma Aldrich, P1860) and 5 μl/ml phosphatase inhibitor (Sigma Aldrich P2745) at times of 0/15/60/120/240 minute from cytokine stimulation.
For RNA array experiments, 4 h following cytokine stimulation cells were washed with PBS before RNA was extracted using the RNeasy Mini Kit (Qiagen, 74101) according to the manufacturers' protocol.

All chemicals used in this study were obtained from Sigma-Aldrich unless otherwise specified. SC514 was purchased from Tocris. Rapamycin was obtained from LC Laboratories. Y27632 dihydrochloride was purchased from Hello Bio. Dynasore was obtained from Ascent Scientific. SARS-CoV-2 (2019-nCoV) spike protein S1 RBD (R319-F541, His-tagged) and vWF ELISA Kit were purchased from Sino Biological. SARS-CoV-2 spike protein S1 mutant sampler set containing wild-type (WT), D614G mutant, N439K mutant, SA variant (K417N/E484 K/N501Y), and SC variant (S13I/W152C/L452R) was obtained from Abenomics. Cy3-conjugated anti-mouse immunoglobulin (Ig)G was purchased from Jackson ImmunoResearch. The anti-ARF1 antibody, rabbit polyclonal, was described previously [39 (link)]. The ARF6(3A-1) mouse monoclonal antibody and the normal goat antibody were obtained from Santa Cruz Biotech. The ACE2 inhibitory antibody (goat polyclonal) and human TNF-α, IL-6, and IL-1β DuoSet ELISA kits were purchased from R&D Systems. 96-well µClear^® half-area black plates with flat bottoms were obtained from Greiner Bio-one. Enhanced chemiluminescence (ECL) Select immunoblotting Detection Reagent was from Cytiva. Restore plus stripping buffer and ultra TMB substrate were obtained from Thermo Fisher Scientific. ChemiDoc XRS imaging system was from Bio-Rad.