Ab 479 na

Human choroidal vascular endothelial cells (HCVECS; #36052-03, Celprogen, USA) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing 4.5 g/l glucose supplemented with 10% (v/v) FBS, 100 U/ml penicillin, and 100 mg/ml streptomycin. The human macrophages were derived from human peripheral blood mononuclear cells and cultured, as the previous description^{22 (link)}. The cells were kept at 37 ^°C in a humidified atmosphere containing 5% CO₂. HCVECs were cultured in the lower well and macrophages were cultured in the upper well of the transwell plate (#CLS3397, Corning, USA) and verified by STR profiling.
The cells were culture in CO₂ incubator (#BBD6220, Thermo Fisher Scientific; 1%O₂, 94%N₂, and 5%CO₂) for 24 h (hypoxia group), HMPL-013 (0.05 μmol/l for 24 h), CCL2-neutralizing antibody (#AB-479-NA, R&D Systems, USA; 5 μg/ml for the last 30 min), recombinant human CCL2 protein (#279-MC, R&D Systems; 100 ng/ml for 24 h), geraniin (macrophage polarization modulator; #PHL80994, Merck; 30 μM for the last 2 h), or LPS (macrophage M1-type polarization agonist; Escherichia coli LPS, serotype 0127:B8; #L3129; Merck, USA; 2 μg/ml for 24 h).

Here, 30% hydrogen peroxide solution (H1009), X-Gal (B4252), potassium hexacyanoferrate(III) (244023), and potassium ferrocyanide (P9387) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Bay11-7082 (S2913) was purchased from Selleck Chemicals (Houston, TX, USA). Antibodies against the following proteins were used in this study: Padi2 (66386–1-Ig; Proteintech, Rosemont, IL, USA), α-tubulin (sc-8035; Santa Cruz Biotechnology, Inc., Dallas, Texas, USA), β-actin (sc-47778; Santa Cruz Biotechnology.), LaminA/C (sc-376248; Santa Cruz Biotechnology), p21 (#2947; Cell Signaling Technology, Inc., Danvers, MA, USA), mouse CCL2 antibody (AB-479-NA; R&D Systems, Inc, Minneapolis, MN, USA), mouse CCL5 antibody (AF478; R&D Systems), mouse CCL7 antibody (AF-456-NA; R&D Systems), and normal goat IgG control (AB-108-C; R&D Systems). The following siRNAs used in the present study were purchased from Origene (Rockville, MD, USA): Padi2 siRNA (SR418983B and C), RelA siRNA (SR427138A), Ccl2 siRNA (SR403037C), Ccl5 siRNA (SR400775A), Ccl7 siRNA (SR400957C), and negative control siRNA (SR30004).

All experiments were conducted in a biosafety level 3 facility. Animal protocols were reviewed and approved by the Institutional Animal Care and Use Committee of the Korea Research Institute of Chemical Technology (permit no. 2021-8A-04-01, protocol no. 8A-M6). Male K18-hACE2 (no. 034860) [B6.Cg-Tg(K18-ACE2)2Prlmn/J] mice were purchased from the Jackson Laboratory. Mice were randomly divided into groups before infection. For SARS-CoV-2 infection, mice were lightly euthanized with isoflurane and injected intranasally with 50 μL of saline containing 2 × 10³ PFU of SARS-CoV-2 wild-type strain or beta variant. Body weight, clinical score, and survival were monitored daily after infection. The initial day of weight loss was defined as the first day with a >5% reduction in initial body weight. Clinical status was scored according to the following grading scale (30 (link)): 0, healthy; 1, ruffled fur but active; 2, ruffled fur with reduced activity; 3, ruffled fur, inactive, and hunched; 4, imminent death; and 5, dead. Mice were sacrificed at 4 and 6 dpi for analysis of viral load and histopathological analysis. For cytokine blockade, beta variant-infected mice were injected intraperitoneally with 5 mg/kg of body weight of CCL2/JE/MCP-1 antibody (AB-479-NA) and normal goat IgG control (AB-108-C) (R&D Systems) at 1, 3, and 6 dpi. Efforts were made to minimize animal suffering.