Raw 264.7 murine macrophage cells

The RAW 264.7 murine macrophage cells and HL-60 human promyelocytic leukemia cells were purchased from the Korean Cell Line Bank. RAW 264.7 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM, Gibco BRL, Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum (FBS, Gibco BRL). HL-60 cells were incubated under the same conditions using RPMI 1640 medium instead of DMEM. In order to induce differentiation into the neutrophil-like cells, HL-60 cells were incubated with 1.25% v/v DMSO for 5 days without media change. Lipopolysaccharide, gelatin, fibronectin, type I collagen, laminin, Sudan black, neutral red, and Griess reagent were purchased from Sigma-Aldrich (St. Louis, MO, USA). Phaseolin was purchased from Molport (CAS: 13401-40-6, Beacon, NY, USA). Polyclonal antibodies against inducible nitric oxide synthase (iNOS), tubulin, nuclear factor kappa B (NF-κB), inhibitor kappa B (I-κB), glyceraldehyde 3-phosphate dehydrogenas (GAPDH), and lamin A were purchased from Santa Cruz Biotechnology. Polyclonal antibodies against endogenous Ninj1 were obtained from Dr. Kyu-Won Kim (Seoul National University, Seoul, Korea).

RAW 264.7 murine macrophage cells were purchased from the Korean Cell Line Bank (Republic of Korea). They were cultured in DMEM containing 10% fetal bovine serum (FBS) supplemented with 1% penicillin-streptomycin at 37°C in a 5% CO₂ humidified incubator (Thermo Fisher Scientific, USA).

RAW 264.7 murine macrophage cells were purchased from the Korean Cell Line Bank (KCLB; Seoul, Korea). The cells were incubated at 37 °C in 5% CO₂ in Dulbecco’s modified Eagle’s media (DMEM; Gibco-BRL, Carlsbad, CA, USA), supplemented with 100 U/mL penicillin, 100 µg/mL streptomycin, and 10% fetal bovine serum (FBS; Gibco-BRL).

RAW 264.7 murine macrophage cells were obtained from the Korean Cell Line Bank (KCLB), and B16F10 mouse melanoma cells were obtained from The Global Bioresource Center (ATCC). Cell cultures were grown in a DMEM medium supplemented with 10% FBS and 1% penicillin–streptomycin (P/S) at 37 °C and 5% CO₂ for 2 and 3 days, respectively.

The strains and plasmids used in this study are listed in Table 1. Unless otherwise noted, the V. vulnificus MO6-24/O (wild type), VvhmpA mutant, and VvhmpA-complemented strain were grown aerobically in Luria-Bertani (LB) medium supplemented with 2.0% (w/v) NaCl (LBS) at 30°C, and their growth was monitored spectrophotometrically at 600 nm (A₆₀₀). The RAW 264.7 murine macrophage cells were obtained from Korean Cell Line Bank (Seoul, South Korea) and grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) and appropriate antibiotics [100 units ml^–1 penicillin G and 100 μg ml^–1 streptomycin (Gibco-BRL, Gaithersburg, MD)] in air supplemented with 5% CO₂ at 37°C.

RAW 264.7 murine macrophage cells (Korean Cell Line Bank, Seoul, Republic of Korea) were cultured using DMEM containing 10% FBS and 1% pen–strep solution and maintained in a humidified incubator at 37 °C with 5% carbon dioxide (CO₂). The cells were seeded into a 96-well plate (4 × 10⁴ cells/well) and incubated for 24 h. Samples were dissolved in DMSO and incubated with the cells at various doses for an additional 24 h. Subsequently, 0.1 µL/mL of EZ-Cytox solution was added to each well and incubated at 37 °C for 3 h. Then, the absorbance was confirmed at 450 nm on a microplate reader (Infinite M200 Pro; Tecan, Krems, Austria).