Cytochalasin d cyt d

The ability of NETs to kill B. parapertussis was evaluated as previously described [42 (link)] with minor modifications. Briefly, 2×10⁵ neutrophils were seeded in 24 well-plates and then incubated with 100 nM PMA for 1 h at 37°C, to induce NETs. Next, neutrophils were treated or not with 100 μg/ml Cytochalasin D (CytD) (Sigma) to inhibit phagocytosis or with 500 mU/ml MNase I, to breakdown NETs, for 30 min. In control experiments a combination of CytD and MNase I was used. Neutrophil were then incubated with bacteria (MOI:100) for 3 h and further treated with MNase I for 20 min to release the bacteria eventually immobilized by NETs. Neutrophils were further subjected to hypotonic lysis by using 0.1% saponin in sterile deionized water and serial dilutions of lysates were rapidly plated onto bBGA to enumerate the CFU. To confirm that NETs can kill B. parapertussis a microscopic analysis using a LIVE/DEAD Cell Viability Assays Kit (Molecular Probes) was performed according to the manufacturer’s instructions.

Cytochalasin D (Cyt D) (Sigma-Aldrich, St. Louis, MO, USA) was used as the inhibitor of actin polymerization. A CCK-8 assay was performed to evaluate the cytotoxicity of Cyt D to EBL cells (Dojindo, Shanghai, China), as previously described [11 (link)]. Briefly, 5 × 10³ cells/well were seeded and allowed to grow for 24 h in 96-well plates and then treated with Cyt D (0.1 μM, 0.5 μM, 1 μM, 2.5 μM, 5 μM, 10 μM, or 20 μM) for 24 h. Cells treated with DMSO were used as the negative control, and cells with no treatment were used as the mock group. Next, each well was incubated with 10 μL CCK-8 for 2 h. The light absorption value at 450 nm was detected by the microplate spectrophotometer (PerkinElmer Victor NIVO 3S, Waltham, MA, USA). Each treatment was carried out in three repeats, and all experiments were performed independently three times. After that, the monolayer EBL cells (2 × 10⁵ cells in a 12-well plate) were treated with the selected concentration of Cyt D for 2 h, followed by the addition of 8 μg/mL rMbovP0145 and incubation for more than 12 h. The total RNA was then extracted, and qRT-PCR was used to analyze the expression of IL-8 mRNA.

The cell lines BJ (hTERT-immortalized human non-transformed fibroblast cells), BJ-LT (SV40 large T-antigen (LT)-transfected BJ cells), BJ-LT-Ras (BJ-LT cell transfected with oncogenic HRas mutant) [24 (link)] were cultured in Dulbecco’s modified Eagle medium (DMEM, Gibco, Life technologies, UK) supplemented with 10% fetal bovine serum (FBS, FB-1090–500, Werner Saveen, Biological Industries, Beit-Haemek Ltd, Israel), 100 U/ml penicillin and 0.1 mg/ml streptomycin (complete medium). The cells were kept at 37 °C in a humid atmosphere containing 5% CO₂. In some experiments 0.5–5 μM cytochalasin D (CytD, Sigma-Aldrich) were used to induce binucleation. Mitotic cells were collected by the shake off method in which exponentially growing cells were washed once with pre-warmed PBS, followed by incubation in complete medium for approximately 2–3 h, and then the loosely attached mitotic cells were detached by tapping the culture flasks. The cells were collected and re-suspended in fresh complete medium for culturing in either ultra-low attachment plates or on plates coated with fibronectin (40 μg/ml). Since BJ-LT cell had a higher tendency to aggregate via cell–cell contacts, 1.2% methylcellulose (M-7027, Sigma-Aldrich) was included in the culture medium when these cell lines were cultured in suspension in ultra-low attachment wells.