Victor nivo 3s

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 cytotoxicity of the membranes was first evaluated by analyzing the effect of their leachables on cell metabolism. For that, cells were seeded in 24-well plates at a density of 1.5 × 10⁵ cells/mL and incubated for 48 h. After the first 24 h, the membranes were cut into small pieces and submerged in culture medium at a concentration of 0.025 g/mL and placed in a 37 °C bath under agitation for 24 h. After, the medium with the leachable was recovered with a syringe and filtered with a 0.45 µm filter. Then, cells were exposed to the leachables in triplicate or to culture medium as a negative control. After 24 h, cell viability was assessed using MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (16%) (CellTiter 96^® AQueous One Solution Cell Proliferation Assay, PROMG3581, Promega, Madison, WI, USA) in a dilution of 1:10 in assay culture medium (DMEM + 0.5% FBS). Cell viability was measured after 3 h by UV-Vis spectroscopy at 490 nm in a microplate reader (HH35L2019044, Victor Nivo 3S, Perkin Elmer, Waltham, MA, USA).

The β‐galactosidase assay was performed using an ONPG method
⁶³ (link),
⁶⁴ (link). The reporter plasmids were transformed into the T. thermophilus HB27ΔTTP0042 strain. Three single colonies were randomly selected for each assay. Transformants carrying different reporter plasmids were grown in the TB medium. Then, cells grown to the log phase (OD₆₀₀ ~1.0) were collected by centrifugation, and cell pellets were resuspended in 10 mM Tris‐HCl at pH 8.0. Next, crude cell lysates were obtained by ultrasonic lysis. Cell debris in the lysates was removed by centrifugation (13,000 rpm, 20°C, 30 min) before the ONPG assay. The protein content of the cellular extracts was determined by the Bradford Protein Assay Reagent (Tiangen, PA102). For each assay, 50 μl of the supernatant was added to 450 μl of reaction buffer, which contained 2.8 mM ONPG and 50 mM sodium phosphate at pH 6.5. Samples were incubated at 65°C for 20 min, and the reaction was stopped by adding an equal volume of 1 M sodium carbonate. The absorbance of o‐nitrophenol was determined by using a microplate reader (Victor Nivo 3S; PerkinElmer) at 420 nm. One unit of specific enzyme activity was defined as 1 nmol ρ‐nitrophenol produced per min per mg total protein.