Wst 1 assay kit

The media and other materials required for culturing cells were purchased from Lonza Inc. (Walkersville, MD, USA). Griess reagent and lipopolysaccharide (LPS: E. coli, serotype O111:B4; L2630) were purchased from Sigma–Aldrich (St. Louis, MO, USA). The WST-1 assay kit was obtained from Daeillab Service Co., Korea. Anti-Toll-like receptor 2 antibody (anti-TLR2), anti-complement receptor 3 antibody (anti-CR3), and anti-Toll-like receptor 4 antibody (anti-TLR4), were obtained from Abcam (Cambridge, MA, USA). Phospho-NF-κB antibody, phospho-p38 (MAPK) antibody, phospho-ERK (MAPK) antibody, and phospho-JNK antibody were purchased from Cell Signalling Technology (Danvers, MA, USA). All chemicals and reagents used in this work were of analytical grade.

To examine if the transduced Tat-PDIA3 proteins protect against DNA damage, NSC-34 cells were pre-treated with 3 μM Tat-PDIA3 protein for 1 h and exposed to 1 mM hydrogen peroxide (H₂O₂) for 3 h. TUNEL and a Cell Death Detection kit (Roche Applied Science) were used to assess cellular damage. Images were analyzed using a fluorescence microscope (Nikon eclipse 80i, Tokyo, Japan).^{43 (link), 45 (link)} Fluorescence levels were measured using a Fluoroskan enzyme-linked immunosorbent assay (ELISA) plate reader (Labsystems Oy) with a 485 nm excitation and 538 nm emission.
The biological activity of Tat-PDIA3 protein was measured by assessing cell viability based on WST-1 assay kit (Daeillab Service, Seoul, South Korea) after exposure to H₂O₂ as described previously.^{43 (link), 45 (link)} NSC-34 cells were plated at a confluence of 70% in a 96 well plate and exposed to Tat-PDIA3 and PDIA3 proteins (0.5–3 μM). After 1 h, cells were treated with 1 mM H₂O₂ for 5 h. Cell viability was measured at 450 nm using an ELISA microplate reader (Labsystems Multiskan MCC/340, Helsinki, Finland) and cell viability was expressed as a percentage of untreated control cells.