Mab210

MDA-MB-231 cells or MCF-7 at the density of 5×10⁵ cells/ml were treated by M protein at concentrations of 60 pmol/ml for 24 h, then the conditioned medium was collected. The conditioned medium was centrifuged at 1000 rpm in 5 minutes, following by another step at 2000 rpm in 20 minutes to remove the cell death and cell debris. In order to inhibit the inflammatory cytokines, neutralizing antibodies were added to conditioned medium, including human anti-TNFα antibody (MAB210, R&D Systems, Minneapolis, MN, USA), anti-IL6 antibody (MAB2061, R&D Systems) and anti-IL8 antibody (MAB208, R&D Systems) at concentrations of 10 μg/ml, 5 μg/ml and 5 μg/ml respectively.

Mouse dorsal fur was removed via shaving and depilation with Veet (Reckitt Benckiser, UK). Mice were placed in restrainers with facial protection and treated once ±250mJ/cm² UVB light using the UV-2 ultraviolet irradiation system (Tyler Research). UVB light was provided by cascade-phosphor ultraviolet generators that emit 310nm UVB radiation.
For irradiation of cultured cells at approximately 80% confluence, growth medium was replaced with PBS and cells were treated with 0-50mJ/cm² UVB light as indicated in specific experiments. PBS was removed immediately following irradiation and replaced with growth medium. Cells were incubated at 37°C for the indicated times.
Inhibitors of cell death (Ac-YVAD-cmk, Sigma Aldrich SML0429; Necrostatin-1, Cayman Chemical 11658; GSK’872, Tocris Bioscience 64-921-0; Z-IETD-FMK, Selleckchem S7314; Z-LEHD-FMK, Selleckchem S7313; Z-VAD-FMK, Selleckchem S7023; UAMC-3203, Selleckchem S8972) were added 30 minutes prior to IFN treatment and again immediately following UVB exposure.
For neutralizing antibody experiments, N/TERTs were treated with isotype control (R&D MAB002, MAB0041) or neutralizing antibodies targeting TRAIL (100ng/ml; R&D MAB375), TNF-α (5µg/ml; R&D MAB210) or FasL (1µg/ml; R&D MAB126) immediately following UVB exposure.