Fig 2: BV6 induces necroptosis in murine macrophages. (a) Wild type and TNFR2-deficient macrophages derived from HoxB8-immortalized MPCs were challenged for 7 h with the indicated combinations of BV6 (10 µM), zVAD-fmk (20 µM) and necrostatin-1 (45 µM). Cells were finally analyzed by western blotting for the presence of TRAF2 and cIAP1. Please note, wild-type cells treated with BV6 and zVAD-fmk were already largely dead when cells were harvested for Western blot analysis. (b) Macrophages derived from HoxB8-immortalized MPCs were stimulated in triplicates (technical replicates) with the indicated concentrations of BV6 in the presence and absence of 20 µM zVAD-fmk and analyzed for viability after 36 h. One of four representative experiments is shown. (c) HoxB8-immortalized MPC-derived macrophages were challenged with the indicated mixtures of 10 µM BV6, 20 µM ZVAD-fmk and 45 µM necrostatin-1 and analyzed for viability after 36 h. Shown are data points with S.E.M. of five independent experiments. (d–f) Macrophages derived from Hoxb8 immortalized MPCs of wild type, TNF- (d), TNFR1- (e) and TNFR2-deficient mice (f) were stimulated with the indicated combinations of BV6 (10 µM) and zVAD-fmk (20 µM). After 36 hours cell viability was quantified using MTT assay or crystal violet staining. Data points derived from six (d and e) or four (f) independent experiments together with mean±S.E.M. are depicted. ***p<0.001

Fig 3: Ultraviolet B (UVB) irradiation enhances TNF receptor type 1 (TNFR1) expression in murine keratinocytes. PAM212 cells were cultured in vitro and received UVB irradiation. (A) The mRNA expression levels of TNFR1 and TNFR2 in keratinocytes were quantitated by quantitative reverse transcription polymerase chain reaction. (B) Western blotting was performed for the TNFR1 and TNFR2 proteins in cell lysates. (C,D) The expression of TNFR1 and TNFR2 in keratinocytes was determined by flow cytometry. (E) TNFR1 and TNFR2 expression was detected in keratinocytes by immunofluorescence. Data were from three independent experiments. Data points and error bars represent mean ± SEM. Representative images are shown. **p < 0.01.

Fig 4: The diagram for TWEAK/factor-inducible 14 (Fn14) regulation of Ro52 expression and other downstream signals. Ultraviolet B (UVB) irradiation enhances the expression of both Fn14 and TNF receptor type 1 (TNFR1) in keratinocytes. The binding of TWEAK to Fn14 initiates the formation of Fn14/TNF receptor associated factor 2 (TRAF2)/cellular inhibitor of apoptosis protein 1 (cIAP1) complex, which further interacts with the TNFR1 partner and triggers cell apoptosis as well as surface expression of Ro52. Fn14/TRAF2/cIAP1 complex can also activate the nuclear factor-kappa B (NF-?B) and phosphatidylinositide 3-kinase (PI3K)/Akt pathways to upregulate the expression of Ro52 and inflammatory cytokines, the latter of which recruit macrophages. Infiltrating macrophages secrete proinflammatory cytokines including TWEAK and interferon-a, which together aggravate immune responses in cutaneous lupus erythematosus.