Additional file 2 of Tumour necrosis factor alpha promotes secretion of 14-3-3η by inducing necroptosis in macrophages

Additional file 2: Figure S1. Endogenous 14-3-3η levels in macrophages are not affected upon treatment with IL-1β, IL-6/sIL-6R, and IL-21. Macrophages were cultured in the presence or absence of IL-1β (10 ng/ml; n = 3), IL-6/sIL-6R (10 ng/ml; n = 3), or IL-21 (10 ng/ml; n = 3) for 24 h. WCL prepared from macrophages were then analysed by IB using specific antibodies against 14-3-3η or β-actin. Figureure S2. Nec-1, but not TOF, blocks TNF-α–induced macrophage death. Macrophages were cultured with TNF-α (100 ng/ml; n = 3) in the presence or absence of nec-1 (20 nM; n = 3) or TOF (300 nM; n = 3) for 24 h and analysed by TEM. Representative images from three independent experiments are shown. Scale bar, 5 μm (upper panel), 2 μm (lower panel). Figure S3. TNF inhibitors do not block macrophage death caused by diamide or TNF-α. Macrophages were cultured with diamide (1 mM; n = 3) or TNF-α (100 ng/ml; n = 3) in the presence or absence of ETN (100 μg/ml) or ADA (100 μg/ml) for 24 h and analysed by TEM. Representative images from three independent experiments are shown. Scale bar, 5 μm (upper panel), 2 μm (lower panel). Figure S4. TNF inhibitors do not block RA macrophage death caused by TNF-α. HD or RA macrophages were cultured in the presence or absence of TNF-α (100 ng/ml; n = 3) with or without ETN (100 μg/ml) or ADA (100 μg/ml) for 24 h and analysed by TEM. Representative images from three independent experiments are shown. Scale bar, 5 μm (upper panel), 2 μm (lower panel). Figure S5. TNF-α induces phosphorylation of RIP3. Macrophages were cultured with or without TNF-α (10 ng/ml; 24 h; n = 3), diamide (100 nM; 24h; n = 3) (A), and LPS (500 ng/ml; 24 h; n = 3) in the presence or absence of zVAD-FMK (20 μM; n = 3). The cells were stained with specific antibodies against anti-RIP3 (phosphor S227) or phospho-Akt (Ser 473), or isotype control, and with DAPI. Representative images from three independent experiments are shown. Scale bar, 50 μm. Figure S6. IL-1β, IL-6/sIL-6R, and IL-21 fail to phosphorylate MLKL. Macrophages were cultured with or without IL-1β (10 ng/ml; n = 3), IL-6/sIL-6R (10 ng/ml; n = 3), or IL-21 (10 ng/ml; n = 3) for 24 h. WCL prepared from macrophages were analysed by IB using specific antibodies against the total or phosphorylated form of MLKL or β-actin. Quantification data for representative images from three independent experiments (n = 3) are shown. Scale bar, 5 μm (upper panel), 2 μm (lower panel). Figure S7. Nec-1 blocks phosphorylation of MLKL induced by TNF-α. Macrophages were cultured with or without TNF-α (10 ng/ml; 24 h; n = 3) in the presence or absence of nec-1 (20 nM; n = 3). WCL were then obtained, and total or phosphorylated form of MLKL, and β-actin were detected by WB. Representative images from three independent experiments are shown. Figure S8. 14-3-3η is detectable in culture supernatants of macrophages derived from HD and treated with TNF-α, diamide, or LPS. The culture supernatants of macrophages cultured in the presence or absence of diamide, TNF-α (10 ng/ml; 24 h; n = 3), or LPS (500 ng/ml; 24 h; n = 3), and with or without nec-1 (20 nM; n = 3) or TOF (300 nM; n = 3) or zVAD-FMK (20 μM; n = 3), were analysed by WB. Recombinant 14-3-3η was used as a positive control. BSA was used as a loading control and was stained with CBB-R350. Representative images from three independent experiments are shown.