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.

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图S1。经白细胞介素1β（IL-1β）、白细胞介素6/可溶性白细胞介素6受体（IL-6/sIL-6R）及白细胞介素21（IL-21）处理后，巨噬细胞内源性14-3-3η水平无显著变化。实验中，巨噬细胞在添加或不添加IL-1β（10 ng/ml；n=3）、IL-6/sIL-6R（10 ng/ml；n=3）或IL-21（10 ng/ml；n=3）的条件下培养24小时，随后制备巨噬细胞全细胞裂解液（whole cell lysate, WCL），采用抗14-3-3η或β-肌动蛋白（β-actin）的特异性抗体进行免疫印迹（immunoblotting, IB）分析。
图S2。坏死素1（Nec-1）可阻断肿瘤坏死因子α（TNF-α）诱导的巨噬细胞死亡，而托法替布（TOF）无此效应。巨噬细胞在添加或不添加Nec-1（20 nM；n=3）或TOF（300 nM；n=3）的条件下，与TNF-α（100 ng/ml；n=3）共培养24小时，随后通过透射电子显微镜（transmission electron microscopy, TEM）进行分析。展示3次独立实验的代表性图像，标尺：上图5 μm，下图2 μm。
图S3。肿瘤坏死因子抑制剂无法阻断由二酰胺（diamide）或TNF-α诱导的巨噬细胞死亡。巨噬细胞在添加或不添加依那西普（ETN，100 μg/ml）或阿达木单抗（ADA，100 μg/ml）的条件下，分别与二酰胺（1 mM；n=3）或TNF-α（100 ng/ml；n=3）共培养24小时，随后通过TEM分析。展示3次独立实验的代表性图像，标尺：上图5 μm，下图2 μm。
图S4。肿瘤坏死因子抑制剂无法阻断由TNF-α诱导的类风湿关节炎（RA）巨噬细胞死亡。健康供体（HD）巨噬细胞或RA巨噬细胞在添加或不添加TNF-α（100 ng/ml；n=3），以及添加或不添加ETN（100 μg/ml）或ADA（100 μg/ml）的条件下培养24小时，随后通过TEM分析。展示3次独立实验的代表性图像，标尺：上图5 μm，下图2 μm。
图S5。TNF-α可诱导受体相互作用蛋白激酶3（RIP3）磷酸化。巨噬细胞分别在添加或不添加TNF-α（10 ng/ml，培养24小时；n=3）、二酰胺（100 nM，培养24小时；n=3）（A组），以及添加或不添加泛半胱天冬酶抑制剂zVAD-FMK（20 μM；n=3）的条件下，与脂多糖（LPS，500 ng/ml，培养24小时；n=3）共培养。随后使用抗磷酸化丝氨酸227位点RIP3（anti-RIP3 (phosphor S227)）、抗磷酸化丝氨酸473位点蛋白激酶B（phospho-Akt (Ser 473)）的特异性抗体，或同型对照抗体，以及4',6-二脒基-2-苯基吲哚（DAPI）对细胞进行染色。展示3次独立实验的代表性图像，标尺：50 μm。
图S6。IL-1β、IL-6/sIL-6R及IL-21无法诱导混合谱系激酶结构域样蛋白（MLKL）磷酸化。巨噬细胞在添加或不添加IL-1β（10 ng/ml；n=3）、IL-6/sIL-6R（10 ng/ml；n=3）或IL-21（10 ng/ml；n=3）的条件下培养24小时，制备WCL后，采用特异性抗体检测MLKL的总蛋白或磷酸化形式，以及β-actin，进行蛋白质免疫印迹（Western Blot, WB）分析。展示3次独立实验代表性图像的定量数据（n=3），标尺：上图5 μm，下图2 μm。
图S7。Nec-1可阻断TNF-α诱导的MLKL磷酸化。巨噬细胞在添加或不添加Nec-1（20 nM；n=3）的条件下，与TNF-α（10 ng/ml，培养24小时；n=3）共培养，随后制备WCL，通过WB检测MLKL的总蛋白或磷酸化形式，以及β-actin。展示3次独立实验的代表性图像。
图S8。14-3-3η可在HD来源的经TNF-α、二酰胺或LPS处理的巨噬细胞培养上清中被检测到。将巨噬细胞在添加或不添加二酰胺、TNF-α（10 ng/ml，培养24小时；n=3）或LPS（500 ng/ml，培养24小时；n=3），以及添加或不添加Nec-1（20 nM；n=3）、TOF（300 nM；n=3）或zVAD-FMK（20 μM；n=3）的条件下培养，收集培养上清并进行WB分析。以重组14-3-3η作为阳性对照，以牛血清白蛋白（BSA）作为上样对照，采用考马斯亮蓝R350（CBB-R350）进行染色。展示3次独立实验的代表性图像。