r d systems af1145 rrid ab 354628  (R&D Systems)

Journal: Autophagy

Article Title: DCN released from ferroptotic cells ignites AGER-dependent immune responses

doi: 10.1080/15548627.2021.2008692

Figure Lengend Snippet: AGER mediates the response to DCN. (A) Western blot analysis of protein expression by BMDMs after transfection with the indicated shRNAs. (B) ELISA analysis of TNF and IL6 release in the indicated gene knockdown BMDMs following treatment with ferroptotic MEFs ( n = 3 biologically independent samples; * P < 0.05 versus control shRNA group, one-tailed t test; data are presented as means ± SD). (C) Lack of Ager in BMDMs blocks ferroptotic cell-induced the production of the pro-inflammatory TNF and IL6 cytokines ( n = 3 biologically independent samples; two-way ANOVA with Tukey’s multiple comparisons test; data are presented as means ± SD). (D) Anti-AGER neutralizing antibody (Ab; 1 mg/ml), but not anti-TLR4 neutralizing antibody (1 mg/ml), inhibits ferroptotic cell-induced TNF and IL6 release in BMDMs ( n = 3 biologically independent samples; * P < 0.05, two-way ANOVA with Tukey’s multiple comparisons test; data are presented as means ± SD). (E, F) Lack of Ager in BMDMs inhibits DCN-induced TNF and IL6 release in the absence or presence of HMGB1 ( n = 3 biologically independent samples; * P < 0.05 versus WT group, two-way ANOVA with Tukey’s multiple comparisons test; data are presented as means ± SD). (G, H) Analysis of NFKB activity and TNF release in the indicated BMDMs following treatment with ferroptotic cells or DCN in the absence or presence of the NFKB pathway inhibitor BMS-345541 ( n = 3 biologically independent samples; * P < 0.05 versus WT group, two-way ANOVA with Tukey’s multiple comparisons test; data are presented as means ± SD). (I) His-tag affinity pull-down analysis of the binding of DCN to AGER.

Article Snippet: WT and pancreatic gpx4 −/− mice received anti-DCN Ab (20 mg/kg; monoclonal rat IgG2A; R&D Systems, 161026), anti-AGER Ab (20 mg/kg; monoclonal rat IgG2A; R&D Systems, 175410), control IgG (20 mg/kg; R&D Systems) or liproxsatin-1 (10 mg/kg) by i.p. injection 2 h after completion of the cerulein-induced pancreatitis protocol. (A) Pancreatic sections were stained with H&E at 16 h (bar: 100 μm). (B–G) Serum AMY (amylase), pancreatic trypsin activity, pancreatic MPO activity, serum DCN, serum TNF, and serum IL6 were assayed at 8 and 16 h ( n = 5 mice/group; * P < 0.05 versus IgG group, two-way ANOVA with Tukey’s multiple comparisons test; data are presented as means ± SD).

Techniques: Western Blot, Expressing, Transfection, Enzyme-linked Immunosorbent Assay, Knockdown, Control, shRNA, One-tailed Test, Activity Assay, Binding Assay

Journal: Autophagy

Article Title: DCN released from ferroptotic cells ignites AGER-dependent immune responses

doi: 10.1080/15548627.2021.2008692

Figure Lengend Snippet: Inhibiting the DCN-AGER pathway protects against cerulein-induced acute pancreatitis. WT and pancreatic gpx4 −/− mice received anti-DCN Ab (20 mg/kg; monoclonal rat IgG2A; R&D Systems, 161026), anti-AGER Ab (20 mg/kg; monoclonal rat IgG2A; R&D Systems, 175410), control IgG (20 mg/kg; R&D Systems) or liproxsatin-1 (10 mg/kg) by i.p. injection 2 h after completion of the cerulein-induced pancreatitis protocol. (A) Pancreatic sections were stained with H&E at 16 h (bar: 100 μm). (B–G) Serum AMY (amylase), pancreatic trypsin activity, pancreatic MPO activity, serum DCN, serum TNF, and serum IL6 were assayed at 8 and 16 h ( n = 5 mice/group; * P < 0.05 versus IgG group, two-way ANOVA with Tukey’s multiple comparisons test; data are presented as means ± SD).

Article Snippet: WT and pancreatic gpx4 −/− mice received anti-DCN Ab (20 mg/kg; monoclonal rat IgG2A; R&D Systems, 161026), anti-AGER Ab (20 mg/kg; monoclonal rat IgG2A; R&D Systems, 175410), control IgG (20 mg/kg; R&D Systems) or liproxsatin-1 (10 mg/kg) by i.p. injection 2 h after completion of the cerulein-induced pancreatitis protocol. (A) Pancreatic sections were stained with H&E at 16 h (bar: 100 μm). (B–G) Serum AMY (amylase), pancreatic trypsin activity, pancreatic MPO activity, serum DCN, serum TNF, and serum IL6 were assayed at 8 and 16 h ( n = 5 mice/group; * P < 0.05 versus IgG group, two-way ANOVA with Tukey’s multiple comparisons test; data are presented as means ± SD).

Techniques: Control, Injection, Staining, Activity Assay

Journal: Autophagy

Article Title: DCN released from ferroptotic cells ignites AGER-dependent immune responses

doi: 10.1080/15548627.2021.2008692

Figure Lengend Snippet: The DCN-AGER pathway drives antitumor immunity induced by vaccination with ferroptotic cancer cells. (A) The inhibition of DCN and AGER (but not TLR4) with specific blocking antibodies (Ab; 20 mg/kg) abolished the capacity of RSL3-treated tumor cells to vaccinate against KPC cells in C57BL/6 J mice. The percentage of tumor-free mice is indicated ( n = 10 mice/group, * P < 0.05, two-way ANOVA test). (B) The inhibition of TLR4 (but not DCN or AGER) with specific blocking antibodies (20 mg/kg) abolished the capacity of oxaliplatin-treated tumor cells to vaccinate against KPC cells in C57BL/6 J mice. The percentage of tumor-free mice is indicated ( n = 10 mice/group, * P < 0.05, two-way ANOVA test). (C) The vaccination effect of RSL3-induced ferroptotic cell death in KPC cells was not observed in immune-deficient ( rag2 −/− ) mice ( n = 10 mice/group).

Article Snippet: WT and pancreatic gpx4 −/− mice received anti-DCN Ab (20 mg/kg; monoclonal rat IgG2A; R&D Systems, 161026), anti-AGER Ab (20 mg/kg; monoclonal rat IgG2A; R&D Systems, 175410), control IgG (20 mg/kg; R&D Systems) or liproxsatin-1 (10 mg/kg) by i.p. injection 2 h after completion of the cerulein-induced pancreatitis protocol. (A) Pancreatic sections were stained with H&E at 16 h (bar: 100 μm). (B–G) Serum AMY (amylase), pancreatic trypsin activity, pancreatic MPO activity, serum DCN, serum TNF, and serum IL6 were assayed at 8 and 16 h ( n = 5 mice/group; * P < 0.05 versus IgG group, two-way ANOVA with Tukey’s multiple comparisons test; data are presented as means ± SD).

Techniques: Inhibition, Blocking Assay

Journal: Autophagy

Article Title: DCN released from ferroptotic cells ignites AGER-dependent immune responses

doi: 10.1080/15548627.2021.2008692

Figure Lengend Snippet: A model illustrating the role of DCN in the communication between ferroptotic cells and macrophages. DCN can be actively secreted during the early phase of ferroptosis through MCOLN1-dependent secretory autophagy. Increased ROS can further stimulate autophagy, which causes the degradation of anti-ferroptotic proteins (e.g., ferritin , GPX4 , ARNTL , and SLC40A1/ferroportin-1 ) or organelles (e.g., lipid droplets ), eventually leading to the rupture of the plasma membrane and passive release of DCN. Once released, DCN can bind AGER on macrophages to activate NFKB-dependent cytokine production, thus igniting inflammatory and immune responses.

Article Snippet: WT and pancreatic gpx4 −/− mice received anti-DCN Ab (20 mg/kg; monoclonal rat IgG2A; R&D Systems, 161026), anti-AGER Ab (20 mg/kg; monoclonal rat IgG2A; R&D Systems, 175410), control IgG (20 mg/kg; R&D Systems) or liproxsatin-1 (10 mg/kg) by i.p. injection 2 h after completion of the cerulein-induced pancreatitis protocol. (A) Pancreatic sections were stained with H&E at 16 h (bar: 100 μm). (B–G) Serum AMY (amylase), pancreatic trypsin activity, pancreatic MPO activity, serum DCN, serum TNF, and serum IL6 were assayed at 8 and 16 h ( n = 5 mice/group; * P < 0.05 versus IgG group, two-way ANOVA with Tukey’s multiple comparisons test; data are presented as means ± SD).

Techniques: Clinical Proteomics, Membrane