Journal: Stem Cells Translational Medicine

Article Title: Extracellular Vesicles From Mesenchymal Umbilical Cord Cells Exert Protection Against Oxidative Stress and Fibrosis in a Rat Model of Bronchopulmonary Dysplasia

doi: 10.1093/stcltm/szad070

Figure Lengend Snippet: Antibody list.

Article Snippet: Goat anti RAGE , AF1145 , BIO-TECHNE.

Techniques:

Journal: Molecular immunology

Article Title: S100A11 regulates nasal epithelial cell remodeling and inflammation in CRSwNPs via the RAGE-mediated AMPK-STAT3 pathway.

doi: 10.1016/j.molimm.2021.09.014

Figure Lengend Snippet: Fig. 5. S100A11 depends on RAGE-mediated can activate AMPK and inhibit STAT3 signaling pathway. A. The phosphorylation levels of AMPK and STAT3 were detected by Western blot after S100A11 was overexpressed. B. The histogram shows the phosphorylation ratio of AMPK and STAT3 (pAMPK/ AMPK and pSTAT3/ STAT3). C. Western blot showed the expression of cleaved-PARP, CCND1, CCNE, Bcl-2, AMPK and STAT3 phosphorylated proteins after treatment of cells with different concentrations of rh-S100A11 protein (0~10000ug/mL) for 24 h. D. Cell immunofluorescence staining detected the expression of STAT3 phosphorylated protein after treatment with 1000ug/mL rh-S100A11 protein for 24 h (bar = 20um). E. The relative mRNA levels of Fas, Stk35, Tsc2 and Socs3 after cells were treated with 1000ug/mL rh-S100A11 protein or transfected for 24 h. F. RAGE protein inhibitors affect the phosphorylation levels of AMPK and STAT3 caused by rhS100A11. The bar graphs show quantification of the results, with each value represents the mean ± SD of three independent experiments. Statistical significance is shown using the Student’s t-test analysis, *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: Rabbit polyclonal anti− CCND1 antibody (Cat. No. 60,186-1-Ig, 1:2000 diluted), anti-RAGE antibody (Cat. No. 66,833-1-Ig, 1:2000 diluted) and anti− CCNE antibody (Cat. No. 11554− 1-AP, 1:2000 diluted for WB) were purchased from Proteintech.

Techniques: Phospho-proteomics, Western Blot, Expressing, Immunofluorescence, Staining, Transfection

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

Journal: Scientific Reports

Article Title: ATP synthase subunit-β down-regulation aggravates diabetic nephropathy

doi: 10.1038/srep14561

Figure Lengend Snippet: ( A ) RAGE antibody binding to RAGE proteins assay in renal proximal tubular cells. The HK-2 cells were treated with the FITC-labeled RAGE antibody (RAGEab-FITC, 10 μg/ml) for 1 hour. The ability of RAGE antibody binding to RAGE proteins was detected by fluorescence ELISA reader. *RAGEab-biotin: biotin-conjugated RAGE antibody. Agarose-streptavidin: streptavidin-conjugated agarose beads. ( B ) HK-2 cells were incubated with AGE-BSA (30 μg/ml) for 24 hours with or without RAGE antibodies (RAGEab, 10 μg/ml) pretreatment for 1 hour. ( C ) HK-2 cells were transiently transfected with ATP5b siRNA (80 nM) for 6 hours and subsequently treated with AGE-BSA (30 μg/ml) for 24 hours. Protein expressions of collagen IV, fibrinogen γ, α-SMA, CTGF, and ATP5b were determined by Western blotting ( B , C ). Quantification of immunoblotting was determined by densitometric analysis. ( D ) The mRNA expressions of CTGF and TGFβ were determined by real-time PCR. HK-2 cells were exposed to AGEs for 24 hours and then subjected to rela-time PCR assay. The mRNA levels were quantified by densitometry and normalized by GAPDH levels. ( E ) The reporter assay for CTGF gene. HK-2 cells were exposed to AGEs for 24 hours with or without ATP5b siRNA transfection. Dual luciferase reporter assay was performed to observe the effect of ATP5b on CTGF transcriptional activity. Data are presented as means ± SEM (n ≥ 5). * P < 0.05, column 4 versus column 3 ( A ); AGE-BSA versus BSA ( B–D ); AGE-BSA/scramble siRNA versus pCTGF vector ( E ). # P < 0.05, column 5 versus column 4 ( A ); AGE-BSA/RAGEab versus AGE-BSA ( B ); AGE-BSA/ATP5b siRNA versus AGE-BSA/scramble siRNA ( C-E ).

Article Snippet: The supernatant of lysate was incubated with or without biotin-conjugated-RAGE polyclonal antibody (2 μg/ml, #bs-4999R-Biotin, Bioss) in the presence of streptavidin-conjugated agarose beads (#S1638, Sigma-Aldrich) at 4 °C overnight.

Techniques: Binding Assay, Labeling, Fluorescence, Enzyme-linked Immunosorbent Assay, Incubation, Transfection, Western Blot, Real-time Polymerase Chain Reaction, Reporter Assay, Luciferase, Activity Assay, Plasmid Preparation