Journal: Autophagy

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

doi: 10.1080/15548627.2021.2008692

Figure Lengend Snippet: DCN is a DAMP released during ferroptosis. (A) Cell death of HT1080 and HeLa cells following treatment with erastin (5 μM) for 24 h ( n = 3 biologically independent samples; * P < 0.05, one-tailed t test; data are presented as means ± SD). (B) An antibody array technology-based screening of protein release from HT1080 and HeLa cells following treatment with erastin (5 µM) for 24 h. The data are presented as the rank of fold change of protein release. (C) Top 10 released proteins in the setting of panel B. (D) ELISA assay of DCN release in HT1080 cells following treatment with erastin (5 μM), sulfasalazine (500 μM), sorafenib (10 μM), cysteine depletion, RSL3 (0.5 μM), ML162 (0.5 μM), ML210 (5 μM), FIN56 (2.5 μM), staurosporine (200 nM), or CCT137690 (5 μM) in the absence or presence of ferrostatin-1 (1 µM), Z-VAD-FMK (10 µM), or necrosulfonamide (1 μM) for 24 h (data are shown in a heat map as the means of three biologically independent samples). (E–G) HT1080 cells were treated with erastin (5 μM) or RSL3 (5 μM) in the absence or presence of glycine for 3–24 h and the release of DCN, HMGB1, and LDH were assayed by ELISA ( n = 3 biologically independent samples; * P < 0.05, two-way ANOVA with Tukey’s multiple comparisons test; data are presented as means ± SD). (H) Western blot assay of DCN in secreted proteins (SN) and whole cell lysates (WCL) in HT1080 cells following RSL3 (5 μM) treatment for 3 h.

Article Snippet: Commercially available ELISA kits were used to measure the concentrations or activity of HMGB1 (Shino Test Corporation, ST51011), DCN (R&D Systems, DY143, DY1060), IL6 (R&D Systems, M6000B, D6050), TNF (R&D Systems, MTA00B, DTA00D), NFKB (Active Motif, 43,296), MPO (Abcam, ab105136), LDH (Abcam, ab102526), trypsin (Abcam, ab102531), CTSB (Abcam, ab119585), and AMY (Abcam, ab102523) in the indicated samples.

Techniques: One-tailed Test, Ab Array, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Autophagy

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

doi: 10.1080/15548627.2021.2008692

Figure Lengend Snippet: DCN mediates inflammation caused by ferroptotic cells. (A) Ferroptotic cells lacking Dcn, Atg5, or Mcoln1 do not elicit the production of the pro-inflammatory TNF and IL6 cytokine by 1 × 10 6 BMDMs ( n = 3 biologically independent samples; * P < 0.05, two-way ANOVA with Tukey’s multiple comparisons test; data are presented as means ± SD). (B,C) Anti-DCN neutralizing antibody (1 mg/ml) inhibits ferroptotic cells, but not late apoptotic cell- or necroptotic 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). (D,E) Lack of Dcn in late apoptotic or necroptotic cells fails to affect the production of TNF and IL6 by BMDMs ( n = 3 biologically independent samples; data are presented as means ± SD).

Article Snippet: Commercially available ELISA kits were used to measure the concentrations or activity of HMGB1 (Shino Test Corporation, ST51011), DCN (R&D Systems, DY143, DY1060), IL6 (R&D Systems, M6000B, D6050), TNF (R&D Systems, MTA00B, DTA00D), NFKB (Active Motif, 43,296), MPO (Abcam, ab105136), LDH (Abcam, ab102526), trypsin (Abcam, ab102531), CTSB (Abcam, ab119585), and AMY (Abcam, ab102523) in the indicated samples.

Techniques:

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: Commercially available ELISA kits were used to measure the concentrations or activity of HMGB1 (Shino Test Corporation, ST51011), DCN (R&D Systems, DY143, DY1060), IL6 (R&D Systems, M6000B, D6050), TNF (R&D Systems, MTA00B, DTA00D), NFKB (Active Motif, 43,296), MPO (Abcam, ab105136), LDH (Abcam, ab102526), trypsin (Abcam, ab102531), CTSB (Abcam, ab119585), and AMY (Abcam, ab102523) in the indicated samples.

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: Commercially available ELISA kits were used to measure the concentrations or activity of HMGB1 (Shino Test Corporation, ST51011), DCN (R&D Systems, DY143, DY1060), IL6 (R&D Systems, M6000B, D6050), TNF (R&D Systems, MTA00B, DTA00D), NFKB (Active Motif, 43,296), MPO (Abcam, ab105136), LDH (Abcam, ab102526), trypsin (Abcam, ab102531), CTSB (Abcam, ab119585), and AMY (Abcam, ab102523) in the indicated samples.

Techniques: Control, Injection, Staining, Activity Assay

Journal: Journal of Sport and Health Science

Article Title: Decorin, an exercise-induced secretory protein, is associated with improved prognosis in breast cancer patients but does not mediate anti-tumorigenic tissue crosstalk in mice

doi: 10.1016/j.jshs.2024.100991

Figure Lengend Snippet: Systemic delivery of recombinant decorin into SCID mice injected with MDA-MB-231-HM cells. SCID mice injected with the MDA-MB-231-HM cells into mammary pads received 2 different doses of recombinant decorin systemically. (A) Tumor volume, (B) metastatic burden, (C) plasma decorin levels, and (D) tumor decorin levels after the low-dose experiment. Mice were dosed every second day with 100 µg of recombinant decorin or an equal volume of saline (Control) ∼14 days post tumor-cell inoculation. (E) Tumor volume, (F) metastatic burden, (G) plasma decorin levels, and (H) tumor decorin levels after the high-dose experiment. Mice were dosed daily with 10 mg/kg body weight (∼2.5 times the dose of low-dose experiment) of recombinant decorin or an equal volume of saline (Control) ∼7 days post tumor-cell inoculation. Tumor volume was assessed by calliper measurement of primary tumor growth and calculated as (length × width )/2. Metastatic burden was assessed by bioluminescence imaging detection. Plasma decorin levels were measured by ELISA. Tumor decorin levels were measured by western blot using total protein normalization. Two AAV:Dcn gastrocnemius lysates from the overexpression experiments were used as positive controls. Statistical testing was done with two-way analyses of variance or mixed effects model for repeated measures or Student's t test. Data expressed as means ± SEM. ( n = 5–7 mice per group). A.U. = arbitrary unit; con = control; Dcn = decorin; ELISA = enzyme linked immunosorbent assay.

Article Snippet: The concentration of decorin was measured in EV-free plasma with enzyme linked immunosorbent assay (ELISA) (DuoSet, Cat# DY143; R&D Systems, Minneapolis, MN, USA).

Techniques: Recombinant, Injection, Clinical Proteomics, Saline, Control, Imaging, Enzyme-linked Immunosorbent Assay, Western Blot, Over Expression