Journal: Frontiers in Immunology

Article Title: Synergistic effects of platelet-rich fibrin and CTLA4Ig gene-transfected porcine skin on accelerating wound healing in a rat model of deep second-degree burns: a mechanistic study

doi: 10.3389/fimmu.2025.1756818

Figure Lengend Snippet: Temporal expression profiles of antioxidant enzymes CAT and SOD1. Immunofluorescence staining of (A) Catalase (CAT, red) and (B) Superoxide Dismutase 1 (SOD1, red) in wound tissues from the Vaseline group, PRF group, Pigskin group, and PRF+pig skin group at days 4, 7, 14, and 21 post-treatment. Cell nuclei are counterstained with DAPI (blue). The PRF+pig skin group shows the most pronounced and sustained enhancement in the expression of both antioxidant enzymes, particularly during the proliferative and remodeling phases (D7–D21), indicating a reinforced antioxidant defense system. Scale bar = 100 μm.

Article Snippet: After deparaffinization and antigen retrieval (similar to IHC), sections were permeabilized with 0.1% Triton X-100, blocked with 5% BSA, and incubated overnight at 4 °C with primary antibodies against CAT (1:200, Proteintech) and SOD1 (1:200, Proteintech).

Techniques: Expressing, Immunofluorescence, Staining

Journal: Animal Models and Experimental Medicine

Article Title: Adropin modulates pancreatic cell proliferation and glutathione levels in an animal model of type 1 diabetes mellitus

doi: 10.1002/ame2.70092

Figure Lengend Snippet: Impact of adropin on superoxide dismutase expression in pancreatic β‐cells of normoglycemic and diabetic rats. (A) Immunofluorescence labeling with anti‐superoxide dismutase antibody and anti‐insulin antibodies showed expression of superoxide dismutase in pancreatic β‐cells. (B) Quantification of the histological analysis showed a significant (*** p < 0.001) decrease in superoxide dismutase distribution in pancreatic endocrine cells and a significant (**** p < 0.0001) decrease in its localization in β‐cells of rats with diabetes when compared to the normal group. n = 6. Scale bar: 50 μm. Data analysis was done using the ANOVA test.

Article Snippet: Superoxide dismutase (rabbit) (1:500) , Rockland Immunochemicals, USA.

Techniques: Expressing, Immunofluorescence, Labeling

Journal: Animal Models and Experimental Medicine

Article Title: Adropin modulates pancreatic cell proliferation and glutathione levels in an animal model of type 1 diabetes mellitus

doi: 10.1002/ame2.70092

Figure Lengend Snippet: Effect of adropin on superoxide dismutase expression in pancreatic α‐cells of normal and diabetic rats. (A) Immunofluorescence staining using anti‐superoxide dismutase and anti‐glucagon antibodies showed expression of superoxide dismutase in pancreatic α‐cells. (B) Quantification of the histological analysis showed a significant (** p < 0.01) decrease in superoxide dismutase distribution in the pancreatic endocrine cells of diabetic rats compared to the normal group. α‐Cell expression of superoxide dismutase did not change with adropin treatment among all groups. n = 6. Scale bar: 50 μm. Data analysis was done using the ANOVA test.

Article Snippet: Superoxide dismutase (rabbit) (1:500) , Rockland Immunochemicals, USA.

Techniques: Expressing, Immunofluorescence, Staining

Journal: Animal Models and Experimental Medicine

Article Title: Adropin modulates pancreatic cell proliferation and glutathione levels in an animal model of type 1 diabetes mellitus

doi: 10.1002/ame2.70092

Figure Lengend Snippet: Effect of adropin on catalase, superoxide dismutase and total glutathione activities in the serum samples of healthy and diabetic rats. (A) Catalase activity was significantly decreased in diabetic rats compared to normal controls. Adropin slightly increased catalase in DMT group. (B) Superoxide dismutase was slightly increased in DMT compared to the diabetic treated with adropin. (C) Total glutathione was significantly raised with adropin treatment in DMT compared to DMUT. n = 4–6. Data analysis was done using the ANOVA test. * p < 0.05.

Article Snippet: Superoxide dismutase (rabbit) (1:500) , Rockland Immunochemicals, USA.

Techniques: Activity Assay

Journal: Scientific reports

Article Title: Dietary Chlorella vulgaris supplementation modulates health, microbiota and the response to oxidative stress of Atlantic salmon.

doi: 10.1038/s41598-024-72531-8

Figure Lengend Snippet: Fig. 5. Protein abundance of catalase (Cat) in liver (A) and gill (B), as well as superoxide-dismutase 1 (Sod1) in liver (C) and gill (D) of Atlantic salmon fed with C. vulgaris enriched diets for eight weeks. The salmon received either a control diet (CD), a diet with 2% (CV2) or 14% C. vulgaris (CV14) on a daily basis, or a diet containing 14% C. vulgaris once weekly (CV14w). The fish were subsequently exposed to an oxidative stressor via treatment with peracetic acid-based disinfectant and sampled before and 18 h after stress treatment. Samples per organ and diet group represent a pool of four fish per tank (n = 3 tanks) and data is presented as boxplots with median. Statistical significance (p < 0.05) was assessed by multiple comparisons for heterogenous variances and p-values for the ANOVA interaction effect of diet and timepoint are shown in the top right of each panel.

Article Snippet: Primary antibody incubation, with Cat antibody (ab209721, Abcam, Cambridge, UK) and Sod1 antibody (NBP2-24915, Novus Biologicals, Bio-Techne Ltd., Abingdon, UK) both in 1:500 dilution in PBS-T containing 2.5% skim milk, was performed at 4 °C at 150 rpm overnight.

Techniques: Quantitative Proteomics, Control

Journal: Communications Biology

Article Title: Structural mechanisms and insights on multiple nanobodies binding diverse SOD1 epitopes

doi: 10.1038/s42003-025-09293-0

Figure Lengend Snippet: A SOD1; B Nb1; C Nb2; D Nb3; E SOD1-Nb1 complex; F SOD1-Nb2 complex; G SOD1-Nb3 complex. The horizontal axis represents mass-to-charge ratio (m/z), and the vertical axis represents intensity, indicating the specific m/z values at peak intensities.

Article Snippet: SOD1 was labeled with Cyanine5-NHS ester iodide (MCE) at a 1:2 molar ratio at 4 °C overnight, and excess red fluorescent dye was removed using Zeba Spin Desalting columns (ThermoFisher).

Techniques:

Journal: Communications Biology

Article Title: Structural mechanisms and insights on multiple nanobodies binding diverse SOD1 epitopes

doi: 10.1038/s42003-025-09293-0

Figure Lengend Snippet: A The structure of the SOD1-Nb1 complex (PDB ID: 8K33). B The structure of the SOD1-Nb2 complex (PDB ID: 8K3A). C The structure of the SOD1-Nb3 complex (PDB ID: 8K3L). D Structural superimposition of the three nanobodies, with SOD1, Nb1, Nb2, and Nb3 shown in cyan, gray-white, yellow, and purple, respectively (Only depicted Nb1, Nb2, and Nb3 bound to one SOD1 monomer). E – G Surface electrostatic potentials of the SOD1-Nb1, SOD1-Nb2, and SOD1-Nb3 binary complexes (positive in blue, negative in red, and neutral in white). SOD1 is shown in cyan, with Cu and Zn atoms depicted as blue and orange spheres. Nb1, Nb2, and Nb3 are shown in gray-white, yellow, and purple, respectively. CDRs 1-3 of the nanobodies are highlighted in blue, green, and red, respectively.

Article Snippet: SOD1 was labeled with Cyanine5-NHS ester iodide (MCE) at a 1:2 molar ratio at 4 °C overnight, and excess red fluorescent dye was removed using Zeba Spin Desalting columns (ThermoFisher).

Techniques:

Journal: Communications Biology

Article Title: Structural mechanisms and insights on multiple nanobodies binding diverse SOD1 epitopes

doi: 10.1038/s42003-025-09293-0

Figure Lengend Snippet: SOD1 is shown in cyan, Nb1, Nb2, and Nb3 shown in gray-white, yellow, and purple, respectively with CDRs 1-3 of Nb1/Nb2/Nb3 shown in blue, green, and red, respectively. Residues involved in the interactions are depicted as sticks, with yellow dotted lines indicating hydrogen bonds and salt bridges. A Overall view and detailed views of the binding interface between SOD1 and complementarity determining regions (CDRs) of Nb1. B Overall view and detailed views of the binding interface between SOD1 and CDRs of Nb2. C Overall view and detailed views of the binding interface between SOD1 and CDRs of Nb3.

Article Snippet: SOD1 was labeled with Cyanine5-NHS ester iodide (MCE) at a 1:2 molar ratio at 4 °C overnight, and excess red fluorescent dye was removed using Zeba Spin Desalting columns (ThermoFisher).

Techniques: Binding Assay

Journal: Communications Biology

Article Title: Structural mechanisms and insights on multiple nanobodies binding diverse SOD1 epitopes

doi: 10.1038/s42003-025-09293-0

Figure Lengend Snippet: A The overall structure of SOD1, Nb1 and Nb2 complex (PDB ID: 8YAF). B The surface view of the SOD1, Nb1 and Nb2 complex. C The overall structure of SOD1, Nb1, Nb2 and Nb3 complex (PDB ID: 8YAT). D The surface view of SOD1, Nb1, Nb2 and Nb3 complex. E The structure comparison of SOD1, Nb1 and Nb2 complex with SOD1 fibril core. F The surface view of the comparison between SOD1, Nb1 and Nb2 complex with SOD1 fibril core. G The structure comparison of SOD1, Nb1, Nb2 and Nb3 complex with SOD1 fibril core. H The surface view of the comparison between SOD1, Nb1, Nb2 and Nb3 complex with SOD1 fibril core.

Article Snippet: SOD1 was labeled with Cyanine5-NHS ester iodide (MCE) at a 1:2 molar ratio at 4 °C overnight, and excess red fluorescent dye was removed using Zeba Spin Desalting columns (ThermoFisher).

Techniques: Comparison

Journal: Communications Biology

Article Title: Structural mechanisms and insights on multiple nanobodies binding diverse SOD1 epitopes

doi: 10.1038/s42003-025-09293-0

Figure Lengend Snippet: A , B Comparison of SOD1 and SOD1-nanobody binary complexes. C , D Detection of epitope binding with two nanobodies. E , F Detection of epitope binding with three nanobodies. The left panels display the full chromatogram, while the right panels provide an enlarged view of the elution peaks. SOD1, SOD1-Nb1, SOD1-Nb2, and SOD1-Nb3 are represented in black, red, blue, and green, respectively. SOD1-Nb1-Nb2, SOD1-Nb1-Nb3, SOD1-Nb2-Nb3, and SOD1-Nb1-Nb2-Nb3 are shown in purple, orange, light blue, and brown, respectively.

Article Snippet: SOD1 was labeled with Cyanine5-NHS ester iodide (MCE) at a 1:2 molar ratio at 4 °C overnight, and excess red fluorescent dye was removed using Zeba Spin Desalting columns (ThermoFisher).

Techniques: Comparison, Binding Assay

Journal: Communications Biology

Article Title: Structural mechanisms and insights on multiple nanobodies binding diverse SOD1 epitopes

doi: 10.1038/s42003-025-09293-0

Figure Lengend Snippet: A – D Binding of Nb1 to SOD1, SOD1-Nb2, SOD1-Nb3, and SOD1-Nb2-Nb3; E – H Binding of Nb2 to SOD1, SOD1-Nb1, SOD1-Nb3, and SOD1-Nb1-Nb3; I – L Binding of Nb3 to SOD1, SOD1-Nb1, SOD1-Nb2, and SOD1-Nb1-Nb2. Panels show ITC profiles with binding isotherms and corresponding thermodynamic parameters.

Article Snippet: SOD1 was labeled with Cyanine5-NHS ester iodide (MCE) at a 1:2 molar ratio at 4 °C overnight, and excess red fluorescent dye was removed using Zeba Spin Desalting columns (ThermoFisher).

Techniques: Binding Assay

Journal: Communications Biology

Article Title: Structural mechanisms and insights on multiple nanobodies binding diverse SOD1 epitopes

doi: 10.1038/s42003-025-09293-0

Figure Lengend Snippet: A , B Particle size distributions of SOD1 and its nanobody complexes by dynamic light scattering. A Size distributions for SOD1 (black), SOD1-Nb1 (red), SOD1-Nb2 (blue), and SOD1-Nb3 (green); B Size distributions for SOD1 (black), SOD1-Nb1-Nb2 (red), SOD1-Nb1-Nb3 (blue), SOD1-Nb2-Nb3 (green), and SOD1-Nb1-Nb2-Nb3 (purple). C Measurement of SOD1 enzyme activity using the Kit-WST. The activities of SOD1, SOD1-Nb1, SOD1-Nb2, and SOD1-Nb3 are shown in blue, purple, pink, and green, respectively. Error bars represent the standard error from three independent experiments (n = 3). D – G Hydrogen bond distances between Asp124 and metal-binding histidines (His46/His71) in different SOD1 conformational states. D WT SOD1, E Nb1 binding SOD1, F Nb2 binding SOD1, G Nb3 binding SOD1. H Thioflavin T (ThT) fluorescence intensities representing the filament formation of SOD1 and nanobodies binding SOD1. Error bars represent the standard error from three independent experiments (n = 3).

Article Snippet: SOD1 was labeled with Cyanine5-NHS ester iodide (MCE) at a 1:2 molar ratio at 4 °C overnight, and excess red fluorescent dye was removed using Zeba Spin Desalting columns (ThermoFisher).

Techniques: Activity Assay, Binding Assay, Fluorescence

Journal: Communications Biology

Article Title: Structural mechanisms and insights on multiple nanobodies binding diverse SOD1 epitopes

doi: 10.1038/s42003-025-09293-0

Figure Lengend Snippet: A , B Interaction of Nb1-Nb2 tandem nanobodies with different linker lengths to SOD1. C Interaction of Nb2-Nb3 tandem nanobodies with SOD1. D Interaction of Nb1-Nb2-Nb3 tandem nanobodies with SOD1.

Article Snippet: SOD1 was labeled with Cyanine5-NHS ester iodide (MCE) at a 1:2 molar ratio at 4 °C overnight, and excess red fluorescent dye was removed using Zeba Spin Desalting columns (ThermoFisher).

Techniques:

Journal: Autophagy

Article Title: Rilmenidine promotes MTOR-independent autophagy in the mutant SOD1 mouse model of amyotrophic lateral sclerosis without slowing disease progression

doi: 10.1080/15548627.2017.1385674

Figure Lengend Snippet: Autophagy is activated in NSC-34 cells expressing ALS-linked misfolded proteins. (A) Immunoblot analysis of macroautophagy and CMA markers in NSC-34 cells stably transfected with wild-type (WT) or mutant G85R or G93A SOD1. Quantification of (B) LC3-II, (C) SQSTM1, (D) LAMP2A and (E) HSPA8 protein levels from immunoblots normalized to WT SOD1-expressing cells. (F) Immunoblot analysis of macroautophagy and CMA markers in NSC-34 cells stably transfected with WT or mutant Q331K or M337V TARDBP. Quantification of (G) LC3-II, (H) SQSTM1, (I) LAMP2A and (J) HSPA8 protein levels from immunoblots normalized to WT TARDBP-expressing cells. Data represent mean ± SD, n = 5-6 independent experiments, * p <0.05 and ** p <0.01 compared to cells expressing the WT form of relevant protein using one-way ANOVA with Tukey's posthoc test.

Article Snippet: Membranes were blocked with 5% (w:v) skim milk dried powder (Fonterra, 492281) in Tris-buffered saline with Tween-20 (TBST; Tris base 20 mM, NaCl 140 mM, pH 8.0, 0.1% [v:v] Tween-20 [Sigma, P7949]) for 30 min and incubated with antibodies to rabbit LC3B (1:1,000; Sigma-Aldrich, L7543), mouse SQSTM1 (1:500; Abcam, ab56416), sheep SOD1 (1:4,000; Merck, 574597), rabbit LAMP2A (1:500; Abcam, ab18528), mouse HSPA8/HSC70 (1:500; Enzo Lifesciences, ALX-804-067), mouse SNCA (1:1,000; BD Transduction Labs, 610786), rabbit VDAC1 (1:1,000; Abcam ab15895), rabbit MTOR (1:1000; Cell Signaling Technology, 2983), rabbit phospho-MTOR (1:1000; Cell Signaling Technology, 2971), mouse human-specific SOD1 (1:2,000; R&D Systems, MAB3418), mouse misfolded SOD1 (C4F6) (1:250; Medimabs, MM-0070-2-P) or mouse ACTB (1:2,000; Sigma-Aldrich, A5316) antibodies in 3% (w:v) BSA (Sigma-Aldrich, A3912) in TBST overnight at 4°C.

Techniques: Expressing, Western Blot, Stable Transfection, Transfection, Mutagenesis

Journal: Autophagy

Article Title: Rilmenidine promotes MTOR-independent autophagy in the mutant SOD1 mouse model of amyotrophic lateral sclerosis without slowing disease progression

doi: 10.1080/15548627.2017.1385674

Figure Lengend Snippet: Rilmenidine stimulates macroautophagy and clears mutant SOD1 protein from NSC-34 cells. (A) Immunoblot analysis of macroautophagy markers in NSC-34 cells transiently transfected with wild-type (WT) or mutant SOD1 A4V and treated with rilmenidine (Ril) for 18 h post-transfection. Quantification of (B) LC3-II and (C) SQSTM1 protein levels from immunoblots normalized to untreated cells. Data represent mean ± SD, n = 4-6 independent experiments, * p <0.05 compared to untreated cells using one-way ANOVA with Tukey's post hoc test. (D) Photomicrographs of NSC-34 cells transfected with mCherry-GFP-LC3 plasmid and treated with rilmenidine. In rilmenidine-treated cells, there is a significantly increased proportion of mCherry-positive mature autolysosomes without GFP (arrows), indicating fusion of autophagosomes (AP) and lysosomes. (E) Quantification of the percentage of mCherry-positive mature autolysosomes relative to total puncta per cell. Data represent mean ± SD, n = 3 independent experiments, * p <0.05 using an unpaired t-test. (F) Immunoblot analysis of LC3-II levels in NSC-34 cells transfected with mutant SOD1 A4V and treated with 400 nM bafilomycin A 1 (Baf A1) ± 10 µM rilmenidine for 24 h. (G) Quantification of LC3-II protein levels from immunoblots normalized to cells treated with bafilomycin A 1 only. Data represent mean ± SD, n = 3 independent experiments, * p <0.05 compared to bafilomycin A 1 -treated cells using an unpaired t-test. (H) Immunoblot analysis of human SOD1 (HsSOD1) levels in WT or mutant SOD1 A4V -transfected NSC-34 cells treated with rilmenidine for 24 h post-transfection. (I) Quantification of HsSOD1 protein levels from immunoblots normalized to untreated cells. Data represent mean ± SD, n = 5 independent experiments, * p <0.05 compared to untreated cells using one-way ANOVA with Tukey's posthoc test. (J) Effect of rilmenidine or rotenone (positive control) on cell viability determined by MTT reduction assay (expressed as % of untreated cells). Data represent mean ± SD, n = 3 independent experiments. ** p <0.01, *** p <0.001 compared to untreated cells using one-way ANOVA with Tukey's posthoc test.

Article Snippet: Membranes were blocked with 5% (w:v) skim milk dried powder (Fonterra, 492281) in Tris-buffered saline with Tween-20 (TBST; Tris base 20 mM, NaCl 140 mM, pH 8.0, 0.1% [v:v] Tween-20 [Sigma, P7949]) for 30 min and incubated with antibodies to rabbit LC3B (1:1,000; Sigma-Aldrich, L7543), mouse SQSTM1 (1:500; Abcam, ab56416), sheep SOD1 (1:4,000; Merck, 574597), rabbit LAMP2A (1:500; Abcam, ab18528), mouse HSPA8/HSC70 (1:500; Enzo Lifesciences, ALX-804-067), mouse SNCA (1:1,000; BD Transduction Labs, 610786), rabbit VDAC1 (1:1,000; Abcam ab15895), rabbit MTOR (1:1000; Cell Signaling Technology, 2983), rabbit phospho-MTOR (1:1000; Cell Signaling Technology, 2971), mouse human-specific SOD1 (1:2,000; R&D Systems, MAB3418), mouse misfolded SOD1 (C4F6) (1:250; Medimabs, MM-0070-2-P) or mouse ACTB (1:2,000; Sigma-Aldrich, A5316) antibodies in 3% (w:v) BSA (Sigma-Aldrich, A3912) in TBST overnight at 4°C.

Techniques: Mutagenesis, Western Blot, Transfection, Plasmid Preparation, Positive Control, MTT Reduction Assay

Journal: Autophagy

Article Title: Rilmenidine promotes MTOR-independent autophagy in the mutant SOD1 mouse model of amyotrophic lateral sclerosis without slowing disease progression

doi: 10.1080/15548627.2017.1385674

Figure Lengend Snippet: Rilmenidine has no effect on the CMA pathway in NSC-34 cells. (A) Immunoblot analysis of CMA markers and substrate SNCA in NSC-34 cells transiently transfected with wild-type (WT) SOD1 and treated with rilmenidine (Ril) for 18 h post-transfection. Quantification of (B) LAMP2A, (C) HSPA8 and (D) SNCA protein levels from immunoblots normalized to untreated cells. Data represent mean ± SD, n = 3 independent experiments.

Article Snippet: Membranes were blocked with 5% (w:v) skim milk dried powder (Fonterra, 492281) in Tris-buffered saline with Tween-20 (TBST; Tris base 20 mM, NaCl 140 mM, pH 8.0, 0.1% [v:v] Tween-20 [Sigma, P7949]) for 30 min and incubated with antibodies to rabbit LC3B (1:1,000; Sigma-Aldrich, L7543), mouse SQSTM1 (1:500; Abcam, ab56416), sheep SOD1 (1:4,000; Merck, 574597), rabbit LAMP2A (1:500; Abcam, ab18528), mouse HSPA8/HSC70 (1:500; Enzo Lifesciences, ALX-804-067), mouse SNCA (1:1,000; BD Transduction Labs, 610786), rabbit VDAC1 (1:1,000; Abcam ab15895), rabbit MTOR (1:1000; Cell Signaling Technology, 2983), rabbit phospho-MTOR (1:1000; Cell Signaling Technology, 2971), mouse human-specific SOD1 (1:2,000; R&D Systems, MAB3418), mouse misfolded SOD1 (C4F6) (1:250; Medimabs, MM-0070-2-P) or mouse ACTB (1:2,000; Sigma-Aldrich, A5316) antibodies in 3% (w:v) BSA (Sigma-Aldrich, A3912) in TBST overnight at 4°C.

Techniques: Western Blot, Transfection

Journal: Autophagy

Article Title: Rilmenidine promotes MTOR-independent autophagy in the mutant SOD1 mouse model of amyotrophic lateral sclerosis without slowing disease progression

doi: 10.1080/15548627.2017.1385674

Figure Lengend Snippet: Rilmenidine stimulates macroautophagy in H9 human embryonic stem cell-derived spinal motor neurons. Photomicrographs of motor neuron spheres stained for (A) HB9 and (B) ISL1, and dissociated mature motor neurons stained with (C) CHAT and (D) TUBB3 antibodies. (E) Immunoblot analysis of macroautophagy markers in induced motor neurons transiently transfected with wild-type SOD1 and treated with rilmenidine (Ril) for 24 h post-transfection. Quantification of (F) LC3-II and (G) SQSTM1 protein levels from immunoblots normalized to untreated cells. Data represent mean ± SD, n = 5 independent experiments, * p <0.05 compared to untreated cells using an unpaired t-test.

Article Snippet: Membranes were blocked with 5% (w:v) skim milk dried powder (Fonterra, 492281) in Tris-buffered saline with Tween-20 (TBST; Tris base 20 mM, NaCl 140 mM, pH 8.0, 0.1% [v:v] Tween-20 [Sigma, P7949]) for 30 min and incubated with antibodies to rabbit LC3B (1:1,000; Sigma-Aldrich, L7543), mouse SQSTM1 (1:500; Abcam, ab56416), sheep SOD1 (1:4,000; Merck, 574597), rabbit LAMP2A (1:500; Abcam, ab18528), mouse HSPA8/HSC70 (1:500; Enzo Lifesciences, ALX-804-067), mouse SNCA (1:1,000; BD Transduction Labs, 610786), rabbit VDAC1 (1:1,000; Abcam ab15895), rabbit MTOR (1:1000; Cell Signaling Technology, 2983), rabbit phospho-MTOR (1:1000; Cell Signaling Technology, 2971), mouse human-specific SOD1 (1:2,000; R&D Systems, MAB3418), mouse misfolded SOD1 (C4F6) (1:250; Medimabs, MM-0070-2-P) or mouse ACTB (1:2,000; Sigma-Aldrich, A5316) antibodies in 3% (w:v) BSA (Sigma-Aldrich, A3912) in TBST overnight at 4°C.

Techniques: Derivative Assay, Staining, Western Blot, Transfection

Journal: Autophagy

Article Title: Rilmenidine promotes MTOR-independent autophagy in the mutant SOD1 mouse model of amyotrophic lateral sclerosis without slowing disease progression

doi: 10.1080/15548627.2017.1385674

Figure Lengend Snippet: Rilmenidine treatment stimulates MTOR-independent macroautophagy and mitophagy in motor neurons of SOD1 G93A mice. (A) Immunoblot analysis of macroautophagy and CMA markers in lumbar spinal cords from vehicle- or rilmenidine-treated SOD1 G93A mice at 90 days of age. Quantification of (C) LC3-II, (D) SQSTM1 and (E) VDAC1 protein levels from immunoblots normalized to vehicle group. (B) Immunoblot analysis of autophagy markers and phosphorylated (p-MTOR) and total MTOR levels in spinal cords from vehicle- or rilmenidine-treated SOD1 G93A mice at end stage. Quantification of (C) LC3-II, (D) SQSTM1 and (E) VDAC1 protein levels and (F) p-MTOR:MTOR ratio from immunoblots normalized to vehicle group. Data represent mean ± SD, n = 3-5 mice, * p <0.05 compared to vehicle-treated mice using an unpaired t-test.

Article Snippet: Membranes were blocked with 5% (w:v) skim milk dried powder (Fonterra, 492281) in Tris-buffered saline with Tween-20 (TBST; Tris base 20 mM, NaCl 140 mM, pH 8.0, 0.1% [v:v] Tween-20 [Sigma, P7949]) for 30 min and incubated with antibodies to rabbit LC3B (1:1,000; Sigma-Aldrich, L7543), mouse SQSTM1 (1:500; Abcam, ab56416), sheep SOD1 (1:4,000; Merck, 574597), rabbit LAMP2A (1:500; Abcam, ab18528), mouse HSPA8/HSC70 (1:500; Enzo Lifesciences, ALX-804-067), mouse SNCA (1:1,000; BD Transduction Labs, 610786), rabbit VDAC1 (1:1,000; Abcam ab15895), rabbit MTOR (1:1000; Cell Signaling Technology, 2983), rabbit phospho-MTOR (1:1000; Cell Signaling Technology, 2971), mouse human-specific SOD1 (1:2,000; R&D Systems, MAB3418), mouse misfolded SOD1 (C4F6) (1:250; Medimabs, MM-0070-2-P) or mouse ACTB (1:2,000; Sigma-Aldrich, A5316) antibodies in 3% (w:v) BSA (Sigma-Aldrich, A3912) in TBST overnight at 4°C.

Techniques: Western Blot

Journal: Autophagy

Article Title: Rilmenidine promotes MTOR-independent autophagy in the mutant SOD1 mouse model of amyotrophic lateral sclerosis without slowing disease progression

doi: 10.1080/15548627.2017.1385674

Figure Lengend Snippet: Rilmenidine treatment increases autophagosome number in spinal motor neurons of SOD1 G93A mice. (A) LC3 immunohistochemical analysis in lumbar spinal cords of vehicle- and rilmenidine-treated SOD1 G93A mice. In vehicle-treated mice, there is diffuse distribution of autophagosomes in the cytoplasm of RBFOX3/NeuN-positive motor neurons. In rilmenidine-treated mice, there is accumulation of autophagosomes (arrows) in motor neurons. (B) LAMP2 immunohistochemical analysis in lumbar spinal cords of vehicle- and rilmenidine-treated SOD1 G93A mice. In vehicle- and rilmenidine-treated mice, lysosomes (arrows) are distributed in the cytoplasm of motor neurons. Quantification of (C) LC3-positive autophagosomes and (D) LAMP2-positive lysosomes in motor neurons. Data represent mean ± SD, n = 3 mice per group, * p <0.05 compared to vehicle-treated mice using an unpaired t-test.

Article Snippet: Membranes were blocked with 5% (w:v) skim milk dried powder (Fonterra, 492281) in Tris-buffered saline with Tween-20 (TBST; Tris base 20 mM, NaCl 140 mM, pH 8.0, 0.1% [v:v] Tween-20 [Sigma, P7949]) for 30 min and incubated with antibodies to rabbit LC3B (1:1,000; Sigma-Aldrich, L7543), mouse SQSTM1 (1:500; Abcam, ab56416), sheep SOD1 (1:4,000; Merck, 574597), rabbit LAMP2A (1:500; Abcam, ab18528), mouse HSPA8/HSC70 (1:500; Enzo Lifesciences, ALX-804-067), mouse SNCA (1:1,000; BD Transduction Labs, 610786), rabbit VDAC1 (1:1,000; Abcam ab15895), rabbit MTOR (1:1000; Cell Signaling Technology, 2983), rabbit phospho-MTOR (1:1000; Cell Signaling Technology, 2971), mouse human-specific SOD1 (1:2,000; R&D Systems, MAB3418), mouse misfolded SOD1 (C4F6) (1:250; Medimabs, MM-0070-2-P) or mouse ACTB (1:2,000; Sigma-Aldrich, A5316) antibodies in 3% (w:v) BSA (Sigma-Aldrich, A3912) in TBST overnight at 4°C.

Techniques: Immunohistochemical staining

Journal: Autophagy

Article Title: Rilmenidine promotes MTOR-independent autophagy in the mutant SOD1 mouse model of amyotrophic lateral sclerosis without slowing disease progression

doi: 10.1080/15548627.2017.1385674

Figure Lengend Snippet: Rilmenidine treatment worsens disease progression and neurodegeneration in SOD1 G93A mice. (A) Onset of body weight decline and (B) locomotor activity were similar in rilmenidine- and vehicle-treated SOD1 G93A mice. (C) Survival was significantly reduced by rilmenidine treatment compared to vehicle-treated animals. Data represent mean ± SD, n = 10 mice per group, * p <0.05 using the log-rank test. (D) Photomicrographs of ventral horns stained with cresyl violet in lumbar spinal cords of mice at 90 days of age. (E) Motor neuron counts in ventral horns of lumbar spinal cords of vehicle- and rilmenidine-treated mice. Motor neuron numbers in rilmenidine-treated mice were significantly decreased compared to vehicle-treated mice. Data represent mean ± SD, n = 5 mice per group, ** p <0.01 compared to vehicle-treated mice using an unpaired t-test. Immunohistochemical analysis of (F) astrocytes using GFAP and (G) microglia using ITGAM/CD11b in lumbar spinal cords of mice at end stage. Astrocyte and microglial activation appear similar in spinal cords of rilmenidine- and vehicle-treated mice. Images are representative of 3 mice per group.

Article Snippet: Membranes were blocked with 5% (w:v) skim milk dried powder (Fonterra, 492281) in Tris-buffered saline with Tween-20 (TBST; Tris base 20 mM, NaCl 140 mM, pH 8.0, 0.1% [v:v] Tween-20 [Sigma, P7949]) for 30 min and incubated with antibodies to rabbit LC3B (1:1,000; Sigma-Aldrich, L7543), mouse SQSTM1 (1:500; Abcam, ab56416), sheep SOD1 (1:4,000; Merck, 574597), rabbit LAMP2A (1:500; Abcam, ab18528), mouse HSPA8/HSC70 (1:500; Enzo Lifesciences, ALX-804-067), mouse SNCA (1:1,000; BD Transduction Labs, 610786), rabbit VDAC1 (1:1,000; Abcam ab15895), rabbit MTOR (1:1000; Cell Signaling Technology, 2983), rabbit phospho-MTOR (1:1000; Cell Signaling Technology, 2971), mouse human-specific SOD1 (1:2,000; R&D Systems, MAB3418), mouse misfolded SOD1 (C4F6) (1:250; Medimabs, MM-0070-2-P) or mouse ACTB (1:2,000; Sigma-Aldrich, A5316) antibodies in 3% (w:v) BSA (Sigma-Aldrich, A3912) in TBST overnight at 4°C.

Techniques: Biomarker Discovery, Activity Assay, Staining, Immunohistochemical staining, Activation Assay

Journal: Autophagy

Article Title: Rilmenidine promotes MTOR-independent autophagy in the mutant SOD1 mouse model of amyotrophic lateral sclerosis without slowing disease progression

doi: 10.1080/15548627.2017.1385674

Figure Lengend Snippet: Rilmenidine treatment increases the burden of pathological and aggregated SOD1 in motor neurons of SOD1 G93A mice. (A) Immunoblot analysis of soluble SOD1 protein levels in spinal cords of vehicle- and rilmenidine-treated SOD1 G93A mice at end stage. Quantification of (B) human SOD1 (HsSOD1) and (C) misfolded SOD1 (misSOD1) protein levels normalized to vehicle-treated mice. (D) Immunoblot analysis of insoluble SOD1 protein levels in spinal cord pellet fractions of vehicle- and rilmenidine-treated SOD1 G93A mice at end stage. (E) Quantification of HsSOD1 protein level in pellet fraction normalized to vehicle group. Data represent mean ± SD, n = 5 mice per group, ** p <0.01 compared to vehicle-treated mice using an unpaired t-test. (F, H and I) Misfolded SOD1 immunohistochemical analysis of lumbar spinal cords of vehicle- and rilmenidine-treated SOD1 G93A mice at 90 days of age. (F-H) In vehicle-treated mice, misfolded SOD1 shows diffuse staining in the cytoplasm of RBFOX3/NeuN-positive motor neurons (arrows). In rilmenidine-treated mice, SOD1 immunoreactivity is increased with accumulation of SOD1 into cytoplasmic aggregates in motor neurons. SOD1 accumulates into (H) Lewy body-like or (I) skein-like inclusions (arrows) in motor neurons. (G) Quantification of misfolded SOD1 aggregates in motor neurons. Data represent mean ± SD, n = 5 mice per group, ** p <0.01 compared to vehicle-treated mice using an unpaired t-test.

Article Snippet: Membranes were blocked with 5% (w:v) skim milk dried powder (Fonterra, 492281) in Tris-buffered saline with Tween-20 (TBST; Tris base 20 mM, NaCl 140 mM, pH 8.0, 0.1% [v:v] Tween-20 [Sigma, P7949]) for 30 min and incubated with antibodies to rabbit LC3B (1:1,000; Sigma-Aldrich, L7543), mouse SQSTM1 (1:500; Abcam, ab56416), sheep SOD1 (1:4,000; Merck, 574597), rabbit LAMP2A (1:500; Abcam, ab18528), mouse HSPA8/HSC70 (1:500; Enzo Lifesciences, ALX-804-067), mouse SNCA (1:1,000; BD Transduction Labs, 610786), rabbit VDAC1 (1:1,000; Abcam ab15895), rabbit MTOR (1:1000; Cell Signaling Technology, 2983), rabbit phospho-MTOR (1:1000; Cell Signaling Technology, 2971), mouse human-specific SOD1 (1:2,000; R&D Systems, MAB3418), mouse misfolded SOD1 (C4F6) (1:250; Medimabs, MM-0070-2-P) or mouse ACTB (1:2,000; Sigma-Aldrich, A5316) antibodies in 3% (w:v) BSA (Sigma-Aldrich, A3912) in TBST overnight at 4°C.

Techniques: Western Blot, Immunohistochemical staining, Staining

Journal: Autophagy

Article Title: Rilmenidine promotes MTOR-independent autophagy in the mutant SOD1 mouse model of amyotrophic lateral sclerosis without slowing disease progression

doi: 10.1080/15548627.2017.1385674

Figure Lengend Snippet: Rilmenidine-induced SOD1 inclusions accumulate outside the autophagy system in motor neurons of SOD1 G93A mice. (A) Misfolded SOD1 and LC3 immunohistochemical analysis in lumbar spinal cords of vehicle- and rilmenidine-treated SOD1 G93A mice at 90 days of age. Vehicle-treated mice show colocalization of SOD1 and autophagosomes (arrowheads). Large SOD1 aggregates in rilmenidine-treated mice (arrows) do not colocalize with autophagosomes (arrowheads). (B) Misfolded SOD1 and LAMP2 immunohistochemical analysis in lumbar spinal cords of vehicle- and rilmenidine-treated SOD1 G93A mice at 90 days of age. Vehicle-treated mice show colocalization of SOD1 and lysosomes (arrowheads). Large SOD1 aggregates in rilmenidine-treated mice (arrows) do not colocalize with lysosomes (arrowheads).

Article Snippet: Membranes were blocked with 5% (w:v) skim milk dried powder (Fonterra, 492281) in Tris-buffered saline with Tween-20 (TBST; Tris base 20 mM, NaCl 140 mM, pH 8.0, 0.1% [v:v] Tween-20 [Sigma, P7949]) for 30 min and incubated with antibodies to rabbit LC3B (1:1,000; Sigma-Aldrich, L7543), mouse SQSTM1 (1:500; Abcam, ab56416), sheep SOD1 (1:4,000; Merck, 574597), rabbit LAMP2A (1:500; Abcam, ab18528), mouse HSPA8/HSC70 (1:500; Enzo Lifesciences, ALX-804-067), mouse SNCA (1:1,000; BD Transduction Labs, 610786), rabbit VDAC1 (1:1,000; Abcam ab15895), rabbit MTOR (1:1000; Cell Signaling Technology, 2983), rabbit phospho-MTOR (1:1000; Cell Signaling Technology, 2971), mouse human-specific SOD1 (1:2,000; R&D Systems, MAB3418), mouse misfolded SOD1 (C4F6) (1:250; Medimabs, MM-0070-2-P) or mouse ACTB (1:2,000; Sigma-Aldrich, A5316) antibodies in 3% (w:v) BSA (Sigma-Aldrich, A3912) in TBST overnight at 4°C.

Techniques: Immunohistochemical staining

Journal: Autophagy

Article Title: Rilmenidine promotes MTOR-independent autophagy in the mutant SOD1 mouse model of amyotrophic lateral sclerosis without slowing disease progression

doi: 10.1080/15548627.2017.1385674

Figure Lengend Snippet: Rilmendine induces excessive mitophagy and mitochondrial depletion in spinal motor neurons of SOD1 G93A mice. (A) Photomicrographs of NSC-34 cells transfected with mt-Rosella plasmid and treated with CCCP (positive control) or rilmenidine. In CCCP- and rilmenidine-treated cells, there is a significantly increased proportion of dsRed-positive puncta without pHluorin (arrowheads), indicating fusion of mitochondria and lysosomes. (B) Quantification of the percentage of dsRed-positive cells relative to total transfected cells. Data represent mean ± SD, n = 2 independent experiments, **p<0.01 and *** p <0.001 using a one-way ANOVA. (C) TOMM20 immunohistochemical analysis in lumbar spinal cords of vehicle- and rilmenidine-treated SOD1 G93A mice. (D) Quantification of TOMM20 immunoreactivity showing mitochondrial depletion in RBFOX3/NeuN-positive motor neurons of rilmenidine-treated mice Data represent mean ± SD, n = 4 mice per group, * p <0.05 compared to vehicle-treated mice using an unpaired t-test.

Article Snippet: Membranes were blocked with 5% (w:v) skim milk dried powder (Fonterra, 492281) in Tris-buffered saline with Tween-20 (TBST; Tris base 20 mM, NaCl 140 mM, pH 8.0, 0.1% [v:v] Tween-20 [Sigma, P7949]) for 30 min and incubated with antibodies to rabbit LC3B (1:1,000; Sigma-Aldrich, L7543), mouse SQSTM1 (1:500; Abcam, ab56416), sheep SOD1 (1:4,000; Merck, 574597), rabbit LAMP2A (1:500; Abcam, ab18528), mouse HSPA8/HSC70 (1:500; Enzo Lifesciences, ALX-804-067), mouse SNCA (1:1,000; BD Transduction Labs, 610786), rabbit VDAC1 (1:1,000; Abcam ab15895), rabbit MTOR (1:1000; Cell Signaling Technology, 2983), rabbit phospho-MTOR (1:1000; Cell Signaling Technology, 2971), mouse human-specific SOD1 (1:2,000; R&D Systems, MAB3418), mouse misfolded SOD1 (C4F6) (1:250; Medimabs, MM-0070-2-P) or mouse ACTB (1:2,000; Sigma-Aldrich, A5316) antibodies in 3% (w:v) BSA (Sigma-Aldrich, A3912) in TBST overnight at 4°C.

Techniques: Transfection, Plasmid Preparation, Positive Control, Immunohistochemical staining

Journal: Antioxidants

Article Title: A Novel Antioxidant, Hydrogen-Rich Coral Calcium Alters Gut Microbiome and Bile Acid Synthesis to Improve Methionine-and-Choline-Deficient Diet-Induced Non-Alcoholic Fatty Liver Disease

doi: 10.3390/antiox13060746

Figure Lengend Snippet: Antibodies used for Western blots.

Article Snippet: SOD1 , 16 , Novus, NBP1-31204.

Techniques: Western Blot