Journal: Oncology Reports

Article Title: Starvation insult induces the translocation of high mobility group box 1 to cytosolic compartments in glioma

doi: 10.3892/or.2023.8653

Figure Lengend Snippet: Expression of HMGB1. Expression of HMGB1 in (A) normal tissues and (B) and (C) glioma tissues. Arrows indicate cells with cytoplasmic expression; asterisks indicate nuclear expression. Scale bar, 5 µm. HMGB1, high mobility group box 1.

Article Snippet: Cell supernatants were collected and assayed using an HMGB1 ELISA kit (Cusabio Technology, LLC; cat. no. CSB-E08223h) in accordance with the manufacturer's instructions.

Techniques: Expressing

Journal: Oncology Reports

Article Title: Starvation insult induces the translocation of high mobility group box 1 to cytosolic compartments in glioma

doi: 10.3892/or.2023.8653

Figure Lengend Snippet: HMGB1 translocation at the indicated time intervals. (A) HMGB1 staining in U87-MG glioma cells at 0, 0.5, 1, 2, 3 and 4 h following starvation with HBSS. Nuclei were stained with 4′6-diamidino-2-phenylindole. Scale bar, 5 µm. (B) Cytoplasmic immunoreactivity of HMGB1 at the indicated time intervals was determined (n=3, 16–26 cells per replicate). (C) Supernatants of U87-MG glioma cells treated with HBSS at indicated time intervals were collected, and HMGB1 was detected using ELISA (n=3). (D) The cytotoxicity of HBSS was measured using an LDH release assay (n=3). Data represent the mean ± SD; Statistical significance was assessed using one-way ANOVA followed by Tukey's post hoc test. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001. ns, not significant; HMGB1, high mobility group box 1; HBSS, Hank's balanced salt solution; LDH, lactate dehydrogenase.

Article Snippet: Cell supernatants were collected and assayed using an HMGB1 ELISA kit (Cusabio Technology, LLC; cat. no. CSB-E08223h) in accordance with the manufacturer's instructions.

Techniques: Translocation Assay, Staining, Enzyme-linked Immunosorbent Assay, Lactate Dehydrogenase Assay

Journal: Oncology Reports

Article Title: Starvation insult induces the translocation of high mobility group box 1 to cytosolic compartments in glioma

doi: 10.3892/or.2023.8653

Figure Lengend Snippet: Immunocytochemistry HMGB1 with mitrotracker red, CANX, catalase and LC3B. (A) Staining for HMGB1 with mitrotracker red probe, CANX, catalase and LC3B antibodies in U87-MG glioma cells at 3 h following HBSS stimulation. Nuclei were stained with 4′6-diamidino-2-phenylindole. Scale bars, 5 µm. (B) Line fluorescence tracing from images in (A). (C) Manders' overlap coefficient analysis of HMGB1 and each marker protein 1 and 3 h following HBSS stimulation (n=3, 20–33 cells per replicate). Data represent the mean ± SD; an unpaired Student's t-test was used to evaluate statistical significance. **P<0.01 and ****P<0.0001. ns, not significants HMGB1, high mobility group box 1; CANX, calnexin; LC3B, microtubule-associated proteins 1A/1B light chain 3B; HBSS, Hank's balanced salt solution.

Article Snippet: Cell supernatants were collected and assayed using an HMGB1 ELISA kit (Cusabio Technology, LLC; cat. no. CSB-E08223h) in accordance with the manufacturer's instructions.

Techniques: Immunocytochemistry, Staining, Fluorescence, Marker

Journal: Oncology Reports

Article Title: Starvation insult induces the translocation of high mobility group box 1 to cytosolic compartments in glioma

doi: 10.3892/or.2023.8653

Figure Lengend Snippet: HMGB1 with LAMP1, Rab5, Rab7 and GFAP. (A) Staining for HMGB1 with LAMP1, Rab5, Rab7 and GFAP antibodies in U87-MG glioma cells at 3 h following HBSS stimulation. Nuclei were stained with 4′6-diamidino-2-phenylindole. Scale bars, 5 µm. (B) Line fluorescence tracing from images in (A). (C) Manders' overlap coefficient analysis of HMGB1 and each marker protein 1 and 3 h after HBSS stimulation (n=3, 20–33 cells per replicate). Data represent the mean ± SD; an unpaired Student's t-test was used to evaluate the statistical significance. ***P<0.001 and ****P<0.0001. ns, not significant; HMGB1, high mobility group box 1; LAMP1, lysosomal-associated membrane protein 1; GFAP, glial fibrillary acidic protein; HBSS, Hank's balanced salt solution.

Article Snippet: Cell supernatants were collected and assayed using an HMGB1 ELISA kit (Cusabio Technology, LLC; cat. no. CSB-E08223h) in accordance with the manufacturer's instructions.

Techniques: Staining, Fluorescence, Marker, Membrane

Journal: Oncology Reports

Article Title: Starvation insult induces the translocation of high mobility group box 1 to cytosolic compartments in glioma

doi: 10.3892/or.2023.8653

Figure Lengend Snippet: Immunohistochemistry of HMGB1 with (A) ATP5A, (B) LAMP1, (C) Rab5, and (D) GFAP in glioma tissues. Boxed areas in the lower right corner are an enlarged image of the boxed area above. Scale bars in the images with a lower magnification value in (A), (B) and (C) are 10 µm, and 5 µm in the images with a higher magnification. Scale bars for both the lower and higher magnified images in (D) are 10 µm. HMGB1, high mobility group box 1; ATP5A, ATP synthase F1 subunit alpha; LAMP1, lysosomal-associated membrane protein 1; GFAP, glial fibrillary acidic protein.

Article Snippet: Cell supernatants were collected and assayed using an HMGB1 ELISA kit (Cusabio Technology, LLC; cat. no. CSB-E08223h) in accordance with the manufacturer's instructions.

Techniques: Immunohistochemistry, Membrane

Journal: Oncology Reports

Article Title: Starvation insult induces the translocation of high mobility group box 1 to cytosolic compartments in glioma

doi: 10.3892/or.2023.8653

Figure Lengend Snippet: Electron microscopy images of HMGB1 in U87-MG glioma cells treated with HBSS for 3 h. HMGB1 localization was visualized using 10 nm gold particles (indicated by arrows). HMGB1 was localized in the (A) nucleus, (B) cytoplasm, and (C) extracellular space. In the cytoplasm, gold particles were found within or around (D) the mitochondria, (E and J) ER, (F) small vesicles, (G) endosomes, (H) coated vesicles, (I) autolysosomes, and (J) the cytoskeleton. (K) Free gold particles of HMGB1 in the cytoplasm. Scale bar, 200 nm. HMGB1, high mobility group box 1; HBSS, Hank's balanced salt solution; ER, endoplasmic reticulum.

Article Snippet: Cell supernatants were collected and assayed using an HMGB1 ELISA kit (Cusabio Technology, LLC; cat. no. CSB-E08223h) in accordance with the manufacturer's instructions.

Techniques: Electron Microscopy

Journal: Oncology Reports

Article Title: Starvation insult induces the translocation of high mobility group box 1 to cytosolic compartments in glioma

doi: 10.3892/or.2023.8653

Figure Lengend Snippet: HMGB1 is enriched in membrane-bound compartments. (A) Pellets containing nuclei and cytoskeletons (N) and cytoplasmic proteins (C) were collected from the HBSS-treated U87-MG glioma cells and immunoprobed with Lamin B1 (marker for the nuclear proteins), β-actin (marker for the cytosolic proteins) and HMGB1. (B) Pellets containing ER, cM and pM were fractionated and immunoprobed with HMGB1, the ER marker, CANX, and the mitochondria marker, ATP5A. HMGB1 was detected in the membrane-bound compartments. (C) Membrane fractionation scheme. Briefly, U87-MG cells were starved in HBSS for 1 h, collected and then homogenized. Cell lysates were differentially centrifuged at 3,000 × g, 25,000 × g, and 100,000 × g at RT. (D) The expression levels of LC3B and HMGB1 in different fractionations were measured using western blotting. (E) Proteinase K digestion was performed using the 25-k and 100-k membrane fractions. HMGB1, high mobility group box 1; HBSS, Hank's balanced salt solution; ER, endoplasmic reticulum; cM, crude mitochondria; pM, pure mitochondria; CANX, calnexin; ATP5A, ATP synthase F1 subunit alpha; LC3B, microtubule-associated proteins 1A/1B light chain 3B.

Article Snippet: Cell supernatants were collected and assayed using an HMGB1 ELISA kit (Cusabio Technology, LLC; cat. no. CSB-E08223h) in accordance with the manufacturer's instructions.

Techniques: Membrane, Marker, Fractionation, Expressing, Western Blot

Journal: Oncology Reports

Article Title: Starvation insult induces the translocation of high mobility group box 1 to cytosolic compartments in glioma

doi: 10.3892/or.2023.8653

Figure Lengend Snippet: HMGB1 is localized at MAMs in glioma. (A) Paraffin-embedded glioma sections were co-stained for HMGB1 and the ER marker, CANX, as well as the mitochondrial protein, ATP5A. HMGB1 was detected at MAMs, as indicated by arrows. The ‘inset’ image is an enlarged image of the boxed region in the merged image. (B) HBSS-treated U87-MG cells were co-stained for HMGB1, CANX and Mitotracker Red. Arrows indicate the localization of HMGB1 at MAMs. The ‘inset’ image is an enlarged image of the boxed region in the merged image. (C) U87-MG cells were transfected with the HMGB1-EGFP plasmid and then treated with HBSS. ER and mitochondria were labeled with ERtracker Blue and Mitotracker Red, respectively. The ‘inset’ image is an enlarged image of the boxed region in the merged image. White arrows indicate the localization of HMGB1 in MAMs. (D) HBSS-treated U87-MG cells were co-stained for HMGB1 and Sigma1-R. The boxed arean the upper right corner is an enlarge image of the boxed area on the upper left corner. (E) Representative images of HBSS-treated U87-MG cells showing the localization of HMGB1 by immune gold. The boxed area and expanded ER are amplified and outlined, respectively, in (F). M, mitochondria. Scale bars: (A) 1 µm, (B) lower magnified image 3 µm and higher magnified images 2 µm, (C) lower magnified images 5 µm and higher magnified image 1 µm, (D) 2 µm, and inset image 1 µm, (E and F) 100 nm. HMGB1, high mobility group box 1; MAMs, mitochondria-associated endoplasmic reticulum membranes; ER, endoplasmic reticulum; CANX, calnexin; ATP5A, ATP synthase F1 subunit alpha; HBSS, Hank's balanced salt solution; Sigma1-R, sigma 1 receptor.

Article Snippet: Cell supernatants were collected and assayed using an HMGB1 ELISA kit (Cusabio Technology, LLC; cat. no. CSB-E08223h) in accordance with the manufacturer's instructions.

Techniques: Staining, Marker, Transfection, Plasmid Preparation, Labeling, Amplification

Journal: Oncology Reports

Article Title: Starvation insult induces the translocation of high mobility group box 1 to cytosolic compartments in glioma

doi: 10.3892/or.2023.8653

Figure Lengend Snippet: HMGB1 secretion is regulated by autophagy-mediated secretion in glioma cells. (A) U251 glioma cells were pretreated with 0.5 µM WOR and 20 µM CQ for 2 h and then treated with HBSS for 3 h. Whole cell lysates immunoblotted with anti-LC3B and anti-β-actin antibodies. (B) Statistical analysis of LC3B-II/LC3B-I ratio (n=3). (C) U251 glioma cells were treated with 0.5 µM WOR and 20 µM CQ for 2 h and then treated with HBSS for 3 h. The secreted HMGB1 levels were measured using ELISA (n=3). (D) LDH release assay determined the cytotoxicity of HBSS, 0.5 µM WOR and 20 µM CQ (n=3). (E) Treatment with HBSS (3 h), WOR (3 h, 0.5 µM), or CQ (3 h, 20 µM) did not induce the apoptosis of U251 glioma cells, as determined using CCK-8 assay (n=3). (F) U251 glioma cells were treated with 0.5 µM WOR and 20 µM CQ for 2 h and then treated with HBSS for 3 h. Immunostaining of HMGB1 and the autophagosome marker, LC3B, in U251 glioma cells subjected to different treatments. (G) Line fluorescence tracing from images in (F). (H) Manders' overlap coefficient analysis of HMGB1 and LC3B (n=3, 10–15 cells per replicate). Data represent the mean ± SD; one-way ANOVA followed by Tukey's post-hoc test was used to evaluate statistical significance. *P<0.05, **P<0.01 and ****P<0.0001. ns, not significant. HMGB1, high mobility group box 1; WOR, wortmannin; CQ, chloroquine; HBSS, Hank's balanced salt solution; LC3B, microtubule-associated proteins 1A/1B light chain 3B; LDH, Lactate dehydrogenase; CCK-8, Cell Counting Kit-8.

Article Snippet: Cell supernatants were collected and assayed using an HMGB1 ELISA kit (Cusabio Technology, LLC; cat. no. CSB-E08223h) in accordance with the manufacturer's instructions.

Techniques: Enzyme-linked Immunosorbent Assay, Lactate Dehydrogenase Assay, CCK-8 Assay, Immunostaining, Marker, Fluorescence, Cell Counting

Journal: Oncology Reports

Article Title: Starvation insult induces the translocation of high mobility group box 1 to cytosolic compartments in glioma

doi: 10.3892/or.2023.8653

Figure Lengend Snippet: Schematic diagram demonstrating the subcellular localization of HMGB1 in glioma cells. Starvation stress triggers the translocation of HMGB1 from the nucleus to the cytoplasm and the extracellular milieu. In the cytoplasm, HMGB1 can be imported into the mitochondria, ER, MAMs, peroxisomes, autophagosomes, lysosomes, early endosomes, late endosomes and cytoskeleton. Early (inhibited by WOR) and late autophagy (inhibited by CQ) mediated the extracellular secretion of HMGB1. HMGB1, high mobility group box 1; ER, endoplasmic reticulum; MAMs, mitochondria-associated endoplasmic reticulum membranes; WOR, wortmannin; CQ, chloroquine.

Article Snippet: Cell supernatants were collected and assayed using an HMGB1 ELISA kit (Cusabio Technology, LLC; cat. no. CSB-E08223h) in accordance with the manufacturer's instructions.

Techniques: Translocation Assay

Journal: Scientific Reports

Article Title: Klotho inhibits IGF1R/PI3K/AKT signalling pathway and protects the heart from oxidative stress during ischemia/reperfusion injury

doi: 10.1038/s41598-023-47686-5

Figure Lengend Snippet: PI3K and AKT levels in rat hearts. ( A ) PI3K level in the heart tissue; ( B ) Correlation of PI3K and p-IGF1R levels; ( C ) Total AKT level; ( D ) Activation of AKT expressed as the level of p-AKT; AKT—protein kinase B; IGF1R—insulin-like growth factor 1 receptor; p-AKT—phosphorylated protein kinase B; p-IGF1R—phosphorylated insulin-like growth factor 1 receptor; PI3K—phosphoinositide-3-kinase; n aero = 8; n IRI = 12–14; n IRI+Klotho = 7; boxes—25–75% percentile, whiskers—min to max + median.

Article Snippet: The level of PI3K in heart tissue was evaluated with Rat Phosphotylinosital 3 Kinase (PI3K) ELISA Kit (Cusabio, Houston, USA).

Techniques: Activation Assay

Journal: Scientific Reports

Article Title: Klotho inhibits IGF1R/PI3K/AKT signalling pathway and protects the heart from oxidative stress during ischemia/reperfusion injury

doi: 10.1038/s41598-023-47686-5

Figure Lengend Snippet: Inactivation of FOXO3. ( A ) The level of total FOXO3 in the heart tissue; ( B ) Inactivated FOXO3 level expressed as p-FOXO3; ( C ) Correlation of p-FOXO3 level with p-IGF1R and PI3K levels; FOXO3—forkhead box protein O 3; p-FOXO3—phosphorylated forkhead box protein O 3; p-IGF1R—phosphorylated insulin-like growth factor 1 receptor; PI3K—phosphoinositide-3-kinase; n aero = 8–9; n IRI = 13–14; n IRI+Klotho = 7; boxes—25–75% percentile, whiskers—min to max + median.

Article Snippet: The level of PI3K in heart tissue was evaluated with Rat Phosphotylinosital 3 Kinase (PI3K) ELISA Kit (Cusabio, Houston, USA).

Techniques:

Journal: Scientific Reports

Article Title: Klotho inhibits IGF1R/PI3K/AKT signalling pathway and protects the heart from oxidative stress during ischemia/reperfusion injury

doi: 10.1038/s41598-023-47686-5

Figure Lengend Snippet: NOX2 level in heart tissue. ( A ) The level of NOX2 in the heart tissue; ( B ) Correlation of p-IGF1R, PI3K and NOX2 levels; ( C ) Correlation of FOXO3 and NOX2 levels; NOX2—nicotinamide adenine dinucleotide phosphate oxidase 2; p-FOXO3—phosphorylated forkhead box protein O 3; p-IGF1R—phosphorylated insulin-like growth factor 1 receptor; PI3K—phosphoinositide-3-kinase; n aero = 7; n IRI = 13; n IRI+Klotho = 7; boxes—25–75% percentile, whiskers—min to max + median.

Article Snippet: The level of PI3K in heart tissue was evaluated with Rat Phosphotylinosital 3 Kinase (PI3K) ELISA Kit (Cusabio, Houston, USA).

Techniques:

Journal: Scientific Reports

Article Title: Klotho inhibits IGF1R/PI3K/AKT signalling pathway and protects the heart from oxidative stress during ischemia/reperfusion injury

doi: 10.1038/s41598-023-47686-5

Figure Lengend Snippet: The magnitude of oxidative stress. ( A ) The level of ROS/RNS in the heart tissue; ( B ) The level of H 2 O 2 in the heart tissue; ( C ) Correlation of ROS/RNS, p-IGF1R level and PI3K levels; ( D ) Correlation of ROS/RNS, p-FOXO3 and NOX2 levels; DCF—dichlorodihydrofluorescein; H 2 O 2 —hydrogen peroxide; NOX2—nicotinamide adenine dinucleotide phosphate oxidase 2; p-FOXO3—phosphorylated forkhead box protein O 3; p-IGF1R—phosphorylated insulin-like growth factor 1 receptor; PI3K—phosphoinositide-3-kinase; ROS/RNS—reactive oxygen/nitrogen species; n aero = 8; n IRI = 12; n IRI+Klotho = 6; mean ± SD.

Article Snippet: The level of PI3K in heart tissue was evaluated with Rat Phosphotylinosital 3 Kinase (PI3K) ELISA Kit (Cusabio, Houston, USA).

Techniques:

Journal: Scientific Reports

Article Title: Klotho inhibits IGF1R/PI3K/AKT signalling pathway and protects the heart from oxidative stress during ischemia/reperfusion injury

doi: 10.1038/s41598-023-47686-5

Figure Lengend Snippet: The intensity of heart injury. ( A ) LDH activity in coronary effluents as a marker of cell death; ( B ) Correlation of LDH activity, p-IGF1R and PI3K levels; ( C ) Correlation of LDH activity, p-FOXO3 and ROS/RNS levels; DCF—dichlorodihydrofluorescein; LDH—lactate dehydrogenase; mU—milli international enzyme units; p-FOXO3—phosphorylated forkhead box protein O 3; PI3K—phosphoinositide-3-kinase; p-IGF1R—phosphorylated insulin-like growth factor 1 receptor; ROS/RNS—reactive oxygen/nitrogen species; n aero = 5; n IRI = 11; n IRI+Klotho = 6; mean ± SD.

Article Snippet: The level of PI3K in heart tissue was evaluated with Rat Phosphotylinosital 3 Kinase (PI3K) ELISA Kit (Cusabio, Houston, USA).

Techniques: Activity Assay, Marker

Journal: Scientific Reports

Article Title: Klotho inhibits IGF1R/PI3K/AKT signalling pathway and protects the heart from oxidative stress during ischemia/reperfusion injury

doi: 10.1038/s41598-023-47686-5

Figure Lengend Snippet: The potential influence of Klotho on the factors in IGF1R/PI3K/AKT signalling pathway and/or oxidative stress during heart IRI. Akt—protein kinase B; FOXO3 –forkhead box protein O 3; GP X —glutathione peroxidase; IGF1—insulin-like growth factor 1; IGF1R—insulin-like growth factor 1 receptor; IRS—intracellular adaptor protein insulin receptor substrate; NOX2—nicotinamide adenine dinucleotide phosphate oxidase 2; P—phosphorylation; PI3K—phosphoinositide-3-kinase; ROS/RNS—reactive oxygen/nitrogen species; SOD—superoxide dismutase. Figure created with BioRender ( https://biorender.com/ ).

Article Snippet: The level of PI3K in heart tissue was evaluated with Rat Phosphotylinosital 3 Kinase (PI3K) ELISA Kit (Cusabio, Houston, USA).

Techniques:

Journal: Journal of Experimental Pharmacology

Article Title: Discovery of Fungus-Derived Nornidulin as a Novel TMEM16A Inhibitor: A Potential Therapy to Inhibit Mucus Secretion in Asthma

doi: 10.2147/JEP.S427594

Figure Lengend Snippet: Effect of nornidulin on Ca 2+ -dependent mucin secretion in Calu-3 cells. ( A ) Immunofluorescence staining of MUC5AC. Hoechst staining was used to determine the cell area. MUC5AC, a marker of pathogenic mucin in asthma, was labeled to evaluate depletion of intracellular mucin store that indicates mucin secretion. Ionomycin treatment was performed to promote Ca 2+ -dependent mucin secretion. Nornidulin (10 µM) and a TMEM16A inhibitor MONNA were able to suppress ionomycin-induced depletion of intracellular mucin store, suggesting that nornidulin was capable of inhibiting mucin secretion. The scale bars of 50 µm were shown. ( B ) Analyses of intracellular mucin levels. The summary and statistical analyses of data are shown. Results were expressed as % of control ± S.E.M. (n = 4–6). ** p < 0.01 compared with control group (one-way ANOVA). # p < 0.05; ## p < 0.01 compared with ionomycin-treated group (one-way ANOVA).

Article Snippet: The Mouse MUC5AC ELISA Kit (catalog number: CSB-E11040m, CUSABIO, Houston, TX, USA), a sandwich-type ELISA kit, was used.

Techniques: Immunofluorescence, Staining, Marker, Labeling

Journal: Journal of Experimental Pharmacology

Article Title: Discovery of Fungus-Derived Nornidulin as a Novel TMEM16A Inhibitor: A Potential Therapy to Inhibit Mucus Secretion in Asthma

doi: 10.2147/JEP.S427594

Figure Lengend Snippet: Effect of nornidulin in the treatment of inflammation-associated mucus hypersecretion in OVA-challenged mouse model of asthma. ( A ) Timeline and detailed protocol of OVA-challenged mouse model of asthma establishment. ( B ) Effect of nornidulin and dexamethasone (DEX) on percent spleen weight. ( C ) H&E and PAS staining of airway tissues from OVA-challenged mice treated with or without nornidulin. Dexamethasone (DEX) was used as a control of this experiment. The scale bars of 40 µm were shown with the red arrows and the black arrow heads indicating clusters of eosinophils and mucus-producing cells, respectively. ( D ) Inflammatory score and ( E ) % PAS-positive area of airway tissues from OVA-challenged mice treated with or without nornidulin. Dexamethasone (DEX) was used as a control of these experiment. ( F ) Effect of nornidulin on in vivo mucus hypersecretion in OVA-challenged mice. Mucus hypersecretion was evaluated by the levels of MUC5AC, a pathogenic mucin, collected from bronchoalveolar lavage fluid (BALF). Both nornidulin and dexamethasone (DEX) significantly reduced mucus hypersecretion. ( G ) Effect of nornidulin on total immune cell number. BALF was collected and immune cells were observed and counted under microscope. Dexamethasone (DEX), but not nornidulin, significantly reduced total cells in BALF. Results were analyzed from 5–7 independent experiments and shown as a means of control ± S.E.M. * p < 0.05; ** p < 0.01; *** p < 0.001 compared with control group (one-way ANOVA). # p < 0.05; ## p < 0.01; ### p < 0.001; NS, non-statistical difference compared with OVA-challenged groups without any treatment (one-way ANOVA).

Article Snippet: The Mouse MUC5AC ELISA Kit (catalog number: CSB-E11040m, CUSABIO, Houston, TX, USA), a sandwich-type ELISA kit, was used.

Techniques: Staining, In Vivo, Microscopy

Journal: PeerJ

Article Title: Profile of the bile acid FXR-FGF15 pathway in the glucolipid metabolism disorder of diabetic mice suffering from chronic stress

doi: 10.7717/peerj.16407

Figure Lengend Snippet: (A) Body weight weekly in CUMS procedures. (B) Body weight in mice after CUMS procedures. (C) Random blood glucose weekly in CUMS procedures. (D) Fasting blood glucose after CUMS procedures. (E) Serum CHO in mice. (F) Serum TG in mice. (G) Serum NEFA in mice. (H) Serum LDL-C in mice. (I) Serum HDL-C in mice. (J) GHbA1c in mice. Data presented as mean ± SEM, n = 10 per group, # P < 0.05, ## P < 0.01 compared with the Control group, * P < 0.05, ** P < 0.01 compared with the T2DM+CUMS group.

Article Snippet: The concentration of glycated hemoglobin A1c (GHbA1c) in the lysate was measured using the Mouse Glycated Hemoglobin A1c (GHbA1c) ELISA Kit (#CSB-E08141m; CUSABIO, Wuhan, China, https://www.cusabio.com/ ) and the Mouse Insulin ELISA kit (#KE10089; Proteintech Group, Inc., Wuhan, China) following the manufacturer’s instructions in order to determine the red blood cell and serum insulin levels.

Techniques:

Journal: Journal of Nanobiotechnology

Article Title: Magnetic vagus nerve stimulation alleviates myocardial ischemia-reperfusion injury by the inhibition of pyroptosis through the M 2 AChR/OGDHL/ROS axis in rats

doi: 10.1186/s12951-023-02189-3

Figure Lengend Snippet: mVNS treatment attenuated myocardial I/R injury, ventricular fibrillation and inflammatory cytokine release. ( A ) Evans blue and TTC staining was used to detect the infarct size of I/R rats. The area of normally supplied blood was blue, the area at risk (AAR) was red and that of infarct size (INF) was pale. INF/AAR reflects the level of dead myocardium, n = 6 per group, Scale bar: 3 mm. ( B ) Representative echocardiographic images showed heart function 24 h after I/R and quantitative analysis of left ventricular ejection fraction (LVEF) and left ventricular fraction shortening (LVFS) among the different groups, n = 6 per group. ( C ) Effects of mVNS treatment on the myocardial enzyme concentrations in serum by Elisa, n = 12 per group. ( D ) Representative electrocardiographic tracings illustrating the inhibitory effect of mVNS on I/R-induced VF. ( E ) Effect of mVNS on the incidence of VF, n = 12 per group. ( F ) Effect of mVNS on VF duration, n = 12 per group. ( G ) Effect of mVNS on the heart rate, n = 12 per group. ( H ) mVNS treatment decreased inflammatory cell infiltration and IL-6 expression in myocardial tissue, n = 6 per group. Scale bar: 100 μm. ( I ) Measurements of mVNS treatment on the concentration of TNF-α and IL-6 in serum by Elisa, n = 12 per group. Data are mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; NS means not significant between groups. All P values were obtained by one-way ANOVA or Chi square test

Article Snippet: Specific concentrations of serum cTnT, CK-MB, LDH, IL-6, TNF-α were measured by ELISA, using commercial Rat Cardiac Troponin T (cTnT) ELISA kit (CSB-E16443r, CUSABIO, China), Rat Creatine Kinase MB isoenzyme (CK-MB) ELISA kit (CSB-E14403r, CUSABIO, China), Rat L-Lactate Dehydrogenase (LDH) ELISA kit (CSB-E11324r, CUSABIO, China), Rat Interleukin 6 (IL-6) ELISA kit (CSB-E04640r, CUSABIO, China), Rat TNF-α ELISA kit (CSB-E11987r, CUSABIO, China), respectively.

Techniques: Staining, Enzyme-linked Immunosorbent Assay, Expressing, Concentration Assay

Journal: Journal of Nanobiotechnology

Article Title: Magnetic vagus nerve stimulation alleviates myocardial ischemia-reperfusion injury by the inhibition of pyroptosis through the M 2 AChR/OGDHL/ROS axis in rats

doi: 10.1186/s12951-023-02189-3

Figure Lengend Snippet: mVNS treatment attenuated myocardial I/R injury, ventricular fibrillation and inflammatory cytokine release. ( A ) Evans blue and TTC staining was used to detect the infarct size of I/R rats. The area of normally supplied blood was blue, the area at risk (AAR) was red and that of infarct size (INF) was pale. INF/AAR reflects the level of dead myocardium, n = 6 per group, Scale bar: 3 mm. ( B ) Representative echocardiographic images showed heart function 24 h after I/R and quantitative analysis of left ventricular ejection fraction (LVEF) and left ventricular fraction shortening (LVFS) among the different groups, n = 6 per group. ( C ) Effects of mVNS treatment on the myocardial enzyme concentrations in serum by Elisa, n = 12 per group. ( D ) Representative electrocardiographic tracings illustrating the inhibitory effect of mVNS on I/R-induced VF. ( E ) Effect of mVNS on the incidence of VF, n = 12 per group. ( F ) Effect of mVNS on VF duration, n = 12 per group. ( G ) Effect of mVNS on the heart rate, n = 12 per group. ( H ) mVNS treatment decreased inflammatory cell infiltration and IL-6 expression in myocardial tissue, n = 6 per group. Scale bar: 100 μm. ( I ) Measurements of mVNS treatment on the concentration of TNF-α and IL-6 in serum by Elisa, n = 12 per group. Data are mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; NS means not significant between groups. All P values were obtained by one-way ANOVA or Chi square test

Article Snippet: Specific concentrations of serum cTnT, CK-MB, LDH, IL-6, TNF-α were measured by ELISA, using commercial Rat Cardiac Troponin T (cTnT) ELISA kit (CSB-E16443r, CUSABIO, China), Rat Creatine Kinase MB isoenzyme (CK-MB) ELISA kit (CSB-E14403r, CUSABIO, China), Rat L-Lactate Dehydrogenase (LDH) ELISA kit (CSB-E11324r, CUSABIO, China), Rat Interleukin 6 (IL-6) ELISA kit (CSB-E04640r, CUSABIO, China), Rat TNF-α ELISA kit (CSB-E11987r, CUSABIO, China), respectively.

Techniques: Staining, Enzyme-linked Immunosorbent Assay, Expressing, Concentration Assay

Journal: Journal of Nanobiotechnology

Article Title: Magnetic vagus nerve stimulation alleviates myocardial ischemia-reperfusion injury by the inhibition of pyroptosis through the M 2 AChR/OGDHL/ROS axis in rats

doi: 10.1186/s12951-023-02189-3

Figure Lengend Snippet: mVNS regulated myocardial pyroptosis by suppressing OGDHL expression and improving mitochondrial damage in vivo. ( A ) Western blot analysis of protein level of OGDHL (GAPDH as an internal control) and quantification of OGDHL activity in the myocardium of rats after I/R or mVNS treatment, n = 6 per group. ( B ) Western blot analysis of OGDHL expression in heart tissue transfected OGDHL, n = 6 per group. GAPDH as an internal control. ( C - J ) Rats were transfected with AAV9-ctrl or AAV9-cTNT-OGDHL three week before I/R surgery and mVNS tratment. ( C ) Evans blue and TTC staining was used to detect the infarct size of I/R rats. The area of normally supplied blood was blue, the AAR was red and that of INF was pale. INF/AAR reflects the level of dead myocardium., n = 6 per group, Scale bar: 3 mm. ( D ) Representative echocardiographic images showed heart function 24 h after I/R and quantitative analysis of LVEF and LVFS among the different groups, n = 6 per group. ( E ) TUNEL analysis for myocardial pyroptosis. Green, TUNEL-positive nuclei; blue, DAPI-stained nuclei, n = 6 per group. Scale bar: 50 μm. ( F ) Immunohistochemical observation of NLRP3-positive expression, n = 6 per group. Scale bar: 40 μm. ( G ) Western blot analysis of protein levels of NLRP3, Cleaved Caspase-1, Caspase-1, Total GSDMD and Cleaved GSDMD-N in myocardium and quantification, n = 6 per group, GAPDH as an internal control. ( H ) ELISA was used to measure the levels of IL-1β and IL-18 in myocardium, n = 6 per group. ( I ) Representative images of mitochondrial ultrastructure of myocardium with TEM, n = 6 per group. Scale bar: 2 μm. ( J ) Representative images of mtROS production in myocardium and quantification of relative mtROS mean intensity, n = 6 per group. Scale bar: 50 μm. Data are mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; NS means not significant between groups. All P values were obtained by one-way ANOVA

Article Snippet: Specific concentrations of serum cTnT, CK-MB, LDH, IL-6, TNF-α were measured by ELISA, using commercial Rat Cardiac Troponin T (cTnT) ELISA kit (CSB-E16443r, CUSABIO, China), Rat Creatine Kinase MB isoenzyme (CK-MB) ELISA kit (CSB-E14403r, CUSABIO, China), Rat L-Lactate Dehydrogenase (LDH) ELISA kit (CSB-E11324r, CUSABIO, China), Rat Interleukin 6 (IL-6) ELISA kit (CSB-E04640r, CUSABIO, China), Rat TNF-α ELISA kit (CSB-E11987r, CUSABIO, China), respectively.

Techniques: Expressing, In Vivo, Western Blot, Activity Assay, Transfection, Staining, TUNEL Assay, Immunohistochemistry, Enzyme-linked Immunosorbent Assay

Journal: Journal of Nanobiotechnology

Article Title: Magnetic vagus nerve stimulation alleviates myocardial ischemia-reperfusion injury by the inhibition of pyroptosis through the M 2 AChR/OGDHL/ROS axis in rats

doi: 10.1186/s12951-023-02189-3

Figure Lengend Snippet: mVNS regulated myocardial pyroptosis by suppressing OGDHL expression and improving mitochondrial damage in vivo. ( A ) Western blot analysis of protein level of OGDHL (GAPDH as an internal control) and quantification of OGDHL activity in the myocardium of rats after I/R or mVNS treatment, n = 6 per group. ( B ) Western blot analysis of OGDHL expression in heart tissue transfected OGDHL, n = 6 per group. GAPDH as an internal control. ( C - J ) Rats were transfected with AAV9-ctrl or AAV9-cTNT-OGDHL three week before I/R surgery and mVNS tratment. ( C ) Evans blue and TTC staining was used to detect the infarct size of I/R rats. The area of normally supplied blood was blue, the AAR was red and that of INF was pale. INF/AAR reflects the level of dead myocardium., n = 6 per group, Scale bar: 3 mm. ( D ) Representative echocardiographic images showed heart function 24 h after I/R and quantitative analysis of LVEF and LVFS among the different groups, n = 6 per group. ( E ) TUNEL analysis for myocardial pyroptosis. Green, TUNEL-positive nuclei; blue, DAPI-stained nuclei, n = 6 per group. Scale bar: 50 μm. ( F ) Immunohistochemical observation of NLRP3-positive expression, n = 6 per group. Scale bar: 40 μm. ( G ) Western blot analysis of protein levels of NLRP3, Cleaved Caspase-1, Caspase-1, Total GSDMD and Cleaved GSDMD-N in myocardium and quantification, n = 6 per group, GAPDH as an internal control. ( H ) ELISA was used to measure the levels of IL-1β and IL-18 in myocardium, n = 6 per group. ( I ) Representative images of mitochondrial ultrastructure of myocardium with TEM, n = 6 per group. Scale bar: 2 μm. ( J ) Representative images of mtROS production in myocardium and quantification of relative mtROS mean intensity, n = 6 per group. Scale bar: 50 μm. Data are mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; NS means not significant between groups. All P values were obtained by one-way ANOVA

Article Snippet: Specific concentrations of serum cTnT, CK-MB, LDH, IL-6, TNF-α were measured by ELISA, using commercial Rat Cardiac Troponin T (cTnT) ELISA kit (CSB-E16443r, CUSABIO, China), Rat Creatine Kinase MB isoenzyme (CK-MB) ELISA kit (CSB-E14403r, CUSABIO, China), Rat L-Lactate Dehydrogenase (LDH) ELISA kit (CSB-E11324r, CUSABIO, China), Rat Interleukin 6 (IL-6) ELISA kit (CSB-E04640r, CUSABIO, China), Rat TNF-α ELISA kit (CSB-E11987r, CUSABIO, China), respectively.

Techniques: Expressing, In Vivo, Western Blot, Activity Assay, Transfection, Staining, TUNEL Assay, Immunohistochemistry, Enzyme-linked Immunosorbent Assay