Journal: iScience

Article Title: The mechanisms and therapeutic potential of clopidogrel in mitigating diabetic cardiomyopathy in db/db mice

doi: 10.1016/j.isci.2024.109134

Figure Lengend Snippet:

Article Snippet: Mouse LDH ELISA Kit , Elabscience Biotechnology Co., Ltd, China , Cat# E-EL-M0419c.

Techniques: Recombinant, Bicinchoninic Acid Protein Assay, Enzyme-linked Immunosorbent Assay, Activity Assay, Multiple Displacement Amplification, GSSG Assay, Isolation, Reverse Transcription, CCK-8 Assay, ROS Assay, Software

Journal: Acta Neuropathologica Communications

Article Title: Activity of translation regulator eukaryotic elongation factor-2 kinase is increased in Parkinson disease brain and its inhibition reduces alpha synuclein toxicity

doi: 10.1186/s40478-018-0554-9

Figure Lengend Snippet: Effects of eEF2K inhibition on human AS cytotoxicity in differentiated N2A cells. a - b Western blot analysis of p-eEF2 (T56) levels in N2A cells subsequent to transient overexpression of human wild type or mutant A53T AS, with or without siRNA mediated eEF2K knockdown ( a ), and corresponding densitometry analysis ( b ) ( n = 6–9/group from three independent experiments; One-way ANOVA post-hoc Bonferroni test, * p < 0.05, *** p < 0.005; error bars indicate Mean ± S.E.M). c Measurements of cytotoxicity by lactate dehydrogenase-LDH release in the culture medium ( c ) and FACS analysis of propidium iodide-PI staining ( d ) in N2A cells subsequent to transient overexpression of human wild type or mutant A53T AS, with or without siRNA mediated eEF2K knockdown ( n = 9–12/group from three independent experiments; One-way ANOVA post-hoc Bonferroni test, * p < 0.05, ** p < 0.01, *** p < 0.005, NS = not significant; error bars indicate Mean ± S.D.)

Article Snippet: Additional reagents and biochemical assays employed during these studies include: pool of small interference RNAs (SiRNAs) targeting mouse eEF2K (Santa Cruz, #sc-39,012), Cell Titer Glo ATP measurement kit (Promega, #G7570), Lactate dehydrogenase (LDH) fluorometric assay (Novus Biologicals, #NBP2–54851), Seahorse Mito stress test kit (Agilent, #103015–100), 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA) fluorescent ROS reagent (ThermoFisher, #D399), and MitoTracker Green fluorescent reagent for mitochondrial mass (ThermoFisher, #M7514).

Techniques: Inhibition, Western Blot, Over Expression, Mutagenesis, Knockdown, Staining

Journal: Journal of Biochemical and Molecular Toxicology

Article Title: Bradykinin/bradykinin 1 receptor promotes brain microvascular endothelial cell permeability and proinflammatory cytokine release by downregulating Wnt3a

doi: 10.1002/jbt.23213

Figure Lengend Snippet: BK enhanced ROS production, cellular permeability, and inflammation, and reduced cell proliferation and tight junction formation by BMECs. BMECs were treated with BK (low concentration, 100 nM or high concentration, 400 nM). ROS production was determined by flow cytometry, and the respective images are shown (A, left panel). Summarized results from three independent experiments are shown (A, right panel). Cell permeability was tested by sodium fluorescein analysis (B, left panel) and the lucifer yellow assay (B, right panel). Cell proliferation was measured by EdU staining. (C, IL‐6, MCP‐1, and IL‐18 expressions were detected by IF (D) and ELISA (E). (F) The cell apoptosis rate was measured using FCM. (G) LDH levels were determined by ELISA. The levels of claudin‐5, occludin, β‐catenin, Wnt3a, and B1R expression were determined by western blotting (H, left panel). The statistical results of western blot studies conducted in three independent experiments (H, right panel). * p < 0.05; ** p < 0.01; *** p < 0.001 versus blank. B1R, bradykinin 1 receptor; BK, bradykinin; BMECs, brain microvascular endothelial cells; DCF‐A, 2′,7′‐dichlorodihydrofluorescein diacetate; EdU, 5‐ethynyl‐2′‐deoxyuridine; ELISA, enzyme‐linked Immunosorbent assay; FCM, flow cytometry; GAPDH glyceraldehyde‐3‐phosphate dehydrogenase; IF, immunofluorescence; IL‐6, interleukin 6; LDH, lactate dehydrogenase; MCP‐1, monocyte chemoattractant protein‐1; ROS, reactive oxygen species.

Article Snippet: The levels of IL‐6 (Elabscience; Cat. No. E‐EL‐H0102c), IL‐18 (Boster; CAT. No. EK0864), MCP‐1 (Boster; Cat. No. EK0441), and LDH (Elabscience; CAT. No. E‐EL‐H0866c) expression were determined by enzyme‐linked immunosorbent assay (ELISA) according to manufacturer's instructions.

Techniques: Permeability, Concentration Assay, Flow Cytometry, Staining, Enzyme-linked Immunosorbent Assay, Expressing, Western Blot, Immunofluorescence

Journal: Journal of Biochemical and Molecular Toxicology

Article Title: Bradykinin/bradykinin 1 receptor promotes brain microvascular endothelial cell permeability and proinflammatory cytokine release by downregulating Wnt3a

doi: 10.1002/jbt.23213

Figure Lengend Snippet: Wnt3a counteracted the effects of BK on BMECs. BMECs were treated with a high concentration (400 nM) of BK or a high concentration of both BK and Wnt3a (100 ng/ml each). ROS production was measured by flow cytometry, and the respective images are shown (A, left panel). The summary results of three independent experiments are shown (A, right panel). Cell permeability was tested by sodium fluorescein analysis (B, left panel) and the lucifer yellow assay (B, right panel). (C) Cell proliferation was measured by EdU staining. IL‐6, MCP‐1, and IL‐18 expressions were detected by (D) IF and (E) ELISA. (F) Cell apoptosis was detected by using the FCM method. (G) LDH levels were determined by ELISA. The levels of claudin‐5, occludin, β‐catenin, Wnt3a, and B1R expression were determined by western blotting (H, left panel). The statistical results of western blot studies conducted in three independent experiments (H, right panel). * p < 0.05; ** p < 0.01; *** p < 0.001 versus blank. # p < 0.05; ## p < 0.01 versus BK‐high. B1R, bradykinin 1 receptor; BK, bradykinin; BMECs, brain microvascular endothelial cells; DCF‐A, 2′,7′‐dichlorodihydrofluorescein diacetate; EdU, 5‐ethynyl‐2′‐deoxyuridine; ELISA, enzyme‐linked Immunosorbent assay; FCM, flow cytometry; GAPDH glyceraldehyde‐3‐phosphate dehydrogenase; IF, immunofluorescence; IL‐6, interleukin 6; LDH, lactate dehydrogenase; MCP‐1, monocyte chemoattractant protein‐1; ROS, reactive oxygen species.

Article Snippet: The levels of IL‐6 (Elabscience; Cat. No. E‐EL‐H0102c), IL‐18 (Boster; CAT. No. EK0864), MCP‐1 (Boster; Cat. No. EK0441), and LDH (Elabscience; CAT. No. E‐EL‐H0866c) expression were determined by enzyme‐linked immunosorbent assay (ELISA) according to manufacturer's instructions.

Techniques: Concentration Assay, Flow Cytometry, Permeability, Staining, Enzyme-linked Immunosorbent Assay, Expressing, Western Blot, Immunofluorescence

Journal: Journal of Cellular and Molecular Medicine

Article Title: Nesfatin‐1 inhibits myocardial ischaemia/reperfusion injury through activating Akt/ERK pathway‐dependent attenuation of endoplasmic reticulum stress

doi: 10.1111/jcmm.16481

Figure Lengend Snippet: Nesfatin‐1 restoration ameliorates MI/R injury. (A‐E) Mice were intramyocardially administrated with 5 or 10 ng/g bodyweight nesfatin‐1 at 30 min after ischaemia. Each group contains 7 mice. (A) Nesfatin‐1 peptide amount in heart homogenates was measured by ELISA assay. (B) Infarct area in the heart slices was evaluated by TTC staining. (C) The cardiac troponin T level in the serum was assessed using ELISA assay. (D) Myocardial apoptosis was detected by TUNEL staining, and the percentage of apoptotic myocardial cells is depicted on the right. (E) The caspase‐3 activity in heart homogenates was determined by caspase activity assay. Data are mean ± SD (n = 7). Student's t test. *, P < .05; **, P < .01. NS, not significant

Article Snippet: The activity of caspase‐3 was detected by Caspase‐3 Activity Assay Kit (#5723, Cell Signaling Technology), and the LDH activity was assessed by the Rat L‐lactate Dehydrogenase (L‐LDH) ELISA Kit (CSB‐E11324r, CUSABIO) following manufacturer's instructions.

Techniques: Enzyme-linked Immunosorbent Assay, Staining, TUNEL Assay, Activity Assay, Caspase Activity Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: Nesfatin‐1 inhibits myocardial ischaemia/reperfusion injury through activating Akt/ERK pathway‐dependent attenuation of endoplasmic reticulum stress

doi: 10.1111/jcmm.16481

Figure Lengend Snippet: Akt/ERK inhibition significantly reverses nesfatin‐1 activities of reducing ER stress and attenuating MI/R injury. (A‐E) Mice were administrated intramyocardially with 10 ng/g bodyweight nesfatin‐1 combined with or without 1 μg/g bodyweight wortmannin at 30 min after ischaemia. Each group contains seven mice. (A) The protein expression of p‐Akt, Akt, p‐ERK, ERK, CHOP, ATF6, GPR78 and caspase‐12 in heart homogenates was determined by Western blotting analysis. Images are representative of three independent experiments. (B) Infarct area in the heart slices was evaluated by TTC staining. (C) Plasma cardiac troponin T level in the serum was assessed using ELISA assay. (D) Myocardial apoptosis was detected by TUNEL staining, and the percentage of apoptotic myocardial cells is depicted on the right. (E) The caspase‐3 activity in heart homogenates was determined by caspase activity assay. Data are mean ± SD (7). Student's t test. *, P < .05; **, P < .01; NS, not significant

Article Snippet: The activity of caspase‐3 was detected by Caspase‐3 Activity Assay Kit (#5723, Cell Signaling Technology), and the LDH activity was assessed by the Rat L‐lactate Dehydrogenase (L‐LDH) ELISA Kit (CSB‐E11324r, CUSABIO) following manufacturer's instructions.

Techniques: Inhibition, Expressing, Western Blot, Staining, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, TUNEL Assay, Activity Assay, Caspase Activity Assay