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: Cell Proliferation

Article Title: Ammonia Accumulation Drives Intervertebral Disc Degeneration by Triggering Ammonia‐Induced Cell Death Through Lysosome–Mitochondria Crosstalk

doi: 10.1111/cpr.70283

Figure Lengend Snippet: Ammonia stress induces cytotoxicity, apoptosis, and degenerative phenotypes in NPCs. (A) Cell viability of NPCs treated with increasing concentrations of NH 4 Cl for the indicated time points, measured by CCK‐8 assay ( n = 5). (B) Lactate dehydrogenase (LDH) release assay showing increased cytotoxicity in NPCs following NH 4 Cl exposure ( n = 5). (C) Representative SEM images showing ultrastructural alterations in NPCs following ammonia exposure, including membrane shrinkage and cytoplasmic vacuolisation. Scale bar, 20 μm; inset magnification, 5 μm ( n = 3). (D) Bright‐field microscopy images of NPCs stained with Safranin O and Alcian blue showing reduced proteoglycan and acidic glycoprotein production after NH 4 Cl treatment. Scale bar, 100 μm ( n = 3). (E) EdU incorporation assay showing reduced proliferation of NPCs following ammonia exposure. Scale bar, 50 μm ( n = 3). (F) Calcein‐AM/propidium iodide (PI) staining showing increased cell death in NPCs under ammonia stress. Scale bar, 50 μm ( n = 3). (G, H) Western blot analysis and quantitative densitometric analysis of apoptosis‐related proteins (Bax, cleaved‐caspase‐3, cleaved‐caspase‐7, and Bcl‐2) in NPCs treated with NH 4 Cl ( n = 3). (I) TUNEL staining showing increased apoptotic NPCs following ammonia exposure. Scale bar, 50 μm ( n = 3). (J) Flow cytometry analysis quantifying apoptosis rates in NPCs treated with increasing concentrations of NH 4 Cl ( n = 3). (K) Immunofluorescence staining showing increased Bax and cleaved‐caspase‐3 expression in NPCs following ammonia exposure. Scale bar, 25 μm ( n = 3). (L, M) Western blot analysis and quantification of extracellular matrix anabolic markers (Col‐II and SOX‐9) and catabolic enzymes (MMP‐3 and MMP‐13) in NPCs treated with NH 4 Cl ( n = 3). (N) Immunofluorescence staining showing increased expression of senescence‐associated secretory phenotype (SASP) factors IL‐1β and IL‐6 in ammonia‐treated NPCs. Scale bar, 25 μm ( n = 3). (O) Senescence‐associated β‐galactosidase staining showing accumulation of senescent NPCs after ammonia exposure. Scale bar, 50 μm ( n = 3). (P) Immunofluorescence staining showing decreased Aggrecan and increased ADAMTS‐5 expression in NPCs under ammonia stress. Scale bar, 25 μm ( n = 3). (Q, R) Immunohistochemical staining showing increased cleaved‐caspase‐3 and MMP‐13 expression in intervertebral disc tissues from rats subjected to ammonia overload (scale bars: 500 μm; n = 6). Data are presented as mean ± SD. Statistical significance was determined by one‐way ANOVA followed by Tukey's multiple comparisons test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; ns, not significant.

Article Snippet: Culture supernatants were collected, and LDH activity was measured using a commercial LDH Cytotoxicity Assay Kit (Solarbio, China) according to the manufacturer's instructions.

Techniques: CCK-8 Assay, Lactate Dehydrogenase Assay, Membrane, Microscopy, Staining, EdU Assay, Western Blot, Analysis, TUNEL Assay, Flow Cytometry, Immunofluorescence, Expressing, Immunohistochemical staining

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

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