Journal: Brain : a journal of neurology

Article Title: Connexin43 mimetic peptide reduces vascular leak and retinal ganglion cell death following retinal ischaemia.

doi: 10.1093/brain/awr338

Figure Lengend Snippet: Figure 3 Three hours of hypoxia and 6 h of reperfusion lead to significant endothelial cell death in vitro in rat brain microvascular endothelial cells (RBMVEC; A). Non-specific gap junction blocker carbenoxolone, non-specific hemichannel blocker LaCl3, and connexin43 (Cx43) mimetic peptide protected endothelial cells against hypoxic injury, with the number of viable cells significantly higher than no treatment, while scrambled peptide did not have any protective effects. The number of viable cells was expressed as percentage of the control without hypoxia. Hypoxia and reperfusion also lead to significant propidium iodide dye uptake into primary rat brain microvascular endothelial cells indicating open hemichannels (B). Carbenoxolone, LaCl3 and connexin43 mimetic peptide significantly prevented dye uptake compared to no treatment, indicating hemichannel closure, while scrambled peptide did not have any effect. Stars denote statistical significance when compared to the control group or compared between groups in brackets; P 5 0.05.

Article Snippet: Rat brain microvascular endothelial cells (R840K-05a, Cell Applications) were plated into 24-well plates (1 105 cells/well) in rat brain microvascular endothelial cell growth media (R819K-500, Cell Applications) and allowed to settle for 16 h. Medium was then removed and replaced with Dulbecco’s Modified Eagle’s Medium/F12 containing 0.5% foetal bovine serum and 1% glutamine.

Techniques: In Vitro, Control

Journal: Oncology Letters

Article Title: MKP-1 overexpression is associated with chemoresistance in bladder cancer via the MAPK pathway

doi: 10.3892/ol.2020.11741

Figure Lengend Snippet: MKP-1 and MAPK expression in RT112 cells transfected with NC and MKP-1 siRNA. (A) Relative MKP-1 expression in the siNC and siMKP-1 groups was examined using reverse transcription-quantitative PCR. **P<0.01. (B) Representative microscopic images of siNC and siMKP-1 treated cells captured in both 2D and 3D environments under a phase contrast microscope. (C) MKP-1 expression of the siNC and siMKP-1 groups, as determined via western blotting. GAPDH was used as the internal control. (D) Phosphorylated and total ERK1/2 protein expressions of the siNC and siMKP-1 group, as determined via western blotting. GAPDH was used as the internal control. (E) Phosphorylated and total p38 protein expressions of the siNC and siMKP-1 group, as determined via western blotting. GAPDH was used as the internal control. (F) Phosphorylated and total JNK protein expression levels of the siNC and siMKP-1 group, as determined via western blotting. GAPDH was used as the internal control. MKP-1, mitogen activated protein kinase phosphatase-1; NC, negative control; siMKP-1, MKP-1 small interfering RNA; siNC, small interfering negative control.

Article Snippet: 3D Cell Culture Gel (cat. no. P720M-10) was purchased from Col-Tgel Med ( http://www.101bio.com/P720_3D_cell_culture_gel.php ).

Techniques: Expressing, Transfection, Real-time Polymerase Chain Reaction, Microscopy, Western Blot, Negative Control, Small Interfering RNA

Journal: American journal of physiology. Endocrinology and metabolism

Article Title: Hyaluronan 35 prevents endotoxin-mediated dysregulated skeletal muscle proteostasis during ethanol exposure

doi: 10.1152/ajpendo.00283.2025

Figure Lengend Snippet: Studies were done in differentiated C2C12 murine myotubes treated without/with ethanol (EtOH) at/for specified concentrations and times. A-C. Representative immunoblots and densitometry for: A . Puromycin incorporation. B . Phosphorylation of mTOR (p-mTOR Ser2448 ) and downstream signaling target (P70S6K). C . Puromycin incorporation. D . Protein synthesis measured by 3 H phenylalanine incorporation with/without 100mM EtOH for 6h. E . Representative photomicrographs and myotube diameter. Measurements were taken from at least 100 myotubes in each group. F . Cellular membrane stiffness by atomic force microscopy in myotubes with/without 100mM EtOH for 6h. G . Gastrocnemius muscle weights from female pair-fed (PF) and 6%v/v binge mouse model of alcohol-associated liver disease (mALD). (n=4 PF, 6 mALD). H . Contractile responses of quadriceps femoris muscle from C57B/6J pair-fed PF and mALD to different frequencies of stimulation. Both binge and chronic (4-weeks) feeding models were studied. Binge model: N=7 pair-fed (3 male, 4 female), 8 mALD (4 male, 4 female); Chronic model: N=10 in each (4 male/6 female) Myotubes: n=3–4 biological replicates. Data shown as mean±SD. * p<0.05; ** p<0.01; *** p<0.001. Statistical analyses: Panels A-E. One-way ANOVA with Fisher’s least significant difference test. Panel F,G. Student’s t-test for independent groups. Panel H. Contractility studies 2-way mixed effects ANOVA with Fisher’s least significant difference test. To avoid large sample size fallacy, for Panel E, comparisons were made for the average for each replicate in different groups.

Article Snippet: In brief, hiPSC were cultured in mTeSR medium (Stem Cell Technologies, Cambridge, MA) with myogenic induction initiated by sequential culture in skeletal muscle induction medium (AMS Biotechnology, Abingdon, UK), myoblast medium (Amsbio-SKM02) and myotube medium (AMS Biotechnology, Abingdon, UK).

Techniques: Western Blot, Phospho-proteomics, Membrane, Microscopy

Journal: American journal of physiology. Endocrinology and metabolism

Article Title: Hyaluronan 35 prevents endotoxin-mediated dysregulated skeletal muscle proteostasis during ethanol exposure

doi: 10.1152/ajpendo.00283.2025

Figure Lengend Snippet: In vitro studies were done in differentiated C2C12 murine myotubes treated without/with ethanol (EtOH) or lipopolysaccharides (LPS) for specified concentrations for 6h. In vivo studies were done in gastrocnemius muscle from wild-type mice without treatment and in a mouse model of sepsis for 4 or 24h. A,B. Representative immunoblots and densitometries of A. Puromycin incorporation with LPS and B. mTORC1 signaling, including LC3 lipidation in myotubes; C. Gastrocnemius muscle weight; D . Representative immunoblots and densitometry of phosphorylation of P65-NFkB, P70S6K, and RPS6, and LC3 lipidation in gastrocnemius muscle from mice. Panels E-G. Myotube responses to 100mM ethanol, 100ng/mL LPS, and 50mM ethanol and 10ng/mL LPS for 6h. Representative immunoblots and densitometry for E. Puromycin incorporation, F. mTORC1 signaling, including LC3 lipidation. G. Myotube diameter. Statistical analyses for all panels: One-way ANOVA with Fisher’s least significant difference post-hoc testing. Data shown as mean±SD from studies in 3–4 biological replicates in myotubes (Panels A,B,E,F) and gastrocnemius from 3–8 mice. * p<0.05; ** p<0.01; *** p<0.001.

Article Snippet: In brief, hiPSC were cultured in mTeSR medium (Stem Cell Technologies, Cambridge, MA) with myogenic induction initiated by sequential culture in skeletal muscle induction medium (AMS Biotechnology, Abingdon, UK), myoblast medium (Amsbio-SKM02) and myotube medium (AMS Biotechnology, Abingdon, UK).

Techniques: In Vitro, In Vivo, Western Blot, Phospho-proteomics

Journal: American journal of physiology. Endocrinology and metabolism

Article Title: Hyaluronan 35 prevents endotoxin-mediated dysregulated skeletal muscle proteostasis during ethanol exposure

doi: 10.1152/ajpendo.00283.2025

Figure Lengend Snippet: C2C12 myotubes were treated with 100mM ethanol (EtOH) for 6 and 24h, and gastrocnemius muscle was harvested from a mouse model of alcohol-related liver disease (mALD) and pair-fed (PF) controls. Bulk RNAseq and untargeted proteomics were performed, and differentially expressed molecules were identified. A. Dendrograms of hierarchical clustered genes from weighted gene co-expression network analyses (WGCNA) where the y-axis is a dissimilarity index based on topological overlap and the distance between branches representing dissimilarity. Similar branches were grouped into ‘modules,’ signified by each color block below the dendrogram. Box and whisker plots of normalized module Eigengenes from select modules. Table of functional enrichment performed using DAVID Functional Annotation Bioinformatics Microarray Analysis with Kyoto Encyclopedia of Genes and Genomes (KEGG) and REACTOME pathway databases mined. B. Heatmaps of LPS-signaling molecules and HA-metabolism on RNAseq in C2C12 myotubes treated without and with 100mM EtOH. C,D. Real-time PCR quantification of mRNA expression fold change of TLR2, TLR4, and CD44 in ( C ) C2C12 myotubes and ( D ) gastrocnemius muscle from mouse model of alcohol-associated liver disease (mALD) and pair-fed (PF) mice. E-G . Representative immunoblots and densitometry of CD44 expression in ( E ) untreated and ethanol-treated myotubes ( F ) gastrocnemius muscle from PF and mALD mice, and ( G ) phosphorylated P65-NFkB in myotubes without/with TLR4 knockdown. One-way ANOVA with Fisher’s least significant difference post-hoc testing performed. Significance cutoff for differentially expressed molecule was p.adj <0.05 in C2C12 RNAseq and p<0.05 for all other datasets. Myotube data from at least 3 biological replicates and mouse data from 3–4 mice per group. All data expressed as mean±SD. *p<0.05; **p<0.01; ***p<0.001.

Article Snippet: In brief, hiPSC were cultured in mTeSR medium (Stem Cell Technologies, Cambridge, MA) with myogenic induction initiated by sequential culture in skeletal muscle induction medium (AMS Biotechnology, Abingdon, UK), myoblast medium (Amsbio-SKM02) and myotube medium (AMS Biotechnology, Abingdon, UK).

Techniques: Biomarker Discovery, Expressing, Blocking Assay, Whisker Assay, Functional Assay, Microarray, Real-time Polymerase Chain Reaction, Western Blot, Knockdown

Journal: American journal of physiology. Endocrinology and metabolism

Article Title: Hyaluronan 35 prevents endotoxin-mediated dysregulated skeletal muscle proteostasis during ethanol exposure

doi: 10.1152/ajpendo.00283.2025

Figure Lengend Snippet: Studies were done in differentiated C2C12 murine or human induced pluripotent stem cell (hiPSC) derived myotubes treated without/with 100mM ethanol, 100ng/mL LPS, or 50mM ethanol and 10ng/mL LPS for 6h without/with 1 mg/ml HA35 . A . Representative immunoblots and densitometry of puromycin incorporation as a measure of protein synthesis in myotubes in response to 100mM ethanol or 100 ng/ml LPS with and without HA35. B . Representative flow cytometry data from myotubes stained with puromycin-Alexa fluor treated without/with HA35, ethanol, and LPS. C . Representative immunoblots and densitometry of mTORC1 signaling and protein homeostasis regulatory molecules (phosphorylation of p65 (p-P65-NFkB Ser536 ), myostatin, and total and phosphorylated P70S6K (p-P70S6K Thr389 ) and ribosomal protein S6 (p-RPS6 Ser240/244 ). D . Mean myotube diameter from differentiated C2C12 myotubes treated with HA35, 100mM ethanol, and 10ng/ml lipopolysaccharide for 6h. E-G. Representative immunoblots and densitometry of ( E ) puromycin incorporation, ( F ) mTORC signaling, including myostatin expression, and ( G ) quantification of mean myotube diameter in myotubes without/with EtOH+LPS without/with HA35. H. Representative immunoblots and densitometry of p-RPS6 Ser240/244 and p-P70S6K Thr389 in hiPSC-derived myotubes. Data expressed as mean±SD from at least 100 myotubes in each group. Data from 3–4 biological replicates expressed as mean±SD. * p<0.05; ** p<0.01; *** p<0.001.

Article Snippet: In brief, hiPSC were cultured in mTeSR medium (Stem Cell Technologies, Cambridge, MA) with myogenic induction initiated by sequential culture in skeletal muscle induction medium (AMS Biotechnology, Abingdon, UK), myoblast medium (Amsbio-SKM02) and myotube medium (AMS Biotechnology, Abingdon, UK).

Techniques: Derivative Assay, Western Blot, Flow Cytometry, Staining, Phospho-proteomics, Expressing