Journal: International Journal of Molecular Sciences

Article Title: High Glucose-Induced Cardiomyocyte Damage Involves Interplay between Endothelin ET-1/ET A /ET B Receptor and mTOR Pathway

doi: 10.3390/ijms232213816

Figure Lengend Snippet: Effect of high glucose (HG) treatment in H9c2 cells. ( A ) The H9c2 cells were seeded in low glucose DMEM medium (5.5 mM) and treated with various concentrations of D-glucose-containing medium (vc = vehicle control, 20 mM, 30 mM, 50 mM, 60 mM, and 80 mM) for 24 h. Cell viability was assessed by MTT assay. ( B ) The H9c2 cells were seeded in low glucose DMEM medium overnight and incubated with HG medium for 24 h and subjected to 2-NBDG-based glucose uptake assay. The fluorescent intensity was measured at wavelengths λex/λem: 485/530 nm. ( C – F ) The H9c2 cells were treated with HG for 24 h, and total RNA was extracted. The mRNA expression level of GLUT1, GLUT4, ET-1, ET A -R, ET B -R, mTOR, Raptor, Rictor, PGC1α, and TFAM genes were determined by qPCR and normalized to GAPDH and expressed as fold versus non-treated controls (vc) (2 −ΔΔCT ). ( G , H ) The H9c2 cells were treated with HG as above, and proteins were extracted after 24 h of incubation. Protein expressions of p-mTOR/Raptor/Rictor ( G ) and survival markers p-Akt, Akt, Bcl-2, and c-Caspase-3 ( H ) were determined using western blotting. N = 3; Mean ± S.E.M.; * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Protein expressions were detected using specific primary antibodies: mTOR (Santa Cruz Biotechnology, Santa Cruz, CA, USA; sc-517464), p-mTOR (sc-293133), Raptor (sc-518004), Rictor (sc-271081), TFEB (sc-166736), PGC1α (sc-518025), Akt (Cell Signaling Technology, Danvers, MA, USA; cst#9272), p-Akt (cst#4060), Bax (cst#2772), Bcl-2 (cst#2876), c-caspase 3 (cst#9664), and GAPDH (Merck, Darmstadt, Germany; CB1001).

Techniques: Control, MTT Assay, Incubation, Expressing, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: High Glucose-Induced Cardiomyocyte Damage Involves Interplay between Endothelin ET-1/ET A /ET B Receptor and mTOR Pathway

doi: 10.3390/ijms232213816

Figure Lengend Snippet: Inhibition of mTOR improved the survival markers in HG-treated H9c2 cells. The H9c2 cells were seeded in low glucose medium overnight. Next, the cells were transfected with siRNAs against Raptor/Rictor for 24 h, followed by ET-1 (20 nM) treatment for 3 h and by HG treatment for another 24 h. Protein expressions of survival/apoptosis markers p-Akt, Bcl-2, and Bax ( A ); antioxidant markers SOD2, catalase, and HO-1 ( B ); and mitochondrial/autophagy marker PGC1α and TFEB ( C ) were determined using western blot. Protein densitometry analysis was done using Image J software (NIH). N = 3; Mean ± S.E.M.; * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Protein expressions were detected using specific primary antibodies: mTOR (Santa Cruz Biotechnology, Santa Cruz, CA, USA; sc-517464), p-mTOR (sc-293133), Raptor (sc-518004), Rictor (sc-271081), TFEB (sc-166736), PGC1α (sc-518025), Akt (Cell Signaling Technology, Danvers, MA, USA; cst#9272), p-Akt (cst#4060), Bax (cst#2772), Bcl-2 (cst#2876), c-caspase 3 (cst#9664), and GAPDH (Merck, Darmstadt, Germany; CB1001).

Techniques: Inhibition, Transfection, Marker, Western Blot, Software

Journal: Brain

Article Title: Schizophrenia-associated changes in neuronal subpopulations in the human midbrain

doi: 10.1093/brain/awae321

Figure Lengend Snippet: Unsupervised clustering identifies 18 neuronal populations in the human midbrain . ( A ) Experimental design of the study . A schematic representation of the rostral midbrain at the level of the red nucleus and third cranial nerves (created with biorender.com ). The lines indicate where the superior colliculus (SC) and cerebral peduncles (CP) were removed before nuclei isolation, FANS and snRNAseq. ( B ) Unsupervised clustering of 31 669 neuronal nuclei from 14 schizophrenia (SZ) cases and 14 controls ( and ). Each dot indicates a nucleus, colours indicate clusters. ( C ) Average expression per cluster of selected neurotransmitter-, neuropeptide- or interneuron-related genes. ( D ) Bar plot depicting the total number of nuclei obtained per population, with the percentage of the total NEUN+ population enclosed in parentheses. ( E ) Violin plots depicting log-normalized expression levels of cluster markers (log 2 fold-change > 0.25, P -adjusted < 0.05) . ( F ) Violin plot depicting log-normalized expression level of DLX1 , a cluster marker for inh-CCK. ( G ) A schematic representation of the rostral midbrain indicating the periaqueductal grey and the third cranial nerve nucleus with a box and the reticular formation with a dotted box. In these regions the immunohistochemical staings, shown in H , were performed. ( H ) Representative images of immunohistochemical stainings for DLX1 (brown) and GFAP (black) in control midbrain ( n = 5). Scale bar = 50 μm. avg. = average; expr. = expression; FANS = fluorescence-activated nuclear sorting; log-norm. = log-normalized; snRNAseq = single nucleus RNA-sequencing; UMAP = Uniform Manifold Approximation and Projection.

Article Snippet: Briefly, after sucrose density centrifugation, nuclei were incubated with fluorescently-conjugated antibodies directed against neuronal marker NEUN (RBFOX3/NEUN 1B7 AF647 mouse mAB, Novus Biologicals, NBP1-92693AF647) and the transcription factor OLIG2 for the oligodendrocyte lineage (Anti-OLIG2 clone 211F1.1 AF488 mouse mAB, Merck Millipore, MABN50A4).

Techniques: Isolation, Expressing, Marker, Immunohistochemical staining, Control, Fluorescence, RNA Sequencing

Journal: Brain

Article Title: Schizophrenia-associated changes in neuronal subpopulations in the human midbrain

doi: 10.1093/brain/awae321

Figure Lengend Snippet: Schizophrenia-associated shifts in proportions of midbrain neuronal populations and subpopulations . ( A ) Uniform Manifold Approximation and Projection (UMAP) of the clusters split by diagnosis. The colour legend indicates the number of nuclei obtained per population and the percentage of the total NEUN+ population enclosed in brackets. ( B ) Box plot depicting the percentage of exc-VGLUT1 and 2 nuclei detected in midbrain samples of schizophrenia and control cases ( n = 14 per group; ). Outlier cases are labelled with grey diamond shapes, each dot represents a case, and horizontal lines indicate the median. Outliers were defined as proportions outside of the third quartile plus 1.5 times the interquartile range. ( C ) UMAP of the subclusters split by diagnosis. The colour legend indicates the number of nuclei obtained per population and the percentage of the total NEUN+ population enclosed in brackets. ( D ) Box plots depicting the percentage of nuclei detected per case in subclusters that had significantly altered proportions in schizophrenia; and . Outlier cases are labelled with grey diamond shapes and were defined as data points outside the third quartile + 1.5 × interquartile range. ( E ) Total count of differentially expressed genes (DEGs) between schizophrenia and controls within a neuronal population. Numbers above the bar indicate the total count of increased plus decreased gene expression changes in schizophrenia. ( F ) Volcano plot depicting differentially expressed genes (absolute log 2 fold-change > 0.25, P -adjusted < 0.05) between schizophrenia and controls within the neuronal population ‘inh-CCK’. The corresponding information on DEGs is available in . ( G ) Total count of differentially expressed genes between schizophrenia and controls within a neuronal subpopulation. Numbers above the bar indicate the total count of increased plus decreased gene expression changes in schizophrenia. ( H ) Volcano plot depicting DEGs (absolute log 2 fold-change > 0.25, P -adjusted < 0.05) between schizophrenia and controls within the neuronal subpopulation ‘inh-C’. The number of gene labels was reduced to avoid overlapping text; the full information on DEGs is available in . ( I ) Heat map with the number indicating the detected schizophrenia susceptibility genes per neuronal population . The colour indicates the normalized ratio of detected susceptibility genes over the total number of expressed genes per neuronal population. High-confidence schizophrenia susceptibility genes derived from Wang et al .

Article Snippet: Briefly, after sucrose density centrifugation, nuclei were incubated with fluorescently-conjugated antibodies directed against neuronal marker NEUN (RBFOX3/NEUN 1B7 AF647 mouse mAB, Novus Biologicals, NBP1-92693AF647) and the transcription factor OLIG2 for the oligodendrocyte lineage (Anti-OLIG2 clone 211F1.1 AF488 mouse mAB, Merck Millipore, MABN50A4).

Techniques: Biomarker Discovery, Control, Gene Expression, Derivative Assay