2 3 c di am ps 2 rp rp disodium salt  (MedChemExpress)

Journal: The Journal of Clinical Investigation

Article Title: Astrocytes contribute to olanzapine-mediated reversal of kleefstra syndrome-associated neurodevelopmental regression

doi: 10.1172/JCI195803

Figure Lengend Snippet: ( A ) IPS cells generated through CRIPSR/cas9 used in this study. ( B ) Western blot showing the EHMT1 protein levels in control and EHMT1 +/– astrocytes. ( C ) Glutamate uptake ability of control and EHMT1 +/– astrocytes, n = 4 independent cultures ( D ) Representative image of astrocytes for calcium imaging measure using Fluo8-AM. ( E ) Representative traces of control and EHMT1 +/– astrocytes showing spontaneous calcium transients. ( F ) Quantification of AUC of spontaneous calcium transients. n = 139 for control and n = 197 for EHMT1 +/– , 3 biological replicates ( G ) Representative traces of control and EHMT1 +/– astrocytes showing evoked activity upon 50 μM glutamate stimulation. Thick black trace represents the average trace. ( H ) Quantification of AUC of evoked calcium events. n = 21 for control and n = 30 for EHMT1 +/- , 3 biological replicates. ( I ) Principal component analysis (PCA) of RNA-seq data from control (Ctrl) and EHMT1 +/– astrocytes, n = 3 independent cultures. ( J ) Volcano plots showing differentially expressed genes (DEGs) between EHMT1 +/– and control human astrocytes. Relative to the control, significantly up- or downregulated genes are shown above the black dashed line in red and blue (FDR < 0.05; log 2 (FC) > 1; log 2 (FC) < –1).( K ) Heatmap showing relative normalized gene expression across samples. Normalized counts were log 2 -transformed and scaled per gene to Z-scores, representing relative expression levels. Both genes and samples were hierarchically clustered based on Z-scores. ( L ) GO term analysis of molecular function. Data represent means ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001. Unpaired Student’s t test ( C , F and H ).

Article Snippet: Primary antibodies for S100B (287006, synaptic systems), and GFAP (ab7260, Abcam) were diluted in 0,4%T-X and 5% NGS and incubated overnight at 4 °C on a shaker.

Techniques: Generated, Western Blot, Control, Imaging, Activity Assay, RNA Sequencing, Gene Expression, Transformation Assay, Expressing

Journal: The Journal of Clinical Investigation

Article Title: Astrocytes contribute to olanzapine-mediated reversal of kleefstra syndrome-associated neurodevelopmental regression

doi: 10.1172/JCI195803

Figure Lengend Snippet: ( A ) Relative expression of S100B in control and EHMT1 +/– astrocytes using qPCR. Control n = 6, EHMT1 +/– n = 6. ( B ) Relative protein level of S100B in the culture medium of control and EHMT1 +/– astrocytes using ELISA. Control n = 4, EHMT1 +/– n = 4. ( C ) Western blot showing the JAK/STAT3 protein levels in astrocytes in the absence or presence of olanzapine ( D ) quantification of Western Blot, normalized to control. Control n = 4, EHMT1 +/– n = 4. ( E ) Representative images and ( F ) quantification of CellROX fluorescent intensity in control, control + Rotenone and EHMT1 +/– astrocytes. Control n = 34, Control + 100 nM Rotenone n = 34, EHMT1 +/– n = 41. ( G ) olanzapine (100 nM, 1 μM, 10 μM, 100 μM) treatment on EHMT1 +/– astrocytes for 1 week could reduce S100B level in the culture medium in a dose-dependent manner as tested by ELISA. n = 3. Data represent means ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001. One-way ANOVA test and post hoc Bonferroni correction ( F ) and unpaired Student’s t test ( A , B , and D ).

Article Snippet: Primary antibodies for S100B (287006, synaptic systems), and GFAP (ab7260, Abcam) were diluted in 0,4%T-X and 5% NGS and incubated overnight at 4 °C on a shaker.

Techniques: Expressing, Control, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: The Journal of Clinical Investigation

Article Title: Astrocytes contribute to olanzapine-mediated reversal of kleefstra syndrome-associated neurodevelopmental regression

doi: 10.1172/JCI195803

Figure Lengend Snippet: ( A ) Representative traces and ( B ) quantification of EHMT1 +/– astrocytes with ONO2506 showing reduced evoked activity upon 50 μM glutamate stimulation. n = 57 for EHMT1 +/– and n = 63 for EHMT1 +/– with ONO2506. ( C ) Representative raster plots and ( D ) quantification of mean firing rate exhibited by EHMT1 +/– astrocytes cocultured with EHMT1 +/– neurons on MEAs at DIV21 after 24-hour treatment of 100 μM ONO2506. n = 12. ( E ) EHMT1 +/– astrocytes infected lentivirus expressing mcherry and scrambled or S100B shRNA. ( F ) Relative expression of mRNA S100B in EHMT1 +/– astrocytes infected with S100B shRNA compared with astrocytes transfected with scrambled shRNA. n = 6 for each group. ( G ) Representative traces and ( H ) quantification of calcium wave from EHMT1 +/– astrocytes transfected with scrambled shRNA and S100B shRNA upon 50 μM glutamate stimulation. n = 29 for EHMT1 +/– + scrambled shRNA and n = 21 for EHMT1 +/– + S100B shRNA. ( I ) Representative raster plots and ( J ) quantification of mean firing rate exhibited by EHMT1 +/– neurons cocultured with EHMT1 +/– astrocytes transfected with scrambled shRNA or S100B shRNA on MEAs. Dashed line represents mean of untreated control cells, n = 6 for scrambled shRNA. n = 15 for S100B shRNA. Data represent means ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001. Unpaired Student’s t test ( B , F , H , and J ) and paired Student’s t test ( D ).

Article Snippet: Primary antibodies for S100B (287006, synaptic systems), and GFAP (ab7260, Abcam) were diluted in 0,4%T-X and 5% NGS and incubated overnight at 4 °C on a shaker.

Techniques: Activity Assay, Infection, Expressing, shRNA, Transfection, Control