Journal: Frontiers in Immunology

Article Title: Contact-Dependent Granzyme B-Mediated Cytotoxicity of Th17-Polarized Cells Toward Human Oligodendrocytes

doi: 10.3389/fimmu.2022.850616

Figure Lengend Snippet: Th17-polarized secretion of granzyme B induces OL death. Percentage of (A) OLs (n = 4 different OL preps) and (B) MO3.13 cell death as measured by FACS (LIVE/DEAD Amcyan staining) after a 16h incubation with activated granzyme B. Each dot represents one independent experiment, n = 4 independent experiments, Friedmann test with Dunn’s method. (C) Percentage of MO3.13 cell death as measured by FACS (LIVE/DEAD Amcyan staining) after a 16h incubation with Th17-polarized cells pretreated with granzyme blocker Ac-IEPD-CHO (Ac) or DMSO control; each dot represents one donor, n = 5–6 T cell donors, diamond = mean, Wilcoxon test (35 μg/ml), and paired t-test (50 μg/ml). *p < 0.05; **p < 0.01.

Article Snippet: Vesicle-like structures were defined as bright spots of size 200 to 600 nm within the MO3.13 cell/OL and manually counted in Imaris using the spot module.

Techniques: Staining, Incubation, Control

Journal: Acta Neuropathologica

Article Title: Oncohistone interactome profiling uncovers contrasting oncogenic mechanisms and identifies potential therapeutic targets in high grade glioma

doi: 10.1007/s00401-022-02489-2

Figure Lengend Snippet: Reduced interaction with DNA methyltransferases leads to global hypomethylation in H3.3G34R-mutant pHGG. A Bubble plot showing differential enrichment of DNMT1 and DNMT3A with each oncohistone relative to WT control. Size = log2(mutant/WT). Color is a function of -log10 p-value and direction of interaction change. Statistically significant ( p < 0.05) differential interactions have a black border. B Proximity ligation assays in MO3.13 cells between HA-H3 and DNMT1. Scale bar: 20 μm. Results are representative of two biological replicates and show mean foci counts relative to H3.3WT ± standard error. n : EV = 52; H3.3WT = 73; H3.3G34R = 61. p : ANOVA. C Median methylation beta value per sample from pHGG categorized as WT ( n = 98); mutant for K27M (n = 34), G34R ( n = 24), or IDH ( n = 25); or normal brain ( n = 7). The box marks the interquartile range (IQR) and shows the median value. The whiskers extend to 1.5 × IQR. p : ANOVA. D Median methylation beta value per probe from pHGG categorized as WT ( n = 98); mutant for K27M ( n = 34), G34R ( n = 24), or IDH ( n = 25); or normal brain ( n = 7). The box marks the interquartile range (IQR) and shows the median value. The whiskers extend to 1.5 × IQR. p : ANOVA. E Scatter plot of number of differentially methylated regions (DMR) detected in H3.3G34R-mutant ( n = 24) vs WT ( n = 98) tumors assigned to each genomic location category versus the log2-enrichment against an expected background. F Enrichment of DMRs detected in H3.3G34R-mutant ( n = 24) vs WT ( n = 98) tumors around transcription start sites (TSS). G Network of pathways (Reactome, KEGG, Gene Ontology Biological Processes) enriched in genes with DMRs ± 1 kb from the TSS. Node size and color reflects significance and edge thickness reflects number of genes shared between two nodes. H Peptide counts of methyl-binding domain (MBD) containing proteins identified by BioID with H3.3WT or H3.3G34R. p : t test

Article Snippet: Flp-In T-REx HEK293 (Thermo); HEK293T, U87, U343 (ATCC); MO3.13 (Cedarlane); iNHA [ ]; and NHA (ABM) cells were cultured in DMEM (Gibco), supplemented with 10% FBS (Wisent) and 1% penstrep (Invitrogen), at 37 °C in 5% CO2.

Techniques: Mutagenesis, Ligation, Methylation, Binding Assay

Journal: Acta Neuropathologica

Article Title: Oncohistone interactome profiling uncovers contrasting oncogenic mechanisms and identifies potential therapeutic targets in high grade glioma

doi: 10.1007/s00401-022-02489-2

Figure Lengend Snippet: Oncohistones disrupt H3K9 methylation. A Bubble plot showing differential enrichment of indicated H3K9 methylases and demethylases with each oncohistone relative to WT control. Size = log2(mutant/WT). Color represents significance. Statistically significant ( p < 0.05) differential interactions have a black border. B Proximity ligation assays in MO3.13 cells between HA-H3 and SUV39H2. Scale bar: 20 μm. Results are representative of two biological replicates and show mean foci counts relative to H3.3WT ± standard error. n : EV = 14; H3.1WT = 36; H3.1K27M = 67; H3.3WT = 60; H3.3K27M = 62. p : ANOVA. C Ectopic histone-containing nucleosomes were immunoprecipitated (IP) from H3-expressing NHA cells and ectopic and endogenous modifications were assessed by western blotting. < : Ectopic HA-tagged histone. + : endogenous histone. D NHA cells were transduced with indicated constructs and whole cell lysates analyzed by western blotting. E Ectopic histone-containing nucleosomes were immunoprecipitated (IP) from H3-expressing NHA cells and ectopic and endogenous modifications were assessed by western blotting. < : Ectopic HA-tagged histone. + : endogenous histone. F Representative gels from in vitro methylation assays carried out with nucleosomes assembled with H3.3WT, H3.3G34R or H3.3K27M and incubated with increasing amounts of SUV39H2. G Representative gels from in vitro methylation assays carried out with nucleosomes assembled with H3.3WT, H3.3G34R or H3.3K27M and incubated with increasing amounts of EHMT2. H Quantification of SUV39H2 methylation assays from F ( n = 3). p (ANOVA): H3.3WT vs H3.3K27M, p = 0.003; H3.3WT vs H3.3G34R, p = 0.001; H3.3K27M vs H3.3G34R, p = 10 –7 . I Quantification of EHMT2 methylation assays from G ( n = 3). p (ANOVA): H3.3WT vs H3.3K27M, p = 0.006; H3.3WT vs H3.3G34R, p = 0.009; H3.3K27M vs H3.3G34R, p = 10 –6

Article Snippet: Flp-In T-REx HEK293 (Thermo); HEK293T, U87, U343 (ATCC); MO3.13 (Cedarlane); iNHA [ ]; and NHA (ABM) cells were cultured in DMEM (Gibco), supplemented with 10% FBS (Wisent) and 1% penstrep (Invitrogen), at 37 °C in 5% CO2.

Techniques: Methylation, Mutagenesis, Ligation, Immunoprecipitation, Expressing, Western Blot, Transduction, Construct, In Vitro, Incubation

Journal: Journal of Personalized Medicine

Article Title: Protein Succinylation and Malonylation as Potential Biomarkers in Schizophrenia

doi: 10.3390/jpm12091408

Figure Lengend Snippet: Visualization of biological pathways enriched by proteins found with higher (Up) or lower (Down) levels of succinylation (SuccK) or malonylation (MalK), induced by haloperidol (Halo), chlorpromazine (Chlor), and quetiapine (Quet) exposure in MO3.13 cells, determined by the Reactome.org database. The number of gene names for each enriched pathway determines the size of the dot, while the colors represent the FDR for the pathways. Comparisons not listed did not enrich for any pathways with FDR < 1% and pathways containing fewer than a total of 5 gene names were excluded.

Article Snippet: In joint treatments, cells were incubated with MK-801 (final concentration 50 µM) for 4 h before the antipsychotic was added to the same medium (to a concentration of 50 µM) for an additional 4 h. MO3.13 cells were sourced from Cedarlane Cellutions Biosystems, Inc. (product code CLU301).

Techniques: