Journal: Cell & Bioscience

Article Title: ISL1 controls pancreatic alpha cell fate and beta cell maturation

doi: 10.1186/s13578-023-01003-9

Figure Lengend Snippet: Physiological changes and molecular abnormalities in islets of Langerhans associated with diabetic phenotype of Isl1CKO. a Blood glucose levels of Isl1CKO (n = 59 pups), heterozygous ( HET , NeuroD1 Cre /Isl1 f/ + ; n = 77 pups), and controls (n = 138 pups) fed ad libitum after birth at P0-P3. Violin plots indicate median (middle line), 25th, and 75th percentile (dotted lines). Data were analyzed by one-way ANOVA with Tukey’s multiple comparisons test (**** P < 0.0001). b Total pancreatic insulin content per pancreatic tissue (ng/mg) at P0 (n = 6 pancreases per genotype), P15 (n = 13 pancreases per control, 14 pancreases per Isl1CKO ), and in the adult mice (n = 19 pancreases per control, n = 16 pancreases per Isl1CKO ) fed ad libitum. Data are presented as mean ± SEM, Student’s t test (* P = 0.017, **** P < 0.0001). c-f Representative sections from the control and Isl1CKO pancreas immunostained for glucagon (GCG), insulin (INS), PDX1 (marker of β cells), or alpha amylase (marker for exocrine cells) at P0 demonstrate reduced endocrine tissue and abnormalities in the formation of pancreatic islets in Isl1CKO with lower production of INS, missing GCG + cells, and reduced expression of PDX1. g , h Immunolabeling for proliferating cell nuclear antigen Ki67 (red) shows proliferating GCG + and INS + cells in the islets of the Isl1CKO and control pancreas at P0. i Total number of INS + and GCG + cells in 80-μm sections of the pancreas (n = 5 pancreases per genotype), and the percentage of INS + cells expressing Ki67 (n = 5 mice per genotype) and phosphorylated histone H3 (pHH3; n = 4 mice per genotype). See also Additional file : Fig. S3. Data are presented as mean ± SD, unpaired t test (**** P < 0.0001, *** P < 0.001, ns = not significant). j-o Immunostaining of α- and β-cell markers (PDX1, INS, GCG, PAX6, and NKX6.1) in the pancreatic sections at P9 shows abnormalities in β cells, including reduced production of INS, variable expression levels of PDX1 with some cells expressing INS but not PDX1 (arrowheads in k indicate INS + cells without PDX1 expression). GCG producing α cells are lost in the Isl1CKO islets of Langerhans. Arrows in m indicate the unusual GCG + cells with a missing expression of PAX6, a marker of both β and α cells. p , q At P35, compare to the control pancreatic islets, the expression of INS and PDX1 is reduced, and GCG + producing cells are missing in the Isl1CKO islets. r , s Representative confocal microscopy images of double staining with anti-PDX1 and anti-ISL1 antibodies show that the expression of ISL1 is not detected in nuclei of endocrine cells of pancreatic islets of Isl1CKO in contrast to the control. Arrowheads indicate nuclei with ISL1 and PDX1 co-expression in the control pancreatic islet. Note reduced PDX1 expression in Isl1CKO . Scale bars: 50 μm. HS, Hoechst nuclear staining

Article Snippet: CUT&Tag validated primary antibodies anti-H3K4me3 (Active Motif, #39,159, 1:100), Anti-H3K27me3 (Active Motif, #39155, 1:100), anti-ISL1 (Developmental Hybridoma Bank, #39.4D5, 1:50), normal rabbit IgG negative control (EpiCypher, #13–0042, 0.5 µg/reaction), anti-mouse secondary antibody (EpiCypher, #13–0048, 0.5 µg/reaction), and anti-rabbit secondary antibody (EpiCypher, #13-0047, 0.5 µg/reaction).

Techniques: Control, Marker, Expressing, Immunolabeling, Immunostaining, Confocal Microscopy, Double Staining, Staining

Journal: Cell & Bioscience

Article Title: ISL1 controls pancreatic alpha cell fate and beta cell maturation

doi: 10.1186/s13578-023-01003-9

Figure Lengend Snippet: Aberrant α-cell lineage development during primary transition in the Isl1CKO pancreas. a Schematic presentation of the pancreas formation, which begins with the independent budding of the dorsal and ventral buds at the posterior region of the foregut. These two buds, surrounded by the mesenchyme, eventually fuse after rotation of the gut to form the pancreatic endoderm. b , c Representative higher-magnification images of whole-mount immunolabeling show a loss of ISL1 and glucagon (GCG) expressing cells in the dorsal pancreas of Isl1CKO at E10.5. d-g Whole-mount immunolabeling shows the formation of GCG + clusters in the dorsal (DP) and ventral pancreatic buds (VP) at E11.5. Higher-magnification images show DP with GCG + clusters. h Quantification of the GCG + area in the PDX1 + domain shows the reduced size of GCG + clusters in the dorsal pancreas of Isl1CKO (n = 8 pancreases) compared to controls (n = 9 pancreases). Data are presented as mean ± SD, Unpaired t -test, ** P = 0.0069). i, j Representative images of whole-mount immunolabeling of the pancreas show endocrine cells co-expressing GCG and insulin (INS) in the control (arrowheads) but not in the Isl1CKO pancreas (asterisks indicate autofluorescent red blood cells). k Quantitative RT-PCR analyses of mRNA levels of Gcg and selected transcription factors in the E12.5 pancreas. Data are presented as mean ± SEM (n = 8 pancreases per genotype), Unpaired t -test (**** P < 0.0001, *** P < 0.001, ** P < 0.01, ns = not significant). Scale bars: 50 μm

Article Snippet: CUT&Tag validated primary antibodies anti-H3K4me3 (Active Motif, #39,159, 1:100), Anti-H3K27me3 (Active Motif, #39155, 1:100), anti-ISL1 (Developmental Hybridoma Bank, #39.4D5, 1:50), normal rabbit IgG negative control (EpiCypher, #13–0042, 0.5 µg/reaction), anti-mouse secondary antibody (EpiCypher, #13–0048, 0.5 µg/reaction), and anti-rabbit secondary antibody (EpiCypher, #13-0047, 0.5 µg/reaction).

Techniques: Immunolabeling, Expressing, Control, Quantitative RT-PCR

Journal: Cell & Bioscience

Article Title: ISL1 controls pancreatic alpha cell fate and beta cell maturation

doi: 10.1186/s13578-023-01003-9

Figure Lengend Snippet: Isl1 elimination induces transcriptomic and epigenetic changes altering the composition of the endocrine cell population in the developing pancreas. a Overview of study design. b Volcano plot showing differentially expressed genes between FACS-sorted pancreatic endocrine cells from Isl1CKO (n = 5) and control E14.5 embryos (n = 6) identified by RNA sequencing (Padj < 0.05 displayed as –log 10 and log 2 fold change − 1 and 0.585). Thresholds are indicated by dotted lines. Complete list of differentially expressed genes in Supplementary Dataset 1a. c A simplified schematic overview of the different pancreatic cell types represented in single cell RNA-seq used as a reference for the cell type deconvolution of our bulk cell RNA-seq data. d The deconvolved cell type proportions in E14.5 endocrine population from our bulk RNA-seq data. The proportion of major cell types is shown as an average and per individual samples. Data are presented as mean ± SD, Unpaired t -test (**** P < 0.0001, *** P = 0.0007). See also Additional file : Fig. S4. e Heatmaps represent the top 30 enriched genes in endocrine progenitors, and in α and β cells in Isl1CKO and control samples based on the transcriptome analyses (*indicates genes with ISL1 binding sites at their promoter regions identified by the ISL1 CUT&Tag-seq, see page 13). Complete list of genes in Supplementary Dataset 1b and 1c, and S2. f Heatmap representation of expression profiles of differentially expressed genes that have differences in H3K4me3 or H3K27me3 enrichment in their promoter regions (± 3 kb from the TSS) between control and Isl1CKO cells. Scale bar for RNA-seq data represents average values calculated from the ratio of rlog of the sample to average rlog of the row (all samples per gene). Scale bars for CUT&Taq-seq data represent average counts per million (cpm; > 25 cpm) per each group. g Genome track view of representative gene loci showing H3K4me3 (green), H3K27me3 (magenta), and negative antibody control IgG (grey) normalized read peaks. EPs, endocrine progenitors

Article Snippet: CUT&Tag validated primary antibodies anti-H3K4me3 (Active Motif, #39,159, 1:100), Anti-H3K27me3 (Active Motif, #39155, 1:100), anti-ISL1 (Developmental Hybridoma Bank, #39.4D5, 1:50), normal rabbit IgG negative control (EpiCypher, #13–0042, 0.5 µg/reaction), anti-mouse secondary antibody (EpiCypher, #13–0048, 0.5 µg/reaction), and anti-rabbit secondary antibody (EpiCypher, #13-0047, 0.5 µg/reaction).

Techniques: Control, RNA Sequencing Assay, Binding Assay, Expressing

Journal: Cell & Bioscience

Article Title: ISL1 controls pancreatic alpha cell fate and beta cell maturation

doi: 10.1186/s13578-023-01003-9

Figure Lengend Snippet: ISL1 modulates expression profiles of endocrine cells in the P9 pancreas. a Volcano plot shows differentially expressed genes (Padj < 0.05 (displayed as –log 10 ) and log 2 fold change − 1 and 0.585) identified by RNA sequencing in 100 FACS-sorted endocrine cells from the P9 pancreas of Isl1CKO (n = 6) in comparison to the control (n = 5). Complete list of genes in Supplementary Dataset 1d. b The most enriched Gene Ontology (GO) biological processes for downregulated and upregulated genes identified by RNA-seq. Complete list of genes for the GO terms in Supplementary Dataset 1e. c Heatmap of insulin secretion genes from the GO analysis shows expression levels for each sample. d Heatmap shows the top upregulated genes in P9 endocrine cells of Isl1CKO that were found enriched in E14.5 endocrine progenitors of Isl1CKO based on deconvolution analyses (Supplementary Dataset 1f). e Venn diagram representing the overlap of genes with differential expression (RNA-seq) and genes with H3K4me3 and/or H3K27me3 marks (CUT&Tag-seq) in their promoter regions (± 3 kb from the TSS) in control and Isl1CKO endocrine cells at P9. f Overlap between H3K4me3 and H3K27me3 modifications in the promoter regions of differentially expressed genes in control and Isl1CKO endocrine cells. g Heatmap shows the selection of differentially expressed genes identified by RNA-seq (average expression level per control and Isl1CKO ) correlating to differences in H3K4me3 and H3K27me3 modification patterns at their promoter regions (± 3 kb from the TSS) between control and Isl1CKO pancreatic endocrine cells at P9. h Genome track view of representative gene loci showing H3K4me3 (green), H3K27me3 (magenta), and negative antibody control IgG (grey) normalized read peaks based on CUT&Tag-seq data. The presence of both marks at the promoter region, representing a poised state, is shown in control cells for Fev, Chga, Chgb, and Abat genes associated with endocrine progenitors. In contrast, a poised state is shown at promoter regions of genes Pyy (α cells); Celsr1, and Glp1r (markers of ß cells) in Isl1CKO but not control cells. A silencing H3K27me3 mark is located at the promoter region of a key ß-cell maturation marker Ucn3 only in Isl1CKO cells

Article Snippet: CUT&Tag validated primary antibodies anti-H3K4me3 (Active Motif, #39,159, 1:100), Anti-H3K27me3 (Active Motif, #39155, 1:100), anti-ISL1 (Developmental Hybridoma Bank, #39.4D5, 1:50), normal rabbit IgG negative control (EpiCypher, #13–0042, 0.5 µg/reaction), anti-mouse secondary antibody (EpiCypher, #13–0048, 0.5 µg/reaction), and anti-rabbit secondary antibody (EpiCypher, #13-0047, 0.5 µg/reaction).

Techniques: Expressing, RNA Sequencing Assay, Comparison, Control, Selection, Modification, Marker

Journal: Cell & Bioscience

Article Title: ISL1 controls pancreatic alpha cell fate and beta cell maturation

doi: 10.1186/s13578-023-01003-9

Figure Lengend Snippet: ISL1 binds regulatory elements of critical developmental genes and reprograms the H3K27me3 landscape in endocrine cells. a Pie chart showing genomic distribution of ISL1 loci. b Venn diagram indicating overlap of ISL1-loci, and of distinctive H3K27me3 loci from pairwise comparisons control and Isl1CKO endocrine cells at E14.5 in promoter regions (Additional file : Dataset S2). c Sequence logos of the significantly enriched motifs against ISL1 peaks (2 kb areas) from Homer FindMotifsGenome analysis. Percent of target sites in ISL1 peaks is indicated. d Venn diagrams illustrating intersection of ISL1-bound genes, genes with differential H3K27me3 depositions from pairwise comparisons of control and Isl1CKO endocrine cells at E14.5, and differentially expressed genes at E14.5 identified from RNA-seq data. e Sequence logos of the significantly enriched motifs against ISL1 peaks containing H3K27me3 peaks (2 kb areas) from Homer FindMotifsGenome analysis. f Genome track view of representative gene loci showing ISL1 and H3K27me3 normalized read peaks based on CUT&Tag-seq data from E14.5 endocrine cells (an arrow indicates a TSS). The presence of both marks at the promoter region indicates overlapping occupancy of silencing methylation signature of H3K27me3 and ISL1. g Venn diagrams indicating overlap of ISL1-bound genes and genes with distinctive H3K27me3 depositions from pairwise comparisons P9 control and Isl1CKO endocrine cells, and differentially expressed genes identified from RNA-seq data at P9 (Additional file : Dataset S3). h and i Genome track view of representative gene loci showing ISL1 and H3K27me3 normalized read peaks based on CUT&Tag-seq data. The presence of both marks at the promoter region indicates overlapping occupancy of silencing methylation signature of H3K27me3 and ISL1 in upregulated (h) and downregulated (i) genes. Samples used for comparison: ISL1-CUT&Tag-seq E14.5 endocrine cells, and H3K27me3-CUT&Tag-seq P9 endocrine cells

Article Snippet: CUT&Tag validated primary antibodies anti-H3K4me3 (Active Motif, #39,159, 1:100), Anti-H3K27me3 (Active Motif, #39155, 1:100), anti-ISL1 (Developmental Hybridoma Bank, #39.4D5, 1:50), normal rabbit IgG negative control (EpiCypher, #13–0042, 0.5 µg/reaction), anti-mouse secondary antibody (EpiCypher, #13–0048, 0.5 µg/reaction), and anti-rabbit secondary antibody (EpiCypher, #13-0047, 0.5 µg/reaction).

Techniques: Control, Sequencing, RNA Sequencing Assay, Methylation, Comparison

Journal: Cell & Bioscience

Article Title: ISL1 controls pancreatic alpha cell fate and beta cell maturation

doi: 10.1186/s13578-023-01003-9

Figure Lengend Snippet: Schematics of changes induced by the elimination of Isl1 in pancreas development

Article Snippet: CUT&Tag validated primary antibodies anti-H3K4me3 (Active Motif, #39,159, 1:100), Anti-H3K27me3 (Active Motif, #39155, 1:100), anti-ISL1 (Developmental Hybridoma Bank, #39.4D5, 1:50), normal rabbit IgG negative control (EpiCypher, #13–0042, 0.5 µg/reaction), anti-mouse secondary antibody (EpiCypher, #13–0048, 0.5 µg/reaction), and anti-rabbit secondary antibody (EpiCypher, #13-0047, 0.5 µg/reaction).

Techniques:

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: eEF2K expression and activity in PD brain. a Quantitation of p-eEF2 (T56) IHC (3,3′-Diaminobenzidine-DAB staining) in postmortem hippocampus- Hip (CA1 and CA2 fields) and midbrain- MB (SN-substantia nigra, and PAG-peri-aqueductal gray matter) sections from 3 control and 6 PD cases (Additional file : Table S1; counts from at least 6 high power fields from each control or PD section; Mann–Whitney test, * p < 0.05, *** p < 0.005; error bars indicate Mean ± S.D.). b - d eEF2K mRNA expression in control and PD striatum ( b ), medial substantia nigra ( b ) and dorsal nucleus of vagus nerve ( d ). The following publicly available transcriptomic profile datasets were analyzed on the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) platform: Striatum ( b )- dataset GEO accession # GSE28894, Illumina human Ref-8 v2.0 expression beadchip platform, probe ID ILMN_1789171, controls n = 15 and PD n = 15; Medial substantia nigra ( c )- dataset GEO accession # GSE8397, Affymetrix Human Genome U133B Array, probe ID 225546_at, controls n = 8 and PD n = 15; Dorsal nucleus of vagus ( d )- dataset GEO accession # GSE43490, Agilent-014850 Whole Human Genome Microarray, probe ID A_24_P716162, controls n = 6 and PD n = 7. (Mann–Whitney test, * p < 0.05; error bars in 3b-d indicate Mean ± S.D.). e Relative eEF2K mRNA expression in human iPSCs derived cultured midbrain control (WT, n = 3) or A53T ( n = 2) mutation carrying organoids (T-test, * p < 0.05; 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: Expressing, Activity Assay, Quantitation Assay, Staining, Control, MANN-WHITNEY, Gene Expression, Microarray, Derivative Assay, Cell Culture, Mutagenesis

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: Brain eEF2K expression and activity in transgenic M83 +/+ PD mice. a eEF2K mRNA levels in whole brain homogenates from transgenic M83 +/+ PD mice intramuscularly (IM) injected bilaterally with phosphate buffered saline (PBS, n = 10) or pre-formed fibrillar (PFF, n = 13) mouse wild type AS. (Mann–Whitney test, *** p < 0.005; error bars indicate Mean ± S.D.). b - c Western blot analysis of p-eEF2 (T56) and p-ASyn (S129) in whole brain homogenates from transgenic M83 +/+ PD mice intramuscularly (IM) injected bilaterally with phosphate buffered saline (PBS) or pre-formed fibrillar (PFF) mouse wild type AS ( b ), and corresponding densitometry analysis ( c ) ( n = 7/group; Mann–Whitney test, * p < 0.05, *** p < 0.005; 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: Expressing, Activity Assay, Transgenic Assay, Injection, Saline, MANN-WHITNEY, Western Blot

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

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 mitochondrial dysfunction and oxidative stress induced by human AS in differentiated N2A cells. a - b Measurements of basal oxygen consumption rate-OCR ( b ) and ATP levels ( c ) 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; Unpaired T-test, * p < 0.05, ** p < 0.01, *** p < 0.005; error bars indicate Mean ± S.D.). c Flow cytometry analysis of reactive oxygen species (ROS), measured by DCFDA staining, in N2A cells subsequent to transient overexpression of human wild type or mutant A53T AS, with or without siRNA mediated eEF2K knockdown ( n = 9/group from three independent experiments; Unpaired T-test, * p < 0.05, ** p < 0.01, *** p < 0.005; 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, Over Expression, Mutagenesis, Knockdown, Flow Cytometry, Staining

Journal: Clinical Epigenetics

Article Title: EZH1 deficiency promotes ferroptosis resistance by activating NRF2 in sepsis-associated liver injury

doi: 10.1186/s13148-025-01892-2

Figure Lengend Snippet: DS3201 improves LPS-induced ferroptosis in liver cells via NRF2. A The structure of DS3201 . B Cell viability by CCK8. C The inhibitory effect of DS3201 on H3K27me3. D Cell morphology under phase contrast microscope in control group, LPS group, LPS + DS3201 group, LPS + DS3201 + ML385 group, magnification: 200x. E CCK8 detected the cell viability of the four groups. F Flow cytometry DCFH-DA staining was used to detect intracellular ROS in the four groups. G Flow cytometry 7-AAD was used to detect the cell viability of the four groups. H Flow cytometry JC-1 staining was used to detect mitochondrial membrane damage in four groups of cells. I Flow cytometry C11 BODIPY staining detected intracellular lipid peroxides in the four groups. N = 3 for each group, * P < 0.05, * * P < 0.01, * * * P < 0.001

Article Snippet: Hepatocytes were treated with Erastin (HY-15763, MCE), LPS O111:B4 (32,160,405, Sigma-Aldrich), Ferrostatin-1 (Fer-1) (HY-15763, MCE), DS3201 (HY-109108, MCE), and ML385 (HY-100523, MCE).

Techniques: Microscopy, Control, Flow Cytometry, Staining, Membrane

Journal: Clinical Epigenetics

Article Title: EZH1 deficiency promotes ferroptosis resistance by activating NRF2 in sepsis-associated liver injury

doi: 10.1186/s13148-025-01892-2

Figure Lengend Snippet: EZH1 inhibitor DS3201 increases the expression and nuclear translocation of NRF2. A The transcription of Gpx4 , Slc7a11 , and Cox2 in the control group, LPS-treated group, LPS + DS3201 group, and LPS + DS3201 + ML385 group was detected using qRT PCR. B The expression of GPX4, xCT, and COX2 in the whole proteins of the four groups. C The four groups’ relative expression of GPX4, xCT, and COX2 in the whole cell proteins. D NRF2 protein levels in the whole cell proteins. E The expression level of NRF2 in the cytoplasm of four groups relative to β-actin. F NRF2 and H3K27me3 proteins in the liver nuclei of four groups. G The expression levels of NRF2 and H3K27me3 in the liver nuclei relative to Histone H3. N = 3 for each group, ns: no significant difference, * P < 0.05, * * P < 0.01, * * * P < 0.001

Article Snippet: Hepatocytes were treated with Erastin (HY-15763, MCE), LPS O111:B4 (32,160,405, Sigma-Aldrich), Ferrostatin-1 (Fer-1) (HY-15763, MCE), DS3201 (HY-109108, MCE), and ML385 (HY-100523, MCE).

Techniques: Expressing, Translocation Assay, Control, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: Upregulation of sphingosine-1-phosphate receptor 3 on fibroblast-like synoviocytes is associated with the development of collagen-induced arthritis via increased interleukin-6 production

doi: 10.1371/journal.pone.0218090

Figure Lengend Snippet: (A) Development of collagen-induced arthritis in WT and S1P 3 knockout (S1P 3 -KO) mice. Arthritis was evaluated three times per week during Days 21–42. Disease severity was measured by assessing the clinical score of all four paws, in which the maximum possible score for an individual mouse was 16. Statistical analysis was performed at Days 36, 38, 40, and 42. Results are presented as the mean ± SEM (n = 20 mice per group) and were analyzed by t -test with Bonferroni’s correction. * P < 0.05 and ** P < 0.01 (WT vs. KO mice). (B) Synovial inflammation, bone erosion, and cartilage destruction were examined histologically on H&E-stained sections of ankle joints and assigned scores of 1–4. Results are presented as box-and-whisker plots (n = 20 mice per group) and were analyzed by Mann-Whitney U-test. The middle hash, the box, and the whiskers represent the median, interquartile range (IQR), and 10/90 percentile values respectively. * P < 0.05, NS = not significant. (C) Representative images of H&E-stained ankle joint sections are shown at a magnification of 40×. Left panel: WT specimen assigned a score of 3 for all parameters. Right panel: KO specimen assigned a score of 1 for inflammation and 0 for the other parameters. (D) Anti-CII IgG 1 and IgG 2 antibodies in serum from CIA mice were measured in an ELISA. Results are presented as box-and-whisker plots (n = 10 mice per group) and were analyzed by Mann-Whitney U-test. The middle hash, the box, and the whiskers represent the median, IQR, and minimum/maximum values respectively. (E) Expression of TNFα and IL-6 in arthritic synovium was analyzed by real-time RT-PCR. Results are presented as the mean ± SEM (n = 6 mice per group) and were analyzed by t -test. * P < 0.05, NS = not significant.

Article Snippet: Levels of anti-CII IgG 1 and IgG 2 were measured using a Mouse Anti-type II Collagen IgG Subtype Antibody ELISA Kit (Chondrex), according to the manufacturer’s instructions.

Techniques: Knock-Out, Staining, Whisker Assay, MANN-WHITNEY, Enzyme-linked Immunosorbent Assay, Expressing, Quantitative RT-PCR

Journal: Cells

Article Title: Mitochondrial ROS–ER Stress Axis Governs IL-10 Production in Neutrophils and Regulates Inflammation in Murine Chlamydia pneumoniae Lung Infection

doi: 10.3390/cells14191523

Figure Lengend Snippet: Live C. pneumoniae infection in mouse neutrophils elicits strong IL-10 production via TLR2. ( A ) Neutrophils were infected with C. pneumoniae (strain AR-39) at MOI = 1. mRNA was isolated from neutrophils after 24 h of infection. Expression of the indicated gene was confirmed by real-time PCR. ( B ) IL-10 levels in the supernatant were measured by ELISA. The supernatant from neutrophils infected with AR-39 at the specified MOI was collected after 24 h. ( C ) Neutrophils were infected with live or heat-killed C. pneumoniae strain AR-39 or strain CWL-029 at MOI = 1. The culture medium was collected 24 h post-infection. IL-10 in the medium was measured by ELISA. ( D ) Neutrophils from WT mice were infected with MOI = 1 AR-39 and cultured with TLR2 inhibitor C29 (100 μM), TLR4 inhibitor TAK-242 (1 μM), or TLR9 inhibitor E6446 (500 nM). Culture medium was collected 24 h after infection, and IL-10 levels were measured by ELISA. ( E ) Neutrophils from WT mice were infected with MOI = 1 AR-39 and co-cultured with or without TLR2 inhibitor C29 (100 μM) for 24 h, then stained with FITC-conjugated anti-chlamydia antibody. The bar length was 10 μm. ( F ) Neutrophils infected with MOI = 1 AR-39 were cultured with TLR2 inhibitor C29. After 24 h, total RNA was isolated, and C. pneumoniae 16S ribosomal RNA was quantified by real-time PCR. ( G ) Neutrophils from WT and TLR2 −/− mice were infected with MOI = 1 AR-39. Culture supernatants were collected after 24 h, and IL-10 levels were measured via ELISA. Statistical analyses were performed using an unpaired two-tailed Student’s t -test ( A , F ), a one-way ANOVA followed by Tukey’s multiple comparison test ( B , D ), and a two-way ANOVA followed by Tukey’s multiple comparison test ( C , G ). * p < 0.05, ** p < 0.01, and **** p < 0.0001, Each experiment was conducted at least three times with n ≥ 3, and the data presented are representative of these results. Cpn: C. pneumoniae .

Article Snippet: C. pneumoniae (strain: AR-39, ATCC 53592, and strain: CWL-029, ATCC VR-1310) and Chlamydia muridarum mouse pneumonitis (MoPn) (strain: Nigg II, ATCC VR-123) were purchased from the American Type Culture Collection.

Techniques: Infection, Isolation, Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Cell Culture, Staining, Two Tailed Test, Comparison

Journal: Cells

Article Title: Mitochondrial ROS–ER Stress Axis Governs IL-10 Production in Neutrophils and Regulates Inflammation in Murine Chlamydia pneumoniae Lung Infection

doi: 10.3390/cells14191523

Figure Lengend Snippet: C. pneumoniae infection-induced mitochondrial ROS–ER stress–IRE1α sensor pathway activation leads to IL-10 production by murine neutrophils. B/M-derived neutrophils were infected with C. pneumoniae for 6 h, stained with MitoRed and MitoGreen ( A ) or with MitoSOX ( B ), and analyzed by flow cytometry. ( C ) ER stress-related genes in neutrophils at 12 h post-infection (h.p.i.) with mock or C. pneumoniae were analyzed by quantitative RT-PCR. ( D ) The protein of Bip or IRE1α, expressed in the mock or C. pneumoniae -infected neutrophils at an indicated time point, was assayed by Western blot. ( E , F ) Mock or C. pneumoniae -infected neutrophils were treated with the mtROS inducer Rotenone or the ER stress inhibitor 4-PBA. Neutrophils were collected at 12 h.p.i. and stained with MitoSOX, then analyzed by flow cytometry. ( G ) Mock or C. pneumoniae -infected neutrophils were treated with or without MitoTEMPO for 12 h. Bip and IRE1α proteins were detected by Western blot. ( H ) Mock or C. pneumoniae -infected neutrophils were treated with ER stress inhibitor 4μ8C or MitoTEMPO. mRNA of neutrophils was isolated at 12 h.p.i. and translated to cDNA. Xbp1 was amplified by PCR and then digested with PstI enzyme for 2 h. The ratio of s Xbp1 to total Xbp1 was normalized to the control C. pneumoniae -infected neutrophils. ( I , J ) Mock or C. pneumoniae -infected neutrophils were treated with mtROS scavenger MitoTEMPO or IRE1α inhibitor 4μ8C. Neutrophils were collected at 12 h.p.i., stained with XBP-1S antibody, and then analyzed by flow cytometry. ( K ) Neutrophils infected with C. pneumoniae were treated with the indicated dose of MitoTEMPO. Culture medium was collected 24 hrs after infection. TNFα and IL-10 in the culture medium were measured by ELISA. ( L , M ) Neutrophils infected with C. pneumoniae were treated with ER stress inhibitor 4-PBA (L) or 4μ8C ( M ); culture medium was collected 24 h after infection. IL-10 in the culture medium was measured by ELISA. Statistical analyses were performed using a two-way ANOVA followed by Tukey’s multiple comparison test ( A , B ), an unpaired two-tailed Student’s t -test ( C ), and a one-way ANOVA followed by Tukey’s multiple comparison test ( F , J – M ). * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. Every experiment was conducted with n ≥ 3 and repeated at least three times, and the experiment shown is representative. Cpn: C. pneumoniae .

Article Snippet: C. pneumoniae (strain: AR-39, ATCC 53592, and strain: CWL-029, ATCC VR-1310) and Chlamydia muridarum mouse pneumonitis (MoPn) (strain: Nigg II, ATCC VR-123) were purchased from the American Type Culture Collection.

Techniques: Infection, Activation Assay, Derivative Assay, Staining, Flow Cytometry, Quantitative RT-PCR, Western Blot, Isolation, Amplification, Control, Enzyme-linked Immunosorbent Assay, Comparison, Two Tailed Test

Journal: Cells

Article Title: Mitochondrial ROS–ER Stress Axis Governs IL-10 Production in Neutrophils and Regulates Inflammation in Murine Chlamydia pneumoniae Lung Infection

doi: 10.3390/cells14191523

Figure Lengend Snippet: In vivo, neutrophil-derived IL-10 shapes immune responses in the lungs of mice infected with C. pneumoniae. ( A – C ) C57BL/6J (WT) mice, WT mice treated with 1A8 mAb, or C57BL/6J TLR2 −/− mice were intranasally infected with C. pneumoniae . Bronchoalveolar lavage fluid (BALF) was collected from infected mice at the indicated days after infection. IL-10 ( A ), IL-12p70 ( B ), and IL-6 ( C ) in the BALF were measured by ELISA. ( D – F ) WT mice, 1A8 mAb-treated WT mice, or TLR2 −/− mice were nasally infected with C. pneumoniae . Infiltrated leukocytes in the lungs were isolated at the indicated days after infection and analyzed by flow cytometry. MFI of CD206 on macrophages ( D ), percentage of CD4 + Foxp3 + cells within CD4 + cells ( E ), and percentage of NK1.1 + IFNγ + cells within NK1.1 + cells ( F ). ( G ) C57BL/6J mice, C57BL/6J mice treated with 1A8 abs, or TLR2 −/− mice were nasally infected with C. pneumoniae . Lungs were collected on the indicated days after infection. The infectious burden of C. pneumoniae in the lungs was measured by IFU assay. ( H ) C57BL/6J mice or PK136 abs-treated C57BL/6J mice were intranasally infected with C. pneumoniae. IFU of C. pneumoniae in the lungs was measured. ( I , J ) C57BL/6J mice were nasally infected with C. pneumoniae and treated with solvent or 4-PBA. BALF was collected from infected mice on the indicated days after infection. IL-10 ( I ) and IL-12p70 ( J ) in the BALF were measured by ELISA. Statistical analyses were performed using a two-way ANOVA followed by Tukey’s multiple comparison test ( A – G , I , J ) and an unpaired two-tailed Student’s t -test ( H ). * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. Every experiment was performed with at least three mice and repeated at least three times, and the experiment shown is representative. Cpn: C. pneumoniae .

Article Snippet: C. pneumoniae (strain: AR-39, ATCC 53592, and strain: CWL-029, ATCC VR-1310) and Chlamydia muridarum mouse pneumonitis (MoPn) (strain: Nigg II, ATCC VR-123) were purchased from the American Type Culture Collection.

Techniques: In Vivo, Derivative Assay, Infection, Enzyme-linked Immunosorbent Assay, Isolation, Flow Cytometry, Solvent, Comparison, Two Tailed Test

Journal: Cells

Article Title: Mitochondrial ROS–ER Stress Axis Governs IL-10 Production in Neutrophils and Regulates Inflammation in Murine Chlamydia pneumoniae Lung Infection

doi: 10.3390/cells14191523

Figure Lengend Snippet: In vivo, neutrophil-derived IL-10 reduces lung tissue damage during C. pneumoniae lung infection. ( A – G ) Mice treated with isotype-matched Abs or Ly6G-specific 1A8 Abs were intranasally infected with C. pneumoniae . ( A ) Lung tissues on days 0, 3, and 7 after infection were stained with anti-IL-10 mAb (brown). The scale bar indicates 50 µm. ( B ) The proportion of IL-10-positive staining area was analyzed using ImageJ software. ( C ) Lung tissues on day 3 post-infection were double-stained with anti-IL-10 (brown) and chloroacetate esterase (red) for identifying IL-10-producing neutrophils. The scale bar indicates 50 µm. ( D , E ) H&E staining of the lung tissues on days 3 and 7 after infection and the pathological scores of the lungs from mice treated with isotype-matched control Abs or 1A8 Abs after C. pneumoniae lung infection. The scale bar indicates 1000 µm. ( F ) Mice treated with isotype Abs or 1A8 Abs were then infected with C. pneumoniae . Survival (n = 6) was measured at the indicated days. Statistical analyses were performed using a two-way ANOVA followed by Tukey’s multiple comparison test ( B , E ) and the log-rank test ( F ). * p < 0.05, ** p < 0.01, and **** p < 0.0001. Experiments ( F ) were repeated three times, and the presented experiment is representative. Cpn: C. pneumoniae .

Article Snippet: C. pneumoniae (strain: AR-39, ATCC 53592, and strain: CWL-029, ATCC VR-1310) and Chlamydia muridarum mouse pneumonitis (MoPn) (strain: Nigg II, ATCC VR-123) were purchased from the American Type Culture Collection.

Techniques: In Vivo, Derivative Assay, Infection, Staining, Software, Control, Comparison

Journal: Cells

Article Title: Mitochondrial ROS–ER Stress Axis Governs IL-10 Production in Neutrophils and Regulates Inflammation in Murine Chlamydia pneumoniae Lung Infection

doi: 10.3390/cells14191523

Figure Lengend Snippet: Induction of IL-10 in neutrophils and their roles in lung infection with C. pneumoniae . The induction of IL-10 in C. pneumoniae -infected neutrophils depends on the activation of the TLR2–mtROS–ER stress axis. IL-10 produced by neutrophils early in infection suppresses IL-12 p70 production from macrophages and/or conventional DCs and inhibits Natural Killer (NK) cell activation, while promoting regulatory T cells and M2-like macrophages. These IL-10-producing neutrophils are crucial for maintaining immune balance during C. pneumoniae infection in the lungs. Blocking C. pneumoniae infection-induced IL-10 production from neutrophils through neutrophil depletion (1A8 Ab treatment) or in TLR2 −/− mice enhances immune responses against C. pneumoniae and reduces bacterial burden in the lungs. However, this heightened immune response can also cause excessive inflammation and tissue damage, leading to severe mortality. The symbols +, ++, and +++ indicate the relative degree (low, moderate, and high, respectively) of the various outcomes. Created in BioRender. Ozuru, R. (2025) https://BioRender.com/fzq2h4y .

Article Snippet: C. pneumoniae (strain: AR-39, ATCC 53592, and strain: CWL-029, ATCC VR-1310) and Chlamydia muridarum mouse pneumonitis (MoPn) (strain: Nigg II, ATCC VR-123) were purchased from the American Type Culture Collection.

Techniques: Infection, Activation Assay, Produced, Blocking Assay

Journal:

Article Title: IL-15 enhances the in vivo antitumor activity of tumor-reactive CD8 + T Cells

doi: 10.1073/pnas.0307298101

Figure Lengend Snippet: pmel-1 splenocytes differentially polarize either to a TEM-orTCM-like population when cultured in IL-2 or IL-15, respectively. Splenocytes from a pmel-1 Tg mouse were primed with 1 μM hgp10025–33 peptide and cultured in culture media containing 10 ng/ml rhIL-2 or 10 ng/ml rhIL-15. On d 7, FACS analysis for surface expression of the activation markers CD25, CD44, and CD69 and the lymph node homing markers CD62L and CCR7 was performed. Naïve pmel-1 cells were obtained from splenocytes of an age-matched littermate. Results from a representative experiment are shown after gating on propidium iodide-negative and CD8+ T cells.

Article Snippet: The gp100 – tumor lines EL-4 (American Type Culture Collection) and methylcholanthrene 205 (MCA-205; National Cancer Institute Tumor Repository, Bethesda) were maintained in culture media and used as irrelevant H-2 b targets.

Techniques: Cell Culture, Expressing, Activation Assay

Journal:

Article Title: IL-15 enhances the in vivo antitumor activity of tumor-reactive CD8 + T Cells

doi: 10.1073/pnas.0307298101

Figure Lengend Snippet: Tumor/self-reactive CD8+ T cells cultured in IL-15 or IL-2 polarize to two functionally distinct populations with unique in vitro and in vivo functions. IFN-γ (A), TNF-α (B), IL-10 (C), and IL-2 (D) production by CD8-enriched pmel-1 splenocytes (▴), pmelIL2 (○), and pmelIL15 (▪) cells cocultured for 24 h with EL-4 pulsed with titrated doses of hgp10025–33 peptide. Data shown are representative of at least two independent coculture experiments. (E) Quantitative RT-PCR for IL-2 mRNA in CD8-enriched pmel-1 splenocytes (gray bar), pmelIL2 (open bar), and pmelIL15 cells (black bar) after a 6-h coculture with EL-4 targets pulsed with 1 × 10–7 M hgp10025–33. Data shown are representative of three independent experiments. (F) pmelIL15 cells at d 8 are less cytolytic than pmelIL2 cells directed against hgp10025–33 pulsed MCA-205 targets. Data shown are representative of three independent cytolytic assays. (G) Absolute number of adoptively transferred cells on d 6 in the blood of tumor-bearing, sublethally irradiated WT mice from groups treated with rFPhgp100, IL-2 (36 μg per dose), and 1 × 106 pmelIL2 cells (open bar) or pmelIL15 cells (filled bar). Data shown are mean ± SEM of three mice per group and represent the global maximum in cell number for groups receiving pmelIL2 and pmelIL15 cells. Similar results were also observed in the spleens of treated animals (data not shown). This experiment was performed twice with similar results.

Article Snippet: The gp100 – tumor lines EL-4 (American Type Culture Collection) and methylcholanthrene 205 (MCA-205; National Cancer Institute Tumor Repository, Bethesda) were maintained in culture media and used as irrelevant H-2 b targets.

Techniques: Cell Culture, In Vitro, In Vivo, Quantitative RT-PCR, Irradiation

Journal:

Article Title: IL-15 enhances the in vivo antitumor activity of tumor-reactive CD8 + T Cells

doi: 10.1073/pnas.0307298101

Figure Lengend Snippet: IL-15-cultured tumor-reactive CD8+ T cells treat established B16 melanoma with enhanced efficacy compared with IL-2-cultured cells. WT mice bearing 14-d established s.c. B16 tumors were sublethally irradiated and either were left untreated as controls (⋄) or received adoptive transfer of 1 × 106 pmel-1 cells cultured in 10 ng/ml rhIL-2 (•) or 10 ng/ml rhIL-15 (▪), rFPhgp100 vaccination, and exogenous IL-2 (36 μg per dose). Tumor area (A) and survival (B) in mice receiving pmelIL2 or pmelIL15 cells, rFPhgp100 vaccination, and exogenous IL-2 compared with untreated controls. Data shown are representative of three independent experiments.

Article Snippet: The gp100 – tumor lines EL-4 (American Type Culture Collection) and methylcholanthrene 205 (MCA-205; National Cancer Institute Tumor Repository, Bethesda) were maintained in culture media and used as irrelevant H-2 b targets.

Techniques: Cell Culture, Irradiation, Adoptive Transfer Assay

Journal:

Article Title: IL-15 enhances the in vivo antitumor activity of tumor-reactive CD8 + T Cells

doi: 10.1073/pnas.0307298101

Figure Lengend Snippet: Forced expression of IL-15 by adoptively transferred CD8+ T cells enhances their in vivo antitumor function. (A) FACS analysis for surface expression of CD122 (IL-2 and IL-15Rβ) and CD44 on CD8+ T cells derived from a pmel-IL-15 double Tg mouse or an age-matched pmel-1 single Tg littermate. Percentages shown represent the percentage of gated cells within the respective quadrant. Results from one representative experiment are shown after gating on propidium iodide-negative and CD8+ T cells. (B) WT mice were inoculated with s.c. B16 melanoma, and 14 d later they received adoptive transfer of 5 × 106 pmel-1 or pmel-IL-15 splenocytes, rFPhgp100 vaccination, and exogenous IL-2 (36 μg per dose) or were left untreated as controls. In addition to tumor size, groups that received pmel-IL-15 splenocytes, rFPhgp100, and IL-2 survived significantly longer (P = 0.0016) compared with groups that received pmel-1 splenocytes, rFPhgp100, and IL-2 (data not shown). Data shown are representative of four independent experiments.

Article Snippet: The gp100 – tumor lines EL-4 (American Type Culture Collection) and methylcholanthrene 205 (MCA-205; National Cancer Institute Tumor Repository, Bethesda) were maintained in culture media and used as irrelevant H-2 b targets.

Techniques: Expressing, In Vivo, Derivative Assay, Adoptive Transfer Assay

Journal: Stem Cell Research & Therapy

Article Title: Mesenchymal stem cells combined with liraglutide relieve acute lung injury through apoptotic signaling restrained by PKA/β-catenin

doi: 10.1186/s13287-020-01689-5

Figure Lengend Snippet: Liraglutide reduces apoptosis of hCMSCs via PKA/β-catenin pathway. a Western blot and b RT-qPCR verify the knockdown effects of three Si-GLP-1R in hCMSCs. c , d Western blot was used to detect of β-catenin and p-β-catenin expression under the stimulation of LPS by adding 20 μM H89 or 100 nM Si-GLP-1R and liraglutide. e The expression of apoptotic proteins Bax, Bcl-2, cleaved caspase-9, and cleaved caspase-3 was detected by western blot with PKA inhibitor H89 and liraglutide. f The expression of GLP-1R and apoptotic proteins Bax, Bcl-2, cleaved caspase-9, and cleaved caspase-3 were detected by western blot with Si-GLP-1R and liraglutide. Error bars represent mean ± SD from three independent experiments. Compared with Si-con group, *** P < 0.001

Article Snippet: To verify the PKA/β-catenin pathway, MSCs were exposed to 10 nM liraglutide or 100 nM SiRNA or 20 μM PKA inhibitor H89 (MCE, Cat. No. 130964-39-5, USA) on the premise of exposure to 30 μg/mL LPS.

Techniques: Western Blot, Quantitative RT-PCR, Knockdown, Expressing

Journal: Nucleic Acids Research

Article Title: The Drosophila MLR COMPASS complex is essential for programming cis -regulatory information and maintaining epigenetic memory during development

doi: 10.1093/nar/gkaa082

Figure Lengend Snippet: MLR regulates hormone-dependent enhancer activation and repression during development. ( A ) Cmi was depleted ( GawB69B-Gal4>cmi-IR) and transcripts analyzed by RNAseq. Stages include W3L blue gut (BG; pre-ecdysone) and clear gut (CG; elevated ecdysone). Image modified from . ( B ) Venn diagram comparing changes in gene expression associated with cmi knockdown at the BG and CG stages. The analysis identified genes showing increased expression ( P -value ≤ 0.05, fold change ≥ 1.3) compared to wild type ( GawB69B-Gal4 ) at the BG stage that subsequently show reduced expression at the CG stage. ( C ) GO enrichment of the 336 genes shown in (B) filtered for RPKM values of ≥5 in wild type animals at each stage. GO groups are listed in descending order of P -value. ( D ) Cmi represses hormone responsive genes prior to activation. Ecdysone responsive genes show elevated BG expression upon cmi knockdown compared to wild type. Activation of these genes at the CG stage is attenuated when cmi is depleted. ( E ) Enrichment peak comparison of Cmi and H3K4me1 (whole W3L) with Nipped-B (wing discs). Co-localized peaks are highlighted in blue. ( F ) shRNAi depletion of Nipped-B results in derepression of Eip74EF at the BG stage. ( G ) Depletion of Cmi leads to increased RNAP II in the Eip74EF 5′ region ( E74A promoter). ( H ) Two-stage model of MLR functions in enhancer activation and enhancer memory. (Left) Enhancer activation is associated with recruitment of the MLR complex to chromatin by sequence-specific transcription factors, monomethylation of H3K4 by Trr, recruitment or stabilization of CBP/p300 by Cmi and acetylation of H3K27. (Right) Previously active enhancers are maintained in an inactive or silenced (poised) state, stabilized by cohesin complexes and retaining the MLR complex in the absence of H3K27ac. See text for additional details.

Article Snippet: Rabbit αH3K27ac (39 133), αH3K27me3 (39 155), αH3K4me1 (39297) and αH3K4me3 (39 915) were from Active Motif.

Techniques: Activation Assay, Modification, Gene Expression, Knockdown, Expressing, Comparison, Sequencing

Journal: Nucleic Acids Research

Article Title: The Drosophila MLR COMPASS complex is essential for programming cis -regulatory information and maintaining epigenetic memory during development

doi: 10.1093/nar/gkaa082

Figure Lengend Snippet: Cmi is enriched on active and inactive enhancers. ChIP-seq analyses of Cmi in Drosophila S2 cells. ( A ) Cmi is significantly associated with tissue-specific (red) and constitutively active genes (yellow) . ( B ) Cmi enrichment near transcription start sites (H3K4me2/me3, H3K9ac;State 1) and enhancers (H3K4me1, H3K18ac and H3K27ac;State 3) . ( C ) Cmi is highly enriched on active/open enhancers in multiple tissues (S2 and OSC cells) and tissue-specific enhancers based on STARR-seq analyses. ( D ) Cmi shows developmental stage-specific genome enrichments, with strong overlap between W3L and WPP and lower overlap between embryos (E0–8 h) and S2 cells. ( E ) Genes near W3L and WPP-specific binding regions are enriched for roles in metamorphosis and ecdysone-regulated processes. ( F ) Late stage (W3L and WPP) binding regions significantly overlap with ecdysone-induced enhancers.

Article Snippet: Rabbit αH3K27ac (39 133), αH3K27me3 (39 155), αH3K4me1 (39297) and αH3K4me3 (39 915) were from Active Motif.

Techniques: ChIP-sequencing, Binding Assay

Journal: Nucleic Acids Research

Article Title: The Drosophila MLR COMPASS complex is essential for programming cis -regulatory information and maintaining epigenetic memory during development

doi: 10.1093/nar/gkaa082

Figure Lengend Snippet: Cmi regulates ecdysone-dependent gene activation. ( A ) Venn diagram depiction of Cmi localization during development. ChIP enrichment peaks were compared between stages, with strongest overlap between the W3L and WPP stages. The majority of embryonic peaks (E0–8hrs) were not present at the later stages. ( B ) Developmentally dynamic enrichment of Cmi at multiple locations within the Eip74EF locus. Embryo 0–8 h (E0–8), S2 cells, wandering third instar larvae (W3L) and white prepupae (WPP). ( C ) Eip74EF transcription scaled to maximum expression (modENCODE) coincides with ecdysone titers in embryos and late L3 larvae. ( D ) Eip74EF activation is reduced following Cmi or Trr shRNAi depletion during late larval development. RT-qPCR in wild type ( GawB69B-Gal4 ) and knockdown of cmi and trr at the blue gut (BG), clear gut (CG), white prepupal (WPP) and early pupal (EP) stages (see Figure for staging). Shown is the fold induction relative to the BG stage for each genotype. ( E ) Fold expression of Eip74EF relative to wild type ( GawB69B-Gal4 ) at each developmental stage. ( F ) Localization of Cmi, H3K4me1, H3K27ac and RNAP II enrichments at the W3L stage. Recurrent Cmi enrichment regions (A-G) are indicated. ( G ) Cmi knockdown in the posterior (P) wing compartment ( en-Gal4>cmi-IR ) causes reductions in H3K4me1. Left panels, DAPI (top) and αCmi (bottom). Right panels, DAPI and αH3K4me1. * P -value ≤0.05 by an unpaired Student's t test.

Article Snippet: Rabbit αH3K27ac (39 133), αH3K27me3 (39 155), αH3K4me1 (39297) and αH3K4me3 (39 915) were from Active Motif.

Techniques: Activation Assay, Expressing, Quantitative RT-PCR, Knockdown

Journal: Nucleic Acids Research

Article Title: The Drosophila MLR COMPASS complex is essential for programming cis -regulatory information and maintaining epigenetic memory during development

doi: 10.1093/nar/gkaa082

Figure Lengend Snippet: The Cmi/Trr complex regulates enhancer function. ( A ) Cmi and Trr co-localize on Eip74EF enhancers in S2 cells. Ecdysone responsive enhancers (e1–e6) were mapped by STARR-seq and DHS-seq, +/− ecdysone . Histone modifications H3K4me1, H3K27ac (modENCODE), Trr and Cmi enrichments in S2 cells (regions A–G). Asterisks, positions of H3K4me3 peaks. Red bars below the Cmi track indicate locations of RNAP II. Region D contains both activation and repression functions (R/E). ( B ) Eip74EF transcription is induced by addition of 20-OH ecdysone (20-HE;1μM). ( C ) Co-localization of Cmi and Trr across the Eip74EF locus by ChIP-qPCR. Regions correspond to the major Cmi peaks (A–E). Enrichments are reduced at some locations upon addition of 20-HE for 6 h. ( D , E ) Enrichments of Cmi and Trr relative to normal S2 cells on the indicated enhancer regions following trr (D) and cmi knockdown (E). ( F–I ) Changes in H3K4me1 and H3K27ac enrichments at ecdysone responsive enhancers (e1–e6) normalized to histone H3 following cmi and trr knockdown, (+/−) 20-HE. H3K4me1 (F, G) and H3K27ac (H, I) are reduced on some enhancers following knockdown (–) 20-HE (F, H) and (+) 20-HE (G, I). * P -value ≤0.05 by an unpaired Student's t test. Error bars represent mean ± SD.

Article Snippet: Rabbit αH3K27ac (39 133), αH3K27me3 (39 155), αH3K4me1 (39297) and αH3K4me3 (39 915) were from Active Motif.

Techniques: Activation Assay, ChIP-qPCR, Knockdown

Journal: Nucleic Acids Research

Article Title: The Drosophila MLR COMPASS complex is essential for programming cis -regulatory information and maintaining epigenetic memory during development

doi: 10.1093/nar/gkaa082

Figure Lengend Snippet: Cmi is required for stable CBP association and H3K27ac of chromatin. ( A ) ChIP-seq co-localization of Cmi, CBP and H3K27ac during the W3L stage in the chromosome region containing Eip74EF and Eip75B . ( B ) Cmi interacts with CBP by co-precipitation in vivo. Soluble nuclear lysates were prepared from embryos expressing an HA epitope-tagged Cmi ( GawB69B> UAS:HA-Cmi ) and αHA precipitated proteins were probed with αCBP antibodies. ( C ) Cmi is required for normal levels of H3K27 acetylation. Depletion of cmi (GawB69B-Gal4>cmi-IR ) in W3L larvae is associated with reduced H3K27ac, but not H3K27me3. Native histone extracts were tested with αH3K27ac or αH3K27me3 by immunoblot. Controls for histone loading (0.1X to 1X) were examined using αH3. Band intensities were measured using Image J relative to the OregonR 1X level, normalized to H3 for each genotype. (D–F) Salivary gland polytene chromosomes from wild type OregonR or Sgs3-Gal4>cmi-IR knockdown larvae. Chromosomes were visualized by staining with DAPI. ( D ) CBP localization. Inset boxes show higher magnification views of chromosome 3R. ( E ) H3K27ac localization. Inset boxes are higher magnification views of the Eip74EF region. ( F ) H3K27me3 localization. Inset boxes are magnified views of chromosome 3L.

Article Snippet: Rabbit αH3K27ac (39 133), αH3K27me3 (39 155), αH3K4me1 (39297) and αH3K4me3 (39 915) were from Active Motif.

Techniques: ChIP-sequencing, In Vivo, Expressing, Western Blot, Knockdown, Staining