Journal:

Article Title: Macrorestriction Fingerprinting of " Streptococcus milleri " Group Bacteria by Pulsed-Field Gel Electrophoresis

doi:

Figure Lengend Snippet: Sources and identities of 54 infection-related SMG isolates used in this study

Article Snippet: Sources and identities of 54 infection-related SMG isolates used in this study table ft1 table-wrap mode="anchored" t5 TABLE 2 caption a7 Reference strain Species identification Clinical source 10c S. anginosus Plaque 18c S. anginosus Plaque 20c S. anginosus Plaque 28c S. intermedius Plaque 33c S. intermedius Plaque 30c S. intermedius Tongue 5′c S. constellatus Plaque 23′c S. intermedius Plaque 38c S. anginosus Tongue 41c S. anginosus Tongue 45c S. intermedius Tongue 46c S. anginosus Tongue 40c S. intermedius Tongue 42c S. anginosus Tongue 43c S. anginosus Tongue 25′c S. intermedius Plaque 69C S. intermedius Plaque 48C S. constellatus Tongue 75C S. anginosus Plaque 72C S. constellatus Tongue 35C S. intermedius Plaque 34C S. constellatus Tongue 83C S. intermedius Plaque 84C S. intermedius Plaque 57C S. constellatus Tongue 56C S. anginosus Tongue 52C S. anginosus Throat 31C S. intermedius Plaque 11C S. intermedius Tongue 10C S. anginosus Tongue 19C S. anginosus Plaque 20C S. intermedius Plaque 17C S. anginosus Tongue 16C S. anginosus Plaque 62r S. anginosus Plaque 23r S. anginosus Plaque 4dw S. intermedius Plaque Open in a separate window Sources and identities of 37 SMG commensal strains isolated from healthy sites A total of 91 clinical SMG isolates were examined in the study, including 3 type strains, S. anginosus NCTC 10713 (ATCC 12395); S. constellatus NCTC 11325 (ATCC 27823), and S. intermedius NCTC 11324 (ATCC 27335) (Table ).

Techniques: Infection

Journal: Nature Communications

Article Title: Activation of the RSAD2-YTHDF1 axis in smooth muscle causes inflammatory bowel disease via intercellular mitochondrial transfer

doi: 10.1038/s41467-025-67707-3

Figure Lengend Snippet: a Venn diagram showing the overlap between differentially expressed genes (DEGs) from RNA-seq and mitochondria-related proteins from MitoCarta 3.0. b Heatmap of 43 mitochondria-related genes in WT and Sm22α −/− mice, n = 4 mice per group. c Rsad2 mRNA levels in mouse colonic tissue, n = 7 mice per group. d , e Western blot analysis and quantification of RSAD2 protein levels in mouse colonic tissue, n = 4 mice per group. f Immunofluorescence staining of RSAD2 (red) with DAPI counterstaining (blue) in mouse colonic tissue. Scale bar, 100 μm. g Quantification of RSAD2 immunofluorescence intensity ( f ), n = 7 mice per group. h Rsad2 mRNA levels in primary CSMCs, n = 7 biological replicates. i , j Western blot analysis and quantification of RSAD2 protein in CSMCs, n = 6 biological replicates. k Immunofluorescence staining for RSAD2 (red), and mitochrondria (green) in primary CSMCs. Nuclei were counterstained with DAPI (blue). Scale bar, 25 μm. l Colocalization analysis using Pearson’s correlation coefficient from ( k ), n = 7 biological replicates. m , n siRNA-mediated knockdown of SM22α in WT CSMCs, followed by Western blot analysis and quantification of RSAD2 levels, n = 6 biological replicates. o , p Western blot analysis and quantification of RSAD2 levels in Sm22α −/− CSMCs infected with Ad-EGFP and Ad-SM22α-EGFP, n = 6 biological replicates. q , r Western blot analysis and quantification of RSAD2 levels in WT CSMCs with or without tumor necrosis factor-α (TNF) treatment, n = 6 biological replicates. Data are presented as mean ± SEM of three independent experiments. P values were determined by 2-tailed Student’s t test. Source data are provided as a file.

Article Snippet: Cyagen Biosciences (S-KO-11081) provided C57BL/6 J wild-type (WT) mice and Rsad2 −/− mice.

Techniques: RNA Sequencing, Western Blot, Immunofluorescence, Staining, Knockdown, Infection

Journal: Nature Communications

Article Title: Activation of the RSAD2-YTHDF1 axis in smooth muscle causes inflammatory bowel disease via intercellular mitochondrial transfer

doi: 10.1038/s41467-025-67707-3

Figure Lengend Snippet: a Immunofluorescence staining of mitochondria (green) in CSMCs with or without RSAD2 overexpression (OE or CON; lanes 1-2). Nuclei were counterstained with DAPI (blue). Mitochondrial superoxide levels were detected by MitoSox (red) in WT CSMCs with or without RSAD2 overexpression (lane 3). Scale bar, 25 μm. b Quantification analysis of mitochondrial morphology from confocal microscopy images ( a ), n = 15 biological replicates. c Relative mtDNA copy number in WT CSMCs with or without RSAD2 overexpression assessed by qRT-PCR, n = 6 biological replicates. d Intracellular ATP levels in CSMCs measured by ATP assay, n = 6 biological replicates. e , f Western blot analysis and quantification of DRP1 protein levels, n = 6 biological replicates. g Immunofluorescence staining of mitochondria (green) in TNF-treated WT and Rsad2 −/− CSMCs, with DAPI (blue) counterstaining (lanes 1-2). Mitochondrial superoxide levels were detected by MitoSox (red) in TNF treated WT and Rsad2 −/− CSMCs (lane 3). Scale bar, 25 μm. h Quantification analysis of mitochondrial morphology from confocal microscopy images ( g ), n = 15 biological replicates. i Relative mtDNA copy number in TNF-treated WT and Rsad2 −/− CSMCs measured by qRT-PCR, n = 6 biological replicates. j Intracellular ATP levels in TNF-treated WT and Rsad2 −/− CSMCs, n = 6 biological replicates. k , l Western blot analysis and quantification of DRP1 protein levels, n = 6 biological replicates. Data are presented as mean ± SEM of three independent experiments. P values were determined by 2-tailed Student’s t- test. Source data are provided as a file.

Article Snippet: Cyagen Biosciences (S-KO-11081) provided C57BL/6 J wild-type (WT) mice and Rsad2 −/− mice.

Techniques: Immunofluorescence, Staining, Over Expression, Confocal Microscopy, Quantitative RT-PCR, ATP Assay, Western Blot

Journal: Nature Communications

Article Title: Activation of the RSAD2-YTHDF1 axis in smooth muscle causes inflammatory bowel disease via intercellular mitochondrial transfer

doi: 10.1038/s41467-025-67707-3

Figure Lengend Snippet: a Drp1 mRNA expression in TNF-treated WT and Rsad2 −/− CSMCs, n = 6 biological replicates. b Drp1 mRNA stability in TNF-treated WT and Rsad2 −/− CSMCs following actinomycin D treatment for the indicated times, n = 5 biological replicates. c Immunoblot analysis of ubiquitinated RSAD2 in CSMCs treated with 5 μM MG132 for 6 hours; IgG served as a control. d Total m 6 A RNA levels in TNF-treated WT and Rsad2 −/− CSMCs, n = 6 biological replicates. e MeRIP-qPCR analysis of Drp1 m 6 A modification in TNF-treated WT and Rsad2 −/− CSMCs, n = 6 biological replicates. f RNA pull-down assay followed by immunoblot demonstrating RSAD2 binding to Drp1 mRNA. g MeRIP-qPCR analysis of Drp1 m 6 A levels, n = 5 biological replicates. h MeRIP-qPCR analysis of Drp1 m 6 A levels in WT CSMCs transducted with siNC or siYTHDF1, n = 6 biological replicates. i Western blot analysis of RSAD2 levels in WT CSMCs transducted with siNC or siYTHDF1, n = 5 biological replicates. j RIP-qPCR analysis of YTHDF1 binding to Drp1 mRNA, n = 6 biological replicates. k RNA pull-down assay followed by immunoblot demonstrating YTHDF1 binding to Drp1 mRNA, n = 5 biological replicates. Data are presented as mean ± SEM of three independent experiments. P values were determined by 2-tailed Student’s t test ( a , b , h and i ) or one-way ANOVA followed by multiple comparisons test ( d , e , g , j and k ). Source data are provided as a file.

Article Snippet: Cyagen Biosciences (S-KO-11081) provided C57BL/6 J wild-type (WT) mice and Rsad2 −/− mice.

Techniques: Expressing, Western Blot, Control, Modification, Pull Down Assay, Binding Assay

Journal: Nature Communications

Article Title: Activation of the RSAD2-YTHDF1 axis in smooth muscle causes inflammatory bowel disease via intercellular mitochondrial transfer

doi: 10.1038/s41467-025-67707-3

Figure Lengend Snippet: a , b Co-immunoprecipitation (CoIP) demonstrating RSAD2-YTHDF1 interaction, n = 5 biological replicates. c Western blot analysis showing enhanced RSAD2-YTHDF1 interaction upon TNF stimulation, n = 6 biological replicates. d TNF stimulation or RSAD2 deficiency did not alter YTHDF1 protein abundance, n = 6 biological replicates. e Immunoprecipitation of YTHDF1 followed by detection of its methylation using Pan me1/me2, n = 5 biological replicates. f WT and Rsad2 −/− CSMCs, with or without TNF treatment, were subjected to YTHDF1 immunoprecipitation or IgG control, followed by Pan me1/me2 detection, n = 5 biological replicates. g Colonic tissues from DSS-treated Sm22α fl/fl and sm Sm22α −/− mice were analyzed as in f , n = 5 mice per group. h Colonic tissues from DSS-treated WT and Rsad2 −/− mice were analyzed similarly, n = 5 mice per group. i Schematic of full-length and truncated RSAD2 constructs. j Proximity ligation assay (PLA) showing RSAD2-YTHDF1 interactions in HEK293A cells co-transfected with HA-YTHDF1 and the indicated RSAD2 constructs. PLA signals are shown in red and nuclei in blue (DAPI). n = 5 biological replicates. k COIP of HEK293A cells expressing HA-YTHDF1 and the indicated RSAD2 constructs, followed by HA immunoblotting, n = 5 biological replicates. l YTHDF1 methylation in cells treated as in ( k ), detected using the Pan me1/me2 antibody, n = 5 biological replicates. m CoIP of HEK293A cells expressing HA-YTHDF1 and either the SAM domain or GFP control, followed by HA detection, n = 5 biological replicates. n YTHDF1 methylation in cells treated as in ( m ), n = 5 biological replicates. o WT CSMCs transfected with the SAM domain or GFP were subjected to YTHDF1 RIP followed by qPCR quantification of Drp1 mRNA enrichment, n = 6 biological replicates. p DRP1 protein levels in WT CSMCs expressing the SAM domain or GFP. Data are presented as mean ± SEM from three independent experiments. ns, not significant. P values were determined by 2-tailed Student’s t test ( c , g , h , o and p ) or one-way ANOVA followed by multiple comparisons ( d, f ). Source data are provided as a file.

Article Snippet: Cyagen Biosciences (S-KO-11081) provided C57BL/6 J wild-type (WT) mice and Rsad2 −/− mice.

Techniques: Immunoprecipitation, Western Blot, Quantitative Proteomics, Methylation, Control, Construct, Proximity Ligation Assay, Transfection, Expressing

Journal: Cell Reports

Article Title: RUNX1-ETO Depletion in t(8;21) AML Leads to C/EBPα- and AP-1-Mediated Alterations in Enhancer-Promoter Interaction

doi: 10.1016/j.celrep.2019.08.040

Figure Lengend Snippet: RUNX1-ETO and the Genome Organization in t(8;21) AML (A) Contact matrix across the whole genome. Each pixel represents a 10-Mb section of the genome. Color intensity represents interaction frequency. The left-hand plot shows a Capture HiC interaction matrix generated with data from Kasumi-1 cells transfected with mismatch control siRNA (siMM) for 4 days; the right-hand plots shows an interaction matrix from RUNX1-ETO-depleted Kasumi-1 cells transfected with the specific siRNA (siRE). (B) Contact matrix across chromosome 3 at 10-Mb resolution. The heatmap shows the raw interactions on chromosome 3 using Kasumi-1 cells transfected with siMM (left) and siRE (right); a UCSC track highlighting the DHS pattern is shown below each heatmap together with the CHi-C first principle component (PC1) plot (see below). (C) UCSC genome browser screenshot shows a first principle component plot for Capture HiC siMM and siRE samples plotted along with RUNX1-ETO ChIP data ( Ptasinska et al., 2014 ) and DNaseI-seq control (siMM) and knockdown (siRE) data from Kasumi-1 cells for a 70-Mb regions on chromosome 11. (D) Percentage of DHSs in active and inactive chromatin compartments in Kasumi-1 cells transfected with siMM and siRE. (E) Percentage of DHSs found at day 10 of knockdown participating in promoter-enhancer interactions (determined at day 4 of knockdown) detected in all active chromatin regions of siMM cells or siRE cells (right two panels), and specific to siMM or siRE cells (left two panels), indicating that the majority of specific DHSs are already present at day 4.

Article Snippet: 4 μg antibody ETO (Santa Cruz) or 4 μg antibody AML1-ETO (15310197, Diagenode), or RUNX1 (Ab23980, Abcam) or 4μg antibody C/EBPα (A2814, Santa Cruz) or 2μg antibody LBD1 (96799, Abcam) or 2μg antibody LMO2 (AF2726, R&D) or 2μg antibody CTCF (70303, Abcam) or 2μg JUND (sc74, Santa Cruz) was added to 10 μL 100 mM sodium phosphate, 0.5% BSA and incubated with protein G beads at 4°C for 1 hour.

Techniques: Generated, Transfection

Journal: Cell Reports

Article Title: RUNX1-ETO Depletion in t(8;21) AML Leads to C/EBPα- and AP-1-Mediated Alterations in Enhancer-Promoter Interaction

doi: 10.1016/j.celrep.2019.08.040

Figure Lengend Snippet: Differential Promoter-Enhancer Interactions after RUNX1-ETO Depletion Are Driven by Differential TF Binding (A) Heatmap representing the correlation of normalized interaction ratios across chr3 at 5-kb resolution, showing the correlation of CHiC peaks in regions specific to DHS peaks that are depleted after RUNX1-ETO knockdown. Each pixel represents a 5-kb section of the genome. The left panel shows the interaction heatmap for siMM and the right panel for siRE cells. Positive correlations are shown as red; negative correlation as blue squares. To determine statistically significant interactions, reads from replicates 1 and 2 were merged. (B) Heatmap representing the correlation of normalized interaction ratios across chr3 at 5-kb resolution and showing the correlation of CHi-C peaks in DHS peaks that are newly formed after RUNX1-ETO (R/E) gene knockdown. For all other features, see (A). (C) DNaseI cleavage patterns within specific distal footprints predicted by Wellington ( Piper et al., 2013 ). Upper strand cut sites are shown in red and lower strand cut sites in green within a 200-bp window centered on each footprint (gap) for siMM- and siRE-specific footprints. (D) Analysis of overrepresented binding motifs within each footprint class as defined in (C). (E) Left panel: time course of DHS development after 2, 4, and 10 days of RUNX1-ETO depletion (see scheme in Figure S1 E). Normalized tag counts are ranked alongside day-10 knockdown (KD) and control-specific (bottom) counts; common and siRE-specific DHS are indicated on the left. Alongside the same genomic coordinates, C/EBPα, JUND, LDB1, CTCF, RUNX1-ETO, LMO2, PU.1, and RUNX1 ChIP-seq reads from Kasumi-1 cells with or without RUNX1-ETO depletion are plotted as indicated (middle panel). The right panel shows the expression levels of the genes linked to the associated DNaseI-seq sites (right panel). (F) UCSC browser screenshot depicting interactions between the CCND2 promoter and surrounding DHS (shown as arcs) together with the indicated ChIP-seq data before and after RUNX1-ETO knockdown. Changing interactions are shown in red, and their associated DHS/ChIP peaks are highlighted using a vertical shaded bar. (G) The same analysis as in (F) for the CITED2 locus.

Article Snippet: 4 μg antibody ETO (Santa Cruz) or 4 μg antibody AML1-ETO (15310197, Diagenode), or RUNX1 (Ab23980, Abcam) or 4μg antibody C/EBPα (A2814, Santa Cruz) or 2μg antibody LBD1 (96799, Abcam) or 2μg antibody LMO2 (AF2726, R&D) or 2μg antibody CTCF (70303, Abcam) or 2μg JUND (sc74, Santa Cruz) was added to 10 μL 100 mM sodium phosphate, 0.5% BSA and incubated with protein G beads at 4°C for 1 hour.

Techniques: Binding Assay, ChIP-sequencing, Expressing

Journal: Cell Reports

Article Title: RUNX1-ETO Depletion in t(8;21) AML Leads to C/EBPα- and AP-1-Mediated Alterations in Enhancer-Promoter Interaction

doi: 10.1016/j.celrep.2019.08.040

Figure Lengend Snippet: The Cooperation of Constitutive and Inducible TFs Is Associated with Differential Interactions (A) Log p values of the differential interactions were plotted ranked from high to low for control and RUNX1-ETO-depleted cells. Red represents an increase in interaction strength and blue represents a decrease. Alongside, the DNaseI-seq fold difference between control and RUNX1-ETO knockdown cells as well as ChIP-seq density profiles for C/EBPα, JUND, LDB1, CTCF, RUNX1-ETO, LMO2, and PU.1 are plotted from Kasumi-1 cells, transfected with either siMM or siRE as indicated. The panels below show the average profiles of the binding of the indicated TFs plotted around the peak summit for control and RUNX1-ETO-depleted cells. Red, ChIP signal specific for peaks with increased interactions; blue, ChIP signal specific for peaks with decreased interactions. (B) Determination of enriched motifs for other TFs in ChIP-seq peaks specific for control and RUNX1-ETO-depleted cells. Motif enrichment was first identified using HOMER and then filtered against digital footprinting data from day 10 of knockout to ensure that these binding motifs were functional. Enrichment scores were subjected to unsupervised clustering for each of the indicated motifs (on the right). The heatmap depicts the degree of motif enrichment with highly enriched motifs shown in red. Peaks were overlaid with the DHS that show new interactions (red brackets at the bottom) or whose interactions are lost (blue brackets). Enrichment scores were calculated by the level of motif enrichment in the unique peaks, as compared to motif enrichment in RUNX1-ETO peaks. Bottom panels: percentage of peaks showing differential interaction with TFs binding to these sites as determined by ChIP-seq (control cells, blue; RUNX1-ETO-depleted cells, red). (C) Bar plots illustrating the distribution of distances between the binding sites of the indicated TFs as determined by ChIP-seq. We measured the changing distance between RUNX1 peaks in siMM and siRE cells and C/EBPα peaks in siMM (top left) and siRE cells (top right), as well as the distance between RUNX1 peaks and JUND control peaks (bottom left) and JUND after R/E KD (bottom right). (D) Bootstrapping analysis of the significance of co-localizing of footprinted motifs within day-10 DHSs for sites that are either lost (left panel) or gained (right panel) after RUNX1-ETO depletion as compared to the rest of the genome. The heatmap shows the significance of motifs co-localizing within 50 bp as compared to sampling by chance. (E) Heatmap highlighting the percentage of day-4 Kasumi-1 DHSs with interactions found in different patient groups indicating the similarity between cell-line and primary t(8;21) data. The t(8;21) and FLT3-ITD DHS/CHi-C patient data were downloaded from GEO: GSE108316 ( Assi et al., 2019 ).

Article Snippet: 4 μg antibody ETO (Santa Cruz) or 4 μg antibody AML1-ETO (15310197, Diagenode), or RUNX1 (Ab23980, Abcam) or 4μg antibody C/EBPα (A2814, Santa Cruz) or 2μg antibody LBD1 (96799, Abcam) or 2μg antibody LMO2 (AF2726, R&D) or 2μg antibody CTCF (70303, Abcam) or 2μg JUND (sc74, Santa Cruz) was added to 10 μL 100 mM sodium phosphate, 0.5% BSA and incubated with protein G beads at 4°C for 1 hour.

Techniques: ChIP-sequencing, Transfection, Binding Assay, Footprinting, Knock-Out, Functional Assay, Sampling

Journal: Cell Reports

Article Title: RUNX1-ETO Depletion in t(8;21) AML Leads to C/EBPα- and AP-1-Mediated Alterations in Enhancer-Promoter Interaction

doi: 10.1016/j.celrep.2019.08.040

Figure Lengend Snippet: Differentially Expressed Genes after RUNX1-ETO Knockdown Are Regulated by Different TF Networks (A) Top panel: data analysis strategy. Transcriptional network of downregulated (blue) non-TF (effector) genes after RUNX1-ETO knockdown (top rows) connected to genes encoding TF families (bottom rows) as determined by digital footprinting and CHi-C. Arrows going outward can come from any TF family within a group; incoming arrows are specific for each gene. (B) Node and edge attributes.

Article Snippet: 4 μg antibody ETO (Santa Cruz) or 4 μg antibody AML1-ETO (15310197, Diagenode), or RUNX1 (Ab23980, Abcam) or 4μg antibody C/EBPα (A2814, Santa Cruz) or 2μg antibody LBD1 (96799, Abcam) or 2μg antibody LMO2 (AF2726, R&D) or 2μg antibody CTCF (70303, Abcam) or 2μg JUND (sc74, Santa Cruz) was added to 10 μL 100 mM sodium phosphate, 0.5% BSA and incubated with protein G beads at 4°C for 1 hour.

Techniques: Footprinting