Journal: bioRxiv

Article Title: Phenotypic screening converges on CDK9 inhibition as a therapeutic strategy in translocation renal cell carcinoma

doi: 10.1101/2025.08.25.672235

Figure Lengend Snippet: (A) Outline of the chromatin displacement assay workflow. (B) Application of chromatin displacement assay to BRD4, using the tool compound JQ1. HEK293T cells were treated with JQ1 dose titration (3 nM to 10 μM). Cells were subject to in situ cell extraction (or not, bottom vs. top rows) and two color IF staining for BRD4 (green) and nucleus (red) was performed. Dose-dependent displacement of BRD4 from chromatin by JQ1 was quantified and plotted for each of 6 individual runs. Data are presented as mean ± SD. (C) Visualization of TFE3 staining and localization in HEK293T cells, with or without Torin1 treatment (which induces nuclear TFE3 localization), and with or without extraction. TFE3 cytoplasmic or nuclear intensity is quantified based on the IF images (mean ± SD, n=6). Note that cytoplasmic signal (white arrow) is dramatically reduced by extraction while nuclear (i.e. chromatin-bound) signal (blue arrow) is not. P -values computed by unpaired t-test; ****P<0.0001. (D) Visualization of ASPL-TFE3 fusion staining and localization in FUUR1 tRCC cells. Cytoplasmic and nuclear staining of the TFE3 fusion with or without extraction was quantified based on the IF images (mean ± SD, n=6) similar to panel (C). Note that TFE3 fusions are constitutively nuclear, as compared with WT TFE3, which shuttles between the cytoplasm and nucleus. P -values computed by unpaired t-test; *P<0.05, ****P<0.0001. (E) Composition of epigenetic modulator (“Epi-Mod”) compound library (n = 121 compounds) used for pilot screening in TFE3 chromatin displacement assay. (F) Replicate-Replicate scatterplot of Epi-Mod library screen using chromatin displacement assay in FUUR1 cells. Select inhibitor classes are highlighted: HDACi, green; HATi, red; HKMTi, blue. (G) Top, IF images showing displacement of ASPL-TFE3 fusion by panobinostat (10 μM) (with extraction condition). Staining: TFE3, green; Nucleus: Red. Quantification of dose-dependent displacement of TFE3, BRD4 and nuclear staining by panobinostat. Data is presented as mean ± SD, n=2.

Article Snippet: For immunofluorescence staining, the fixed cells were blocked (PBS + 1% BSA) for 30 minutes at room temperature and stained with primary antibodies for 1 hour at 37°C at the following dilutions: rabbit anti-human TFE3 antibody (Millipore Sigma #ZRB1272) at 1:1000 and mouse anti-human BRD4 antibody (Santa Cruz Biotechnology #sc518021) at 1:200.

Techniques: Titration, In Situ, Extraction, Staining, Drug discovery

Journal: bioRxiv

Article Title: Phenotypic screening converges on CDK9 inhibition as a therapeutic strategy in translocation renal cell carcinoma

doi: 10.1101/2025.08.25.672235

Figure Lengend Snippet: (A) Screening funnel of 25,000 compounds leading to 4 hits after dose response validation studies. (B) Replicate-Replicate scatterplot for run 4 (out of 5 total runs) of chromatin displacement screening of 25,000 compounds in in FUUR1 cells. Chromatin displacers, red; chromatin retention, green (see methods for hit selection from the 5 separate runs). Primary hits from this run are labeled in red or green; validated hits are named. (C) Dose-dependent increase in nuclear signal of TFE3 with BRD6866 treatment (chromatin retention) in a chromatin displacement assay (CDA) in FUUR1 cells. Nuclear signal (DAPI) as well as nuclear IF signal for TFE3 and BRD4 were quantified and plotted (mean ± SD, n=3). (D) Dose-dependent decrease in nuclear signal of TFE3 with BRD7659 (chromatin displacement) in a CDA in FUUR1 cells. Nuclear signal (DAPI) as well as nuclear IF signal for TFE3 and BRD4 were quantified and plotted (mean ± SD, n=3). (E) Chemical structure of BRD6866 and BRD7659. (F) RNA Seq of UOK109 cells after 16h treatment with BRD6866 at 1μM. Differential transcriptomics analysis using a volcano plot is shown. Antiapoptotic genes with known sensitivity to CDK9 inhibition, MCL1 and XIAP are labeled . RNA Seq schematic created using BioRender.com. (G) Hallmark gene set pathway enrichment analysis upon BRD6866 treatment was compared to two recent studies with CDK9 inhibition (CDK9i) and CDK9 degradation (CDK9d) ( , ). (H) CDK activity profiling assay (Reaction Biology) showing extent of inhibition of various CDK/cyclin pairs by BRD6866 (10 μM). Loss of activity for each CDK and cyclin pairs by BRD6866 was compared to DMSO control. CDK9/cyclin pairs are bolded. The cyclin interacting motif PFTAIRE defines a subgroup of CDKs that does not fall under named categories.

Article Snippet: For immunofluorescence staining, the fixed cells were blocked (PBS + 1% BSA) for 30 minutes at room temperature and stained with primary antibodies for 1 hour at 37°C at the following dilutions: rabbit anti-human TFE3 antibody (Millipore Sigma #ZRB1272) at 1:1000 and mouse anti-human BRD4 antibody (Santa Cruz Biotechnology #sc518021) at 1:200.

Techniques: Biomarker Discovery, Selection, Labeling, RNA Sequencing, Inhibition, Activity Assay, Control

Journal: Nature Communications

Article Title: Zfp260 choreographs the early stage osteo-lineage commitment of skeletal stem cells

doi: 10.1038/s41467-024-54640-0

Figure Lengend Snippet: a Workflow of in vivo labeling strategy using TAM and the experimental design. b PCA indicating the variations of transcriptomes among Lin - ZsGreen + cells isolated from BF-Ctrl, BF-cKO, MSFL-Ctrl and MSFL-cKO groups. c GO and KEGG enrichment analysis of FACS-RNA-seq data. d Heatmap of replicate data for H3K4me1 and H3K27ac enrichment as detected by CUT&Tag. n = 3 from 3 biological replicates. e GO-biological process enrichment analysis of differentially enriched super-enhancers. f Heatmap of replicate data for Zfp260-V5 enrichment detected by ChIP-seq. n = 2 from 2 biological replicates. g Top enriched de novo motifs of Zfp260-V5 enriched genes. h Distribution of peaks in the genome. i GO and KEGG enrichment analysis of Zfp260-V5 enriched genes. j Screening strategy for the potential master downstream regulator. k Transcripts Per Kilobase (TPM) of Runx2 expression level from fracture and MSFL derived Lin - ZsGreen + cells. n = 3 from 3 biological replicates of RNA-seq data. l Genome browser view of peaks enriched for H3K4me1, Brd4, H3K27ac, and Zfp260-V5 over the Runx2 gene locus on chromosome 17 (left) with the magnified super-enhancer region displayed on the right. Primers 1 and 2 indicated the primer sets for the subsequent ChIP-qPCR detection. m Co-IP was performed to examine the condensates for the super-enhancer via immortalized PSCs. n = 3 from 3 biological replicates. n , o mIHC co-staining for Zfp260 (purple) with Brd4 (gold), Med1 (cyan), and P300 (gray) in the homeostatic and osteogenic states of PSCs. The yellow dotted line indicated the route for the subsequent fluorescence intensity measurements. n = 3 from 3 biological replicates. p Fluorescence intensity measurements along the route, with black triangles indicating the merged signals of the four channels. q , r ChIP-qPCR assays for H3K27ac and Brd4 binding via immortalized PSCs. n = 6 from 2 biological replicates. Two-way ANOVA. Scalebars: 5 μm. All data in this figure are represented as mean ± SD. Source data and exact p values are provided in the Source Data file.

Article Snippet: The primary antibodies used in mIHC (dilution 1:400 for all antibodies) included goat anti-mouse/human/rat Itgav (AF1219, Novus Biologicals), mouse anti-mouse/rat CD90 (NB100-65543, Novus Biologicals), mouse anti-mouse/human CD105 (NBP2-22122, Novus Biologicals), rabbit anti-human/mouse/rat CD200 (AF2724, Novus Biologicals), rabbit anti-mouse/human/rat Runx2 (ab236639, Abcam), rabbit anti-mouse/human/rat Sox9 (ab185966, Abcam), rabbit anti-mouse/human Alpl (MA5-24845, Invitrogen), rabbit anti-mouse/human/rat Zfp260 (ABE295, Merck), mouse anti-human/mouse/rat p300 (NB100-616, Novus Biologicals), rabbit anti-human/mouse MED1 (NB100-2574, Novus Biologicals), rabbit anti-human/mouse BRD4 (NBP2-76393, Novus Biologicals), mouse anti-human/mouse/rat Prkca (NB600-201, Novus Biologicals), rabbit anti-V5 tag (13202, CST), mouse anti-Collagen type I (67288-1-Ig, proteintech), rabbit anti-Collagen type II (28459-1-AP, proteintech).

Techniques: In Vivo, Labeling, Isolation, RNA Sequencing, ChIP-sequencing, Expressing, Derivative Assay, ChIP-qPCR, Co-Immunoprecipitation Assay, Staining, Fluorescence, Binding Assay

Journal: Nature Communications

Article Title: Zfp260 choreographs the early stage osteo-lineage commitment of skeletal stem cells

doi: 10.1038/s41467-024-54640-0

Figure Lengend Snippet: a GST-pull down assay of PSC’s whole cell lysate (WCL). The red dotted box indicated the regions for M/S analysis. b GST-Zfp260 specially enriched kinases with high HT sequest scores. c Co-IP of 293 T cell line. d Co-IP of immortalized PSCs. e Co-staining image for Zfp260 and Prkca in PSCs. The white dotted circle indicated the nucleus (Left). The white dotted line indicated the route for the fluorescence intensity measurements (right). f Representative images of immunofluorescence of Zfp260 with osteogenic induction, with the MFI/cytosolic MFI calculated (right). g Separation of nuclear (NE) and cytosolic extracts (CE) followed by Western blot. h In vitro Phos-Assay was performed by Phospho-PAGE. i Co-IP, Phos-PAGE, and SDS-PAGE were jointly performed in immortalized PSCs. The red dotted rectangle indicating the Y173, S182, and S197 residues. j Suggested binding mode of Zfp260 and Prkca by AlphaFold2. k The shortest distance between Zfp260-aa173 and the catalytic domain (CD) of Prkca (Z173-CD), aa182 and CD (Z182-CD), and aa197 and CD (Z197-CD) were calculated. l Co-IP was performed in immortalized PSCs, with statistical analysis (right). m Representative images of immunofluorescence of Zfp260-V5 before and after osteogenic induction (left), with the nuclear MFI/cytosolic MFI evaluated (right). n Separation of NE and CE, followed by Western blot. o Representative ARS staining images of PSCs. Scalebar: 2 mm. p–r ChIP-qPCR assays for Zfp260-V5, Brd4, and H3K27ac binding. n = 6 from 2 biological replicates. For ( a , d , e , h , i ), experiments were conducted independently 3 times, consistently producing similar results. For ( c , g , h , i , l , n , o ), n = 3 from 3 biological replicates. For ( f , m ), n = 30 from 3 biological replicates, with 10 randomly selected cells calculated per replicate. For ( e , f , m ), scale bars: 5 μm. Two-way ANOVA. Box plots display the minimum and maximum values, with the center line representing the median, and the bounds of the box representing the 25th to 75th percentiles. Other data in this figure are represented as mean ± SD. Source data and exact p values are provided in the Source Data file.

Article Snippet: The primary antibodies used in mIHC (dilution 1:400 for all antibodies) included goat anti-mouse/human/rat Itgav (AF1219, Novus Biologicals), mouse anti-mouse/rat CD90 (NB100-65543, Novus Biologicals), mouse anti-mouse/human CD105 (NBP2-22122, Novus Biologicals), rabbit anti-human/mouse/rat CD200 (AF2724, Novus Biologicals), rabbit anti-mouse/human/rat Runx2 (ab236639, Abcam), rabbit anti-mouse/human/rat Sox9 (ab185966, Abcam), rabbit anti-mouse/human Alpl (MA5-24845, Invitrogen), rabbit anti-mouse/human/rat Zfp260 (ABE295, Merck), mouse anti-human/mouse/rat p300 (NB100-616, Novus Biologicals), rabbit anti-human/mouse MED1 (NB100-2574, Novus Biologicals), rabbit anti-human/mouse BRD4 (NBP2-76393, Novus Biologicals), mouse anti-human/mouse/rat Prkca (NB600-201, Novus Biologicals), rabbit anti-V5 tag (13202, CST), mouse anti-Collagen type I (67288-1-Ig, proteintech), rabbit anti-Collagen type II (28459-1-AP, proteintech).

Techniques: Pull Down Assay, Co-Immunoprecipitation Assay, Staining, Fluorescence, Immunofluorescence, Western Blot, In Vitro, SDS Page, Binding Assay, ChIP-qPCR

Journal: Cells

Article Title: Bromodomain-Containing 4 Is a Positive Regulator of Interleukin-34 Production in the Gut

doi: 10.3390/cells13201698

Figure Lengend Snippet: In IBD, BRD4 expression correlates with interleukin (IL)-34 content. ( A ). Representative Western blots showing BRD4, IL-34, and β-actin in total proteins extracted from colonic samples of 3 controls (CTR), 3 UC patients, and 3 CD patients. The example is representative of 2 experiments in which 6 CTRs, 6 UC patients, and 6 CD patients were analyzed. Note that one CD patient and one UC patient had similar levels of BRD4 and IL-34. ( B ). Correlation between the protein expression of BRD4 and IL-34 in UC patients (black circles) and CD patients (grey circles) as evaluated by Western blotting of mucosal samples ( p = 0.001, Pearson’s test coefficient r = 0.8). ( C ). Representative images of immunofluorescence staining of colon sections taken from 1 CTR, 1 patient with UC, and 1 patient with CD, and analyzed for the expression of BRD4 (green), IL-34 (red) and DAPI (blue) (20× magnification). The example is representative of 3 separate experiments in which sections of 3 colonic CTRs, 3 UC patients, and 3 CD patients were analyzed. The lower right inset shows higher magnification (40×).

Article Snippet: BRD4 and IL-34 were detected using a rabbit anti-human BRD4 (final dilution: 1:1000, Novus Biological, Milan, Italy) or mouse anti-human IL-34 (final dilution: 1:1000, Abcam, Cambridge, UK), respectively, followed by horseradish peroxidase-conjugated secondary IgG monoclonal antibodies (final dilution: 1:20000, Dako, Milan, Italy).

Techniques: Expressing, Western Blot, Immunofluorescence, Staining

Journal: Cells

Article Title: Bromodomain-Containing 4 Is a Positive Regulator of Interleukin-34 Production in the Gut

doi: 10.3390/cells13201698

Figure Lengend Snippet: The percentages of live CD45+LPMCs expressing IL-34 or BRD4 are higher in IBD than in controls. ( A , B ). LPMCs were isolated from 5 IBD patients (3 UC and 2 CD patients) and 4 CTRs and analyzed by flow cytometry for IL-34 ( A ) or BRD4 ( B ). The positive cells were selected in the gate of live CD45+LPMCs. Staining results with an isotype control antibody for IL-34 ( A ) or BRD4 ( B ) are also shown. The right panels indicate the mean ± SEM of the percentages of IL-34 or BRD4-expressing CD45+ LPMCs.

Article Snippet: BRD4 and IL-34 were detected using a rabbit anti-human BRD4 (final dilution: 1:1000, Novus Biological, Milan, Italy) or mouse anti-human IL-34 (final dilution: 1:1000, Abcam, Cambridge, UK), respectively, followed by horseradish peroxidase-conjugated secondary IgG monoclonal antibodies (final dilution: 1:20000, Dako, Milan, Italy).

Techniques: Expressing, Isolation, Flow Cytometry, Staining, Control

Journal: Cells

Article Title: Bromodomain-Containing 4 Is a Positive Regulator of Interleukin-34 Production in the Gut

doi: 10.3390/cells13201698

Figure Lengend Snippet: In IBD, most of the IL-34-positive CD45+LPMCs express BRD4, and the percentages of CD45+ LPMCs co-expressing IL-34 and BRD4 are significantly higher than in the controls. ( A ). LPMCs were isolated from 5 IBD patients (3 UC and 2 CD patients) and analyzed by flow cytometry for IL-34 and BRD4. The gated IL-34-expressing live CD45+ LPMCs were analyzed for BRD4 expression. One of five representative experiments is shown. The right panel indicates the mean ± SEM of the percentages of IL-34-expressing CD45+ LPMCs either positive or not for BRD4. ( B ). LPMCs were isolated and analyzed as above. The gated BRD4-expressing live CD45+ LPMCs were analyzed for IL-34 expression. One of five representative experiments is shown. The right panel indicates the mean ± SEM of the percentages of BRD4-expressing CD45+ LPMCs either positive or not for IL-34. ( C ). LPMCs were isolated from 5 IBD patients (3 UC and 2 CD patients) and 4 CTRs and analyzed by flow cytometry for the expression of BRD4 and IL-34 in the gate of live CD45+ cells. The right panel indicates the mean ± SEM of the percentages of CD45+ LPMCs co-expressing BRD4 and IL-34 in IBD and controls.

Article Snippet: BRD4 and IL-34 were detected using a rabbit anti-human BRD4 (final dilution: 1:1000, Novus Biological, Milan, Italy) or mouse anti-human IL-34 (final dilution: 1:1000, Abcam, Cambridge, UK), respectively, followed by horseradish peroxidase-conjugated secondary IgG monoclonal antibodies (final dilution: 1:20000, Dako, Milan, Italy).

Techniques: Expressing, Isolation, Flow Cytometry

Journal: Cells

Article Title: Bromodomain-Containing 4 Is a Positive Regulator of Interleukin-34 Production in the Gut

doi: 10.3390/cells13201698

Figure Lengend Snippet: IL-34 and BRD4 are co-expressed by several immune cells in both CTR and IBD patients. LPMCs were isolated from 5 IBD patients (3 UC and 2 CD patients) and 4 CTRs and analyzed for the expression of CD16, CD14, CD68, CD11c, CD3, CD56, and CD19 in live CD45+ cells expressing BRD4 and IL-34. The data indicate mean ± SEM of 5 IBD samples and 4 CTR samples.

Article Snippet: BRD4 and IL-34 were detected using a rabbit anti-human BRD4 (final dilution: 1:1000, Novus Biological, Milan, Italy) or mouse anti-human IL-34 (final dilution: 1:1000, Abcam, Cambridge, UK), respectively, followed by horseradish peroxidase-conjugated secondary IgG monoclonal antibodies (final dilution: 1:20000, Dako, Milan, Italy).

Techniques: Isolation, Expressing

Journal: Cells

Article Title: Bromodomain-Containing 4 Is a Positive Regulator of Interleukin-34 Production in the Gut

doi: 10.3390/cells13201698

Figure Lengend Snippet: BRD4 inhibition reduces the expression of IL-34. ( A ). IBD LPMCs were transfected with either a scrambled control oligonucleotide (NC AS) or BRD4 AS for 24 h and BRD4 (black) and IL-34 (grey) RNA transcripts were analyzed by real-time PCR. Levels were normalized to β-actin. Data indicate mean ± SEM of 4 independent experiments. ( B ). IBD LPMCs were transfected with either NC AS or BRD4 AS for 48 h and BRD4, IL-34, and β-actin were analyzed by Western blotting. One of 4 independent experiments is shown. The right panels show the quantitative analysis of the BRD4/β-actin ratio and IL-34/β-actin ratio in protein extracts as measured by densitometry scanning of Western blots. Values are expressed in arbitrary units (a.u.) and indicate the mean ± SEM of all experiments. ( C ). IBD LPMCs were treated with JQ1 (200 nM) or vehicle (DMSO) for 24 h, and IL-34 RNA transcripts were analyzed by real-time PCR. Levels were normalized to β-actin. The data indicate mean ± SEM of 4 independent experiments. ( D ). Mice received either regular drinking water (CTR) or dextran sulfate sodium (DSS) and were killed on day 8. BRD4 and IL-34 mRNA expression was evaluated in colonic tissue by real-time PCR and levels were normalized to β-actin. The data indicate mean ± SEM of 6 CTR mice and 9 DSS-treated mice. ( E ). Mice receiving DSS were intraperitoneally given JQ1 (DSS+JQ1) or vehicle (DMSO) on days 3 and 6 and then killed on day 8. IL-34 mRNA expression was evaluated in colonic tissue by real-time PCR and levels were normalized to β-actin. The data indicate mean ± SEM of all samples.

Article Snippet: BRD4 and IL-34 were detected using a rabbit anti-human BRD4 (final dilution: 1:1000, Novus Biological, Milan, Italy) or mouse anti-human IL-34 (final dilution: 1:1000, Abcam, Cambridge, UK), respectively, followed by horseradish peroxidase-conjugated secondary IgG monoclonal antibodies (final dilution: 1:20000, Dako, Milan, Italy).

Techniques: Inhibition, Expressing, Transfection, Control, Real-time Polymerase Chain Reaction, Western Blot

Journal: Cells

Article Title: The Microtubule-Targeting Agent Pretubulysin Impairs the Inflammatory Response in Endothelial Cells by a JNK-Dependent Deregulation of the Histone Acetyltransferase Brd4.

doi: 10.3390/cells12162112

Figure Lengend Snippet: Figure 9. Pretubulysin increases Brd4 enrichment at short treatment times and leads to a time- dependent decrease in Brd4 enrichment at the icam-1 and vcam-1 RNA polymerase II stall site, while increasing H3K9me3 enrichment in the gene body after long treatment times. Enrichment of Brd4 at the RNA polymerase stall sites in the icam-1 (a) or vcam-1 (b) gene and H3K9me3 enrichment in the gene body of icam-1 (c, left) and vcam-1 (c, right). Confluent HUVECs were pre-treated with pretubulysin (PT; 300 nM) and activated with TNF (10 ng/mL) for different durations (2, 6, 16 h; (a,b)) or for 16 h (c) as shown by chromatin immunoprecipitation (ChIP). ChIP was performed with a Brd4 or H3K9me3 antibody and the respective IgG mock antibody. Results were derived based on 2% of input DNA. (n = 3) Data are expressed as mean ± SEM. # p ≤0.05 versus control, * p ≤0.05 versus TNF control.

Article Snippet: Anti-human NFκB-p65 rabbit polyclonal antibody (C15310256), anti-human RNA polymerase II mouse monoclonal antibody (C15200004), anti-human Brd4 rabbit polyclonal antibody (C15410337), negative control mouse IgG antibody (C15400001) and negative control rabbit IgG antibody (C15410206) was obtained from Diagenode (Belgium), anti-human AP-1-cJun polyclonal rabbit antibody (24909-1-AP) was obtained from Proteintech (Planegg-Martinsried, Germany) and anti-human tri-methyl-histone H3 monoclonal rabbit antibody was obtained from Cell Signaling Technologies (Netherlands).

Techniques: Chromatin Immunoprecipitation, ChIP-chip, Derivative Assay, Control

Journal: Cells

Article Title: The Microtubule-Targeting Agent Pretubulysin Impairs the Inflammatory Response in Endothelial Cells by a JNK-Dependent Deregulation of the Histone Acetyltransferase Brd4.

doi: 10.3390/cells12162112

Figure Lengend Snippet: Figure 10. The pretubulysin-induced reduction of Brd4 enrichment in the icam-1 and vcam-1 RNA polymerase stalling sites and the PT-induced decrease in Brd4 protein levels can be reversed by inhibition of JNK. (a) Chromatin immunoprecipitation (ChIP)-based enrichment of Brd4 at the icam-1 and vcam-1 RNA polymerase stalling sites in dependency of JNK inhibition. Confluent HUVECs were pre-treated with JNKIN8 (5 µM) for 30 min, treated with PT (300 nM) for further 30 min and activated with TNF (10 ng/mL) for 6 h. ChIP was performed with an antibody against Brd4 or the respective mock antibody. Results were obtained based on 2% of input. (b) Western blot analysis of the Brd4 protein levels in the nuclear fraction and (c) nuclear Brd4 levels in dependency of JNK inhibition. Confluent HUVECs were pre-treated with pretubulysin (PT; 300 nM) for 30 min and activated with TNF (10 ng/mL) for different durations (2, 6, 16 h; b) or pre-treated with JNKIN8 (5 µM) for 30 min, treated with PT (300 nM) for further 30 min and activated with TNF (10 ng/mL) for 6 h (c). Results were normalized on the respective histone 3 (H3) levels. (d) Western blot analysis of the total Brd4 protein levels (left) or flow-cytometric analysis of the ICAM-1 and VCAM-1 cell surface levels (middle and right) after Brd4 knock-down. Sub-confluent HUVECs were transfected with 100 nM of Brd4 siRNA or non-targeting siRNA and incubated for 48 h, pre-treated with PT (300 nM) for 30 min and activates with TNF (10 ng/mL) for 24 h and were used for flow-cytometry or western blot analysis. Western blot results were normalized on the respective β-actin levels (d, left). (n = 3) Data are expressed as mean ± SEM. # p ≤0.05 versus control, p ≤0.05 versus TNF control. The bar with dot represents statistical significance over the corresponding data groups with • p ≤0.05.

Article Snippet: Anti-human NFκB-p65 rabbit polyclonal antibody (C15310256), anti-human RNA polymerase II mouse monoclonal antibody (C15200004), anti-human Brd4 rabbit polyclonal antibody (C15410337), negative control mouse IgG antibody (C15400001) and negative control rabbit IgG antibody (C15410206) was obtained from Diagenode (Belgium), anti-human AP-1-cJun polyclonal rabbit antibody (24909-1-AP) was obtained from Proteintech (Planegg-Martinsried, Germany) and anti-human tri-methyl-histone H3 monoclonal rabbit antibody was obtained from Cell Signaling Technologies (Netherlands).

Techniques: Inhibition, Chromatin Immunoprecipitation, Western Blot, Knockdown, Transfection, Incubation, Cytometry, Control

Journal: Cells

Article Title: The Microtubule-Targeting Agent Pretubulysin Impairs the Inflammatory Response in Endothelial Cells by a JNK-Dependent Deregulation of the Histone Acetyltransferase Brd4.

doi: 10.3390/cells12162112

Figure Lengend Snippet: Figure 11. Schematic representation of the effects of PT and VIN, COL and PAC in endothelial cells (after treatment with 300 nM; percent decrease or increase in relation to the respective TNF control). Binding of TNF to the TNF receptor leads to intracellular activation of IKK and JNK. Treatment with PT increases the phosphorylation of IKK and subsequent degradation of IκBα, leading to an unimpaired NFκB translocation. Microtubule depolymerization due to PT increases the concentration of free tubulin heterodimers. MAP3K1, which has been shown to be activated by free-tubulin dimers, significantly increases JNK activity. The strongly activated JNK activates cJun in the AP-1 transcription factor, leading to the autoinduction of cJun transcription by activated cJun, increasing the cJun protein levels. NFκB-p65 and AP-1-cJun both bind to the promoters of the CAMs, but the transcriptional activity of both transcription factors is significantly reduced after treatment with the depolymerizing MTAs. Under TNF stimulation, Brd4 (which contains two bromodomains BD1 and BD2) associates with pTEFb in order to release the stalled RNA polymerase II in the non-translated regions downstream of the transcription start sites of the CAM promoters. The increased JNK activity negatively regulates the association of Brd4 with chromatin and likely prevents proper formation of the pTEFb-Brd4 complex. In addition, treatment with PT also reduces the levels of the RNA polymerase in the promoters and stalling regions of the CAMs. The effects of the depolymerizing agents result in decreased expression of CAM mRNA and total CAM protein levels as well as cell surface levels. The decreased CAM levels also significantly reduce the adhesion of leukocytes to the endothelial cells, thereby reducing leukocyte infiltration.

Article Snippet: Anti-human NFκB-p65 rabbit polyclonal antibody (C15310256), anti-human RNA polymerase II mouse monoclonal antibody (C15200004), anti-human Brd4 rabbit polyclonal antibody (C15410337), negative control mouse IgG antibody (C15400001) and negative control rabbit IgG antibody (C15410206) was obtained from Diagenode (Belgium), anti-human AP-1-cJun polyclonal rabbit antibody (24909-1-AP) was obtained from Proteintech (Planegg-Martinsried, Germany) and anti-human tri-methyl-histone H3 monoclonal rabbit antibody was obtained from Cell Signaling Technologies (Netherlands).

Techniques: Control, Binding Assay, Activation Assay, Phospho-proteomics, Translocation Assay, Concentration Assay, Activity Assay, Expressing

Journal: Cells

Article Title: The Microtubule-Targeting Agent Pretubulysin Impairs the Inflammatory Response in Endothelial Cells by a JNK-Dependent Deregulation of the Histone Acetyltransferase Brd4

doi: 10.3390/cells12162112

Figure Lengend Snippet: Pretubulysin increases Brd4 enrichment at short treatment times and leads to a time-dependent decrease in Brd4 enrichment at the icam-1 and vcam-1 RNA polymerase II stall site, while increasing H3K9me3 enrichment in the gene body after long treatment times. Enrichment of Brd4 at the RNA polymerase stall sites in the icam-1 ( a ) or vcam-1 ( b ) gene and H3K9me3 enrichment in the gene body of icam-1 ( c , left) and vcam-1 ( c , right). Confluent HUVECs were pre-treated with pretubulysin (PT; 300 nM) and activated with TNF (10 ng/mL) for different durations (2, 6, 16 h; a and b ) or for 16 h ( c ) as shown by chromatin immunoprecipitation (ChIP). ChIP was performed with a Brd4 or H3K9me3 antibody and the respective IgG mock antibody. Results were derived based on 2% of input DNA. (n = 3) Data are expressed as mean ± SEM. # p ≤ 0.05 versus control, * p ≤ 0.05 versus TNF control.

Article Snippet: Anti-human NFκB-p65 rabbit polyclonal antibody (C15310256), anti-human RNA polymerase II mouse monoclonal antibody (C15200004), anti-human Brd4 rabbit polyclonal antibody (C15410337), negative control mouse IgG antibody (C15400001) and negative control rabbit IgG antibody (C15410206) was obtained from Diagenode (Belgium), anti-human AP-1-cJun polyclonal rabbit antibody (24909-1-AP) was obtained from Proteintech (Planegg-Martinsried, Germany) and anti-human tri-methyl-histone H3 monoclonal rabbit antibody was obtained from Cell Signaling Technologies (Netherlands).

Techniques: Chromatin Immunoprecipitation, ChIP-chip, Derivative Assay

Journal: Cells

Article Title: The Microtubule-Targeting Agent Pretubulysin Impairs the Inflammatory Response in Endothelial Cells by a JNK-Dependent Deregulation of the Histone Acetyltransferase Brd4

doi: 10.3390/cells12162112

Figure Lengend Snippet: The pretubulysin-induced reduction of Brd4 enrichment in the icam-1 and vcam-1 RNA polymerase stalling sites and the PT-induced decrease in Brd4 protein levels can be reversed by inhibition of JNK. ( a ) Chromatin immunoprecipitation (ChIP)-based enrichment of Brd4 at the icam-1 and vcam-1 RNA polymerase stalling sites in dependency of JNK inhibition. Confluent HUVECs were pre-treated with JNKIN8 (5 µM) for 30 min, treated with PT (300 nM) for further 30 min and activated with TNF (10 ng/mL) for 6 h. ChIP was performed with an antibody against Brd4 or the respective mock antibody. Results were obtained based on 2% of input. ( b ) Western blot analysis of the Brd4 protein levels in the nuclear fraction and ( c ) nuclear Brd4 levels in dependency of JNK inhibition. Confluent HUVECs were pre-treated with pretubulysin (PT; 300 nM) for 30 min and activated with TNF (10 ng/mL) for different durations (2, 6, 16 h; b ) or pre-treated with JNKIN8 (5 µM) for 30 min, treated with PT (300 nM) for further 30 min and activated with TNF (10 ng/mL) for 6 h ( c ). Results were normalized on the respective histone 3 (H3) levels. ( d ) Western blot analysis of the total Brd4 protein levels (left) or flow-cytometric analysis of the ICAM-1 and VCAM-1 cell surface levels (middle and right) after Brd4 knock-down. Sub-confluent HUVECs were transfected with 100 nM of Brd4 siRNA or non-targeting siRNA and incubated for 48 h, pre-treated with PT (300 nM) for 30 min and activates with TNF (10 ng/mL) for 24 h and were used for flow-cytometry or western blot analysis. Western blot results were normalized on the respective β-actin levels ( d , left). (n = 3) Data are expressed as mean ± SEM. The bar with dot represents statistical significance over the corresponding data groups with • p ≤ 0.05.

Article Snippet: Anti-human NFκB-p65 rabbit polyclonal antibody (C15310256), anti-human RNA polymerase II mouse monoclonal antibody (C15200004), anti-human Brd4 rabbit polyclonal antibody (C15410337), negative control mouse IgG antibody (C15400001) and negative control rabbit IgG antibody (C15410206) was obtained from Diagenode (Belgium), anti-human AP-1-cJun polyclonal rabbit antibody (24909-1-AP) was obtained from Proteintech (Planegg-Martinsried, Germany) and anti-human tri-methyl-histone H3 monoclonal rabbit antibody was obtained from Cell Signaling Technologies (Netherlands).

Techniques: Inhibition, Chromatin Immunoprecipitation, Western Blot, Transfection, Incubation, Flow Cytometry

Journal: Cells

Article Title: The Microtubule-Targeting Agent Pretubulysin Impairs the Inflammatory Response in Endothelial Cells by a JNK-Dependent Deregulation of the Histone Acetyltransferase Brd4

doi: 10.3390/cells12162112

Figure Lengend Snippet: Schematic representation of the effects of PT and VIN, COL and PAC in endothelial cells (after treatment with 300 nM; percent decrease or increase in relation to the respective TNF control). Binding of TNF to the TNF receptor leads to intracellular activation of IKK and JNK. Treatment with PT increases the phosphorylation of IKK and subsequent degradation of IκBα, leading to an unimpaired NFκB translocation. Microtubule depolymerization due to PT increases the concentration of free tubulin heterodimers. MAP3K1, which has been shown to be activated by free-tubulin dimers, significantly increases JNK activity. The strongly activated JNK activates cJun in the AP-1 transcription factor, leading to the autoinduction of cJun transcription by activated cJun, increasing the cJun protein levels. NFκB-p65 and AP-1-cJun both bind to the promoters of the CAMs, but the transcriptional activity of both transcription factors is significantly reduced after treatment with the depolymerizing MTAs. Under TNF stimulation, Brd4 (which contains two bromodomains BD1 and BD2) associates with pTEFb in order to release the stalled RNA polymerase II in the non-translated regions downstream of the transcription start sites of the CAM promoters. The increased JNK activity negatively regulates the association of Brd4 with chromatin and likely prevents proper formation of the pTEFb-Brd4 complex. In addition, treatment with PT also reduces the levels of the RNA polymerase in the promoters and stalling regions of the CAMs. The effects of the depolymerizing agents result in decreased expression of CAM mRNA and total CAM protein levels as well as cell surface levels. The decreased CAM levels also significantly reduce the adhesion of leukocytes to the endothelial cells, thereby reducing leukocyte infiltration.

Article Snippet: Anti-human NFκB-p65 rabbit polyclonal antibody (C15310256), anti-human RNA polymerase II mouse monoclonal antibody (C15200004), anti-human Brd4 rabbit polyclonal antibody (C15410337), negative control mouse IgG antibody (C15400001) and negative control rabbit IgG antibody (C15410206) was obtained from Diagenode (Belgium), anti-human AP-1-cJun polyclonal rabbit antibody (24909-1-AP) was obtained from Proteintech (Planegg-Martinsried, Germany) and anti-human tri-methyl-histone H3 monoclonal rabbit antibody was obtained from Cell Signaling Technologies (Netherlands).

Techniques: Binding Assay, Activation Assay, Translocation Assay, Concentration Assay, Activity Assay, Expressing