Journal: bioRxiv

Article Title: The proximity-based protein interaction landscape of the transcription factor p65 NF-κB / RELA and its gene-regulatory logics

doi: 10.1101/2024.01.03.574021

Figure Lengend Snippet: (A) The left graph shows the X-ray crystal structure of a p50 / p65 heterodimer bound to DNA as published in (PDB 1kvx), while the right graph shows the entire p65 protein structure including the disordered C-terminal half as calculated by alphafold ( https://alphafold.ebi.ac.uk/entry/Q04206 ). Residues required for dimerization (Phe (F) 213, Leu (L) 215) or DNA binding (Glu (E) 39) are indicated in both structures. (B) Scheme of the HA-tagged p65-miniTurbo fusion proteins that were used to reconstitute p65-deficient HeLa cells under the control of a tetracycline-sensitive promoter. F213 and L215 in p65 wildtype (wt) were mutated to Asp (FL / DD) for dimerization-deficient p65 or E39 to Ile (E / I) for DNA-binding-deficient p65. (C) Principle of proximity-based biotin tagging. (D) Pools of HeLa cells with CRISPR / Cas9-based suppression of endogenous p65 / RELA (Δp65) were transiently transfected (using branched Polyethyleneimine, PEI)) with the constructs shown in (B) and their expression was induced with doxycycline (1 µg / ml) for 17 h. At the end of this incubation, intracellular biotinylation was induced by adding 50 µM biotin for 70 minutes as indicated. Additionally, half of the samples were treated with IL-1α (10 ng / ml) for the last 60 minutes. Cell cultures expressing HA-miniTurbo only (empty vector, EV) or receiving only doxycycline or biotin served as negative controls (indicated by gray font). Parental HeLa cells (p) were included as further controls. Left panel: Cells were lysed and proteins were analyzed by Western blotting for the expression of p65-HA-miniTurbo and HA-miniTurbo using anti p65 and anti HA antibodies. Equal loading was confirmed by probing the blots with anti β-actin antibodies. Right panel: Biotinylated proteins from the same samples were purified on streptavidin agarose beads and biotinylation patterns were visualized by Western blotting using streptavidin-horseradish peroxidase (HRP) conjugates (representative images from two independent experiments). (E) Biotinylated proteins from the experiment shown in (C) and from a second biological replicate were identified by mass spectrometry. Volcano plots show the ratio distributions of Log 2 -transformed mean protein intensity values on the X-axes obtained with wild type p65 or the p65 mutants compared to the empty vector controls in the presence or absence of IL-1α treatment. Y axes show corresponding p values from t-test results. Strong enrichment of the bait p65 / RELA proteins together with the core canonical NF-kB components is shown in red and blue colors, respectively (two biologically independent experiments and three technical replicates per sample). (F) Specific proteins binding to p65 / RELA wild type were defined by significant enrichment (LFC ≥ 2, -log 10 p ≥ 1.3) compared to HA-miniTurbo only and to cells exposed to doxycycline or biotin only (see ). This set of proteins was intersected with proteins enriched in cells expressing p65 mutant proteins (LFC ≥ 2, -log10 p ≥ 1.3). Venn diagrams show the numbers of p65 / RELA interactors and their overlaps before and after IL-1α-treatment, with values in the lower left corners indicating total numbers of interactors. (G) The six protein sets shown in (E) were subjected to parallel overrepresentation pathway analysis using Metascape software . The Venn diagrams show the overlap of the top 100 enriched pathway terms. For IL-1α samples, only 92 terms were enriched. Values in the lower left corners indicate total numbers of unique pathways. (H) The table shows the most strongly enriched pathway categories associated with the p65 / RELA wild type or mutant interactomes. Numbers in brackets indicate the total numbers of p65 / RELA interactors per condition that were subjected to overrepresentation analysis according to (E, F). The mass spectrometry data and bioinformatics analysis results are provided in Supplementary Table 1. See also and . rtTA, reverse tetracycline-controlled transactivator.

Article Snippet: 1 μg of total RNA was prepared by column purification using the NucleoSpin® RNA Kit (Macherey-Nagel; #740955.250) and transcribed into cDNA using 0.5 μl RevertAid Reverse Transcriptase (Fisher Scientific #EP0441), 4 μl 5x reaction buffer, 0.5 μl Random Hexamer Primer, 0.5 mM dNTP mix (10 mM) in a total volume of 20 μl at 25°C for 10 min, 42°C for 1 h and 70°C for 10 min. 1 μl of the reaction mixture was used to amplify cDNA using Taqman® Gene Expression Assays (0.25 μl) (Applied Biosystems) primarily for ACTB (#Hs99999903_m1), GUSB (#Hs99999908_m1), GAPDH (#Hs02758991_g1), IL8 (#Hs00174103_m1), NFKBIA (#Hs00153283_m1), CXCL2 (# Hs00236966_m1), RELA (#Hs01042019_g1) and TaqMan® Fast Universal PCR Master Mix (Applied Biosystems; #4352042).

Techniques: Binding Assay, Control, CRISPR, Transfection, Construct, Expressing, Incubation, Plasmid Preparation, Western Blot, Purification, Mass Spectrometry, Transformation Assay, Mutagenesis, Software

Journal: bioRxiv

Article Title: The proximity-based protein interaction landscape of the transcription factor p65 NF-κB / RELA and its gene-regulatory logics

doi: 10.1101/2024.01.03.574021

Figure Lengend Snippet: (A) Parental HeLa cells or pools of HeLa cells with CRISPR / Cas9-based suppression of endogenous p65 / RELA (Δp65) were transiently transfected with empty vector (EV) encoding HA-miniTurbo (HA-mTb) or with p65 / RELA wild type (wt) fused C-terminally to HA-mTb (p65(wt)-HA-mTb) as described in the legend of . The expression of the constructs was induced with increasing concentrations of doxycycline for 17 h as indicated. At the end of the incubation, half of the cell cultures were treated with IL-1α (10 ng / ml) for 1 h. Cell extracts were analyzed by Western blotting for the expression of the p65-HA-mTb fusion protein or HA-mTb using polyclonal antibodies raised against the C-terminus of p65 / RELA (sc-372) or a monoclonal antibody raised against N-terminal amino acids 1-286 of p65 / RELA (sc-8008), or an anti HA antibody, respectively. Note that the fusion protein is better recognized with the N-terminal antibody preparations. (B) HeLa cells with CRISPR / Cas9-based suppression of endogenous p65 / RELA (Δp65) were transiently transfected with the indicated constructs and their expression was induced with doxycycline at 1 µg / ml for 17 h. On the next day, half of the cell cultures were treated with IL-1α (10 ng / ml) for 1 h. Total RNA was isolated and analyzed by RT-qPCR for expression of the indicated genes. Bar graphs show means ± s.d. from two biologically independent experiments. (C) Cells were transfected as in (A) and expression of the p65 / RELA fusion protein was induced 20 h later with doxycycline (10 ng / ml) for 4 h. In last period of this incubation, half of the cell cultures were treated with IL-1α (10 ng / ml) for 1 h. Cells were lysed and cytosolic (C), soluble nuclear (N1) and insoluble, chromatin nuclear fractions (N2) were analyzed by Western blotting for the expression and distribution of p65(wt)-HA-mTb. Antibodies against RNA polymerase II, tubulin and β-actin were used to control purity of fractions and equal loading.

Article Snippet: 1 μg of total RNA was prepared by column purification using the NucleoSpin® RNA Kit (Macherey-Nagel; #740955.250) and transcribed into cDNA using 0.5 μl RevertAid Reverse Transcriptase (Fisher Scientific #EP0441), 4 μl 5x reaction buffer, 0.5 μl Random Hexamer Primer, 0.5 mM dNTP mix (10 mM) in a total volume of 20 μl at 25°C for 10 min, 42°C for 1 h and 70°C for 10 min. 1 μl of the reaction mixture was used to amplify cDNA using Taqman® Gene Expression Assays (0.25 μl) (Applied Biosystems) primarily for ACTB (#Hs99999903_m1), GUSB (#Hs99999908_m1), GAPDH (#Hs02758991_g1), IL8 (#Hs00174103_m1), NFKBIA (#Hs00153283_m1), CXCL2 (# Hs00236966_m1), RELA (#Hs01042019_g1) and TaqMan® Fast Universal PCR Master Mix (Applied Biosystems; #4352042).

Techniques: CRISPR, Transfection, Plasmid Preparation, Expressing, Construct, Incubation, Western Blot, Isolation, Quantitative RT-PCR, Control

Journal: bioRxiv

Article Title: The proximity-based protein interaction landscape of the transcription factor p65 NF-κB / RELA and its gene-regulatory logics

doi: 10.1101/2024.01.03.574021

Figure Lengend Snippet: (A) Biotinylated proteins from the experiments shown in and from a second biological replicate were identified by mass spectrometry in the presence or absence of IL-1α treatment of cells. Volcano plots show the ratio distributions of Log 2 transformed mean protein intensity values obtained with wild type p65 in the presence of doxycycline and biotin (wt) compared to the empty vector control (EV) or compared with conditions in which only biotin (wt(bio)) or doxycycline (wt(dox)) were added to the cell cultures, to determine false positive values in the absence of expression of fusion protein but facilitated biotinylation, or in the absence of biotinylation but induced expression of the fusion protein, respectively. X-axes show mean ratio value and Y-axes show p values from t-test results. Strong enrichment of the bait p65 / RELA proteins together with the core canonical NF-kB components is shown in red and blue colors, respectively (two biologically independent experiments and three technical replicates per sample). (B) Specific proteins binding to p65 / RELA wild type were defined by significant enrichment (LFC ≥ 2, -log 10 p ≥ 1.3) compared to HA-miniTurbo only and to cells exposed to doxycycline or biotin only as shown in (A). Venn diagrams show the total numbers of specific p65 / RELA interactors and their overlaps before and after IL-1α-treatment. The intersecting 279 (without IL-1α) and 310 (with IL-1α) interactors were pooled, resulting in the set of 366 specific p65 / RELA interactors that was used for further downstream analyses. Numbers in the left lower corner of the boxes indicate the total number of detected interactors.

Article Snippet: 1 μg of total RNA was prepared by column purification using the NucleoSpin® RNA Kit (Macherey-Nagel; #740955.250) and transcribed into cDNA using 0.5 μl RevertAid Reverse Transcriptase (Fisher Scientific #EP0441), 4 μl 5x reaction buffer, 0.5 μl Random Hexamer Primer, 0.5 mM dNTP mix (10 mM) in a total volume of 20 μl at 25°C for 10 min, 42°C for 1 h and 70°C for 10 min. 1 μl of the reaction mixture was used to amplify cDNA using Taqman® Gene Expression Assays (0.25 μl) (Applied Biosystems) primarily for ACTB (#Hs99999903_m1), GUSB (#Hs99999908_m1), GAPDH (#Hs02758991_g1), IL8 (#Hs00174103_m1), NFKBIA (#Hs00153283_m1), CXCL2 (# Hs00236966_m1), RELA (#Hs01042019_g1) and TaqMan® Fast Universal PCR Master Mix (Applied Biosystems; #4352042).

Techniques: Mass Spectrometry, Transformation Assay, Plasmid Preparation, Control, Expressing, Binding Assay

Journal: bioRxiv

Article Title: The proximity-based protein interaction landscape of the transcription factor p65 NF-κB / RELA and its gene-regulatory logics

doi: 10.1101/2024.01.03.574021

Figure Lengend Snippet: (A) Protein interaction network of the 46 known p65 / RELA interactors found by miniTurboID. Edge widths visualize the evidence for experimental interactions deposited in the STRING database . Nodes are colored in red and are arranged according to the enrichment found by proximity labeling in our study. (B) Venn diagram of p65 / RELA interactors in IL-1α or untreated cells revealing a total of 366 unique p65 / RELA interactors, of which 320 (87.4 %) have no documented protein interaction entries in STRING. (C) Overlap of the RELA interactome with 1639 human TFs and 801 epigenetic regulators . (D) Graphs visualizing the top 10 enriched epigenetic regulators. Volcano plots show the ratio distributions of Log 2 transformed mean protein intensity values obtained with wild type p65 / RELA (wt) or with p65 / RELA mutants (FL/DD, E/I) compared to empty vector controls (EV). Only 9 reader proteins were found. (E) Association of enriched epigenetic regulators with known epigenetic complexes according to the annotation provided by . Numbers in brackets show identified components per complex. (F) Venn diagram showing the overlap of enriched TFs in basal or IL-1α-stimulated conditions. (G) Volcano plots visualizing all TFs significantly enriched with wt p65 / RELA (LFC ≥ 2, -log 10 p ≥ 1.3) compared with empty vector control (EV) and the changes obtained with p65 mutants in basal conditions. (H) Distribution of TF families found to be associated with p65 / RELA in basal and IL-1α-stimulated conditions according to the annotation provided by (I) IL-1α-dependent enrichment of all TF belonging to ZBTB and ZNF families as identified by miniTurboID. (J) The top 10 pathway terms according to GO (BP, CC, MF), KEGG, Reactome, STRING clusters and WikiPathways data base entries and the top 10 subcellular localizations associated with the 366 p65 / RELA interactors. Annotations, number of components and false discovery rates (FDR) were retrieved using the STRING plugin of Cytoscape . The mass spectrometry data sets and bioinformatics analysis results are provided in Supplementary Table 1.

Article Snippet: 1 μg of total RNA was prepared by column purification using the NucleoSpin® RNA Kit (Macherey-Nagel; #740955.250) and transcribed into cDNA using 0.5 μl RevertAid Reverse Transcriptase (Fisher Scientific #EP0441), 4 μl 5x reaction buffer, 0.5 μl Random Hexamer Primer, 0.5 mM dNTP mix (10 mM) in a total volume of 20 μl at 25°C for 10 min, 42°C for 1 h and 70°C for 10 min. 1 μl of the reaction mixture was used to amplify cDNA using Taqman® Gene Expression Assays (0.25 μl) (Applied Biosystems) primarily for ACTB (#Hs99999903_m1), GUSB (#Hs99999908_m1), GAPDH (#Hs02758991_g1), IL8 (#Hs00174103_m1), NFKBIA (#Hs00153283_m1), CXCL2 (# Hs00236966_m1), RELA (#Hs01042019_g1) and TaqMan® Fast Universal PCR Master Mix (Applied Biosystems; #4352042).

Techniques: Labeling, Transformation Assay, Plasmid Preparation, Control, Mass Spectrometry

Journal: bioRxiv

Article Title: The proximity-based protein interaction landscape of the transcription factor p65 NF-κB / RELA and its gene-regulatory logics

doi: 10.1101/2024.01.03.574021

Figure Lengend Snippet: (A) Final list of top ranking high confidence interactors p65 / RELA selected for further studies. The heatmap shows the Log 2 transformed mean protein intensity values from technical triplicates of the two biological independent miniTurboID experiments, the enrichment ratio values compared to the empty vector (HA-miniTurbo) control (EV) and the regulation by IL-1α. With the exception of N4BP3, all proteins were identified by at least two peptides. (B) Graph showing that the top 38 p65 / RELA interactors are largely devoid of known protein interactions based on STRING entries. According to STRING, only two factors (CEBPD and FOSL1) interact with p65 /RELA. Node borders visualize the main functional annotations. (C) HeLa cells were transiently transfected for 48 h with 20 nM of siRNAs mixtures for 38 HCI and p65 / RELA, a siRNA targeting luciferase, transfection reagent alone or were left untreated (untr.). Half of the cells per plate were treated for 1 h with IL-1α (10 ng / ml) at the end of the incubation. cDNAs were transcribed in lysates and amplicons for three NF-kB target genes, two housekeeping genes and all 38 HCI p65 / RELA interactors were pre-amplified by linear PCR and then quantified by qPCR. Based on Ct values, mRNA levels were quantified and normalized against GUSB . The effects of knockdowns were calculated separately for basal and IL-1α-inducible conditions against the luciferase siRNA. The heatmap shows hierarchically Kmeans clustered mean ratio values derived from three biologically independent siRNA screens. As a positive control, RELA knockdowns were performed in parallel. Green colors highlight p65 / RELA interactors selected for further analysis. (D) The miniTurboID enrichment of six p65 / RELA interactors (green colors) chosen from (C) is shown. The complete set of data of the screen is provided in Supplementary Table 2. See also .

Article Snippet: 1 μg of total RNA was prepared by column purification using the NucleoSpin® RNA Kit (Macherey-Nagel; #740955.250) and transcribed into cDNA using 0.5 μl RevertAid Reverse Transcriptase (Fisher Scientific #EP0441), 4 μl 5x reaction buffer, 0.5 μl Random Hexamer Primer, 0.5 mM dNTP mix (10 mM) in a total volume of 20 μl at 25°C for 10 min, 42°C for 1 h and 70°C for 10 min. 1 μl of the reaction mixture was used to amplify cDNA using Taqman® Gene Expression Assays (0.25 μl) (Applied Biosystems) primarily for ACTB (#Hs99999903_m1), GUSB (#Hs99999908_m1), GAPDH (#Hs02758991_g1), IL8 (#Hs00174103_m1), NFKBIA (#Hs00153283_m1), CXCL2 (# Hs00236966_m1), RELA (#Hs01042019_g1) and TaqMan® Fast Universal PCR Master Mix (Applied Biosystems; #4352042).

Techniques: Transformation Assay, Plasmid Preparation, Control, Functional Assay, Transfection, Luciferase, Incubation, Amplification, Derivative Assay, Positive Control

Journal: bioRxiv

Article Title: The proximity-based protein interaction landscape of the transcription factor p65 NF-κB / RELA and its gene-regulatory logics

doi: 10.1101/2024.01.03.574021

Figure Lengend Snippet: (A) Scheme illustrating the arrangement of siRNAs and controls on individual cell culture plates and the performance of RT-qPCR measurements in cell extracts without prior RNA purification. A linear PCR amplification step was included to pre-amplify specific transcripts. (B) Confirmation of knockdown of 38 HCI and of RELA mRNAs by RT-qPCR as shown in (A). Bar graphs show mean changes ± s.d. relative to the luciferase siRNA controls (siLuci) from three biologically independent experiments.

Article Snippet: 1 μg of total RNA was prepared by column purification using the NucleoSpin® RNA Kit (Macherey-Nagel; #740955.250) and transcribed into cDNA using 0.5 μl RevertAid Reverse Transcriptase (Fisher Scientific #EP0441), 4 μl 5x reaction buffer, 0.5 μl Random Hexamer Primer, 0.5 mM dNTP mix (10 mM) in a total volume of 20 μl at 25°C for 10 min, 42°C for 1 h and 70°C for 10 min. 1 μl of the reaction mixture was used to amplify cDNA using Taqman® Gene Expression Assays (0.25 μl) (Applied Biosystems) primarily for ACTB (#Hs99999903_m1), GUSB (#Hs99999908_m1), GAPDH (#Hs02758991_g1), IL8 (#Hs00174103_m1), NFKBIA (#Hs00153283_m1), CXCL2 (# Hs00236966_m1), RELA (#Hs01042019_g1) and TaqMan® Fast Universal PCR Master Mix (Applied Biosystems; #4352042).

Techniques: Cell Culture, Quantitative RT-PCR, Purification, Amplification, Knockdown, Luciferase

Journal: bioRxiv

Article Title: The proximity-based protein interaction landscape of the transcription factor p65 NF-κB / RELA and its gene-regulatory logics

doi: 10.1101/2024.01.03.574021

Figure Lengend Snippet: Proximity-ligation assays coupled to immunofluorescence (IF) were performed with HeLa cells or Δp65 HeLa cells lacking endogenous p65 / RELA to demonstrate interactions of p65 / RELA with TFE3 (A), TFEB (B), GLIS2 (C) and ZBTB5 (D) using pairs of antibodies as indicated. PLA-spots are colored in red, while p65 IF is colored in green. Nuclear DNA is counterstained with Hoechst (blue signals). The images show representative fluorescence raw data and the violin plots on the right show quantification from the numbers of cells indicated in brackets. Samples omitting one of the two antibodies or both primary antibodies (ctr) served as negative controls. Solid lines indicate medians and dashed lines indicate 1 st and 3 rd quartiles. Asterisks indicate results from Kruskal-Wallis tests compared to the parental control (****p ≤ 0.0001). obtained by one-way ANOVA.

Article Snippet: 1 μg of total RNA was prepared by column purification using the NucleoSpin® RNA Kit (Macherey-Nagel; #740955.250) and transcribed into cDNA using 0.5 μl RevertAid Reverse Transcriptase (Fisher Scientific #EP0441), 4 μl 5x reaction buffer, 0.5 μl Random Hexamer Primer, 0.5 mM dNTP mix (10 mM) in a total volume of 20 μl at 25°C for 10 min, 42°C for 1 h and 70°C for 10 min. 1 μl of the reaction mixture was used to amplify cDNA using Taqman® Gene Expression Assays (0.25 μl) (Applied Biosystems) primarily for ACTB (#Hs99999903_m1), GUSB (#Hs99999908_m1), GAPDH (#Hs02758991_g1), IL8 (#Hs00174103_m1), NFKBIA (#Hs00153283_m1), CXCL2 (# Hs00236966_m1), RELA (#Hs01042019_g1) and TaqMan® Fast Universal PCR Master Mix (Applied Biosystems; #4352042).

Techniques: Ligation, Immunofluorescence, Fluorescence, Control

Journal: bioRxiv

Article Title: The proximity-based protein interaction landscape of the transcription factor p65 NF-κB / RELA and its gene-regulatory logics

doi: 10.1101/2024.01.03.574021

Figure Lengend Snippet: (A) Schematic illustrating the strategy to analyze the influences of novel p65 / RELA interactors on basal p65 / RELA target genes by combining siRNA-mediated knockdown with transcriptome analysis. (B) HeLa cells were transiently transfected for 48 hours with 20 nM siRNA mixtures against RELA, ZBTB5, S100A8, S100A9 (series 1) or RELA, GLIS2, TFE3, TFEB (series 2) and an siRNA against luciferase (siLuc) as control. Half of the cells were treated with IL-1α (10 ng/ml) for 1 hour at the end of incubation, and Agilent microarray analyses were performed from total RNA. Normalized data were used to identify DEGs based on an LFC ≥ 1 with a -log 10 p value ≥ 1.3. Venn diagrams show the overlap of all DEGs that were affected at least twofold by siRNA knockdown in untreated, basal conditions, with the ratio of siLuc to individual knockdown determined in each case. Red colors mark genes jointly regulated by knockdown of RELA and one of its interactors (two biologically independent experiments). (C) Violin plots show the distribution, medians, and interquartile ranges of normalized expression levels for all constitutively expressed genes and the corresponding changes in the gene subsets defined in that were affected by siRNA knockdown. The number of these genes is indicated in parentheses. (D) Superimposed pairwise correlation analyses of the mean ratio changes of all genes (gray), and gene sets significantly up- or down-regulated by siRNA knockdown (red). Ratio values from RELA knockdown conditions were compared with the knockdown of a RELA interactor in each case. Genes that are jointly regulated by knockdown of RELA and one of its interactors correspond to the Venn diagrams of (B) and are marked in red. Coefficients of correlation (Pearson’s r), corresponding p values and coefficients of determination (r 2 ) rare indicated for all comparisons. The complete set of data is provided in Supplementary Table 3.

Article Snippet: 1 μg of total RNA was prepared by column purification using the NucleoSpin® RNA Kit (Macherey-Nagel; #740955.250) and transcribed into cDNA using 0.5 μl RevertAid Reverse Transcriptase (Fisher Scientific #EP0441), 4 μl 5x reaction buffer, 0.5 μl Random Hexamer Primer, 0.5 mM dNTP mix (10 mM) in a total volume of 20 μl at 25°C for 10 min, 42°C for 1 h and 70°C for 10 min. 1 μl of the reaction mixture was used to amplify cDNA using Taqman® Gene Expression Assays (0.25 μl) (Applied Biosystems) primarily for ACTB (#Hs99999903_m1), GUSB (#Hs99999908_m1), GAPDH (#Hs02758991_g1), IL8 (#Hs00174103_m1), NFKBIA (#Hs00153283_m1), CXCL2 (# Hs00236966_m1), RELA (#Hs01042019_g1) and TaqMan® Fast Universal PCR Master Mix (Applied Biosystems; #4352042).

Techniques: Knockdown, Transfection, Luciferase, Control, Incubation, Microarray, Expressing

Journal: bioRxiv

Article Title: The proximity-based protein interaction landscape of the transcription factor p65 NF-κB / RELA and its gene-regulatory logics

doi: 10.1101/2024.01.03.574021

Figure Lengend Snippet: (A) Schematic illustrating the strategy to analyze the influences of novel p65 / RELA interactors on IL-1α-regulated p65 / RELA target genes by combining siRNA-mediated knockdown with transcriptome analysis. (B) HeLa cells were transiently transfected for 48 h with 20 nM siRNA mixtures against RELA, ZBTB5, S100A8, S100A9 (series 1) or RELA, GLIS2, TFE3, TFEB (series 2) and an siRNA against luciferase (siLuc) as control. Half of the cells were treated with IL-1α (10 ng/ml) for 1 hour at the end of incubation, and Agilent microarray analyses were performed from total RNA. Normalized data were used to identify DEGs based on an LFC ≥ 1 with a -log 10 p value ≥ 1.3. Venn diagrams show the overlap of all DEGs that were affected at least twofold by siRNA knockdown in IL-1α-treated samples, with the ratio of siLuc to individual knockdown determined in each case. Red colors mark genes jointly regulated by knockdown of RELA and one of its interactors (two biologically independent experiments). (C) Violin plots show the distribution, medians, and interquartile ranges of normalized expression levels for all IL-1α-regulated genes and the corresponding changes in the gene subsets defined in that were affected by siRNA knockdown. The number of these genes is indicated in parentheses. Asterisks indicate significant changes as determined by a two-tailed Mann-Whitney test (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001). (D) Superimposed pairwise correlation analyses of the mean ratio changes of all genes (gray), IL-1α-regulated genes (blue), and gene sets significantly up- or down-regulated by siRNA knockdown (red). Ratio values from RELA knockdown conditions were compared with the knockdown of a RELA interactor in each case. Genes that are jointly regulated by knockdown of RELA and one of its interactors correspond to the Venn diagrams of (B) and are marked in red. Coefficients of correlation (Pearson’s r), corresponding p values and coefficients of determination (r 2 ) rare indicated for all comparisons. The complete set of data is provided in Supplementary Table 3.

Article Snippet: 1 μg of total RNA was prepared by column purification using the NucleoSpin® RNA Kit (Macherey-Nagel; #740955.250) and transcribed into cDNA using 0.5 μl RevertAid Reverse Transcriptase (Fisher Scientific #EP0441), 4 μl 5x reaction buffer, 0.5 μl Random Hexamer Primer, 0.5 mM dNTP mix (10 mM) in a total volume of 20 μl at 25°C for 10 min, 42°C for 1 h and 70°C for 10 min. 1 μl of the reaction mixture was used to amplify cDNA using Taqman® Gene Expression Assays (0.25 μl) (Applied Biosystems) primarily for ACTB (#Hs99999903_m1), GUSB (#Hs99999908_m1), GAPDH (#Hs02758991_g1), IL8 (#Hs00174103_m1), NFKBIA (#Hs00153283_m1), CXCL2 (# Hs00236966_m1), RELA (#Hs01042019_g1) and TaqMan® Fast Universal PCR Master Mix (Applied Biosystems; #4352042).

Techniques: Knockdown, Transfection, Luciferase, Control, Incubation, Microarray, Expressing, Two Tailed Test, MANN-WHITNEY

Journal: bioRxiv

Article Title: The proximity-based protein interaction landscape of the transcription factor p65 NF-κB / RELA and its gene-regulatory logics

doi: 10.1101/2024.01.03.574021

Figure Lengend Snippet: (A) Schematic illustrating the strategy to project the protein interactions of all target genes defined by knockdowns of p65 / RELA or its interactors in IL-1α-stimulated cells into combined functional networks. (B) Table summarizing the numbers of mapped IDs (= nodes) corresponding to the gene groups shown in , their protein interactions (= edges) and the protein interaction network enrichment p values as derived from STRING. (C) Cytoscape-derived PPI networks. Nodes are colored and arranged according to the deregulation of the corresponding genes by knockdown of p65 / RELA or its interactors. Edges visualize known protein interactions, including the small number of interactions reported for p65 / RELA, S100A8 / 9, and TFE3 / TFEB. No interactions were found for ZBTB5 and GLIS2.

Article Snippet: 1 μg of total RNA was prepared by column purification using the NucleoSpin® RNA Kit (Macherey-Nagel; #740955.250) and transcribed into cDNA using 0.5 μl RevertAid Reverse Transcriptase (Fisher Scientific #EP0441), 4 μl 5x reaction buffer, 0.5 μl Random Hexamer Primer, 0.5 mM dNTP mix (10 mM) in a total volume of 20 μl at 25°C for 10 min, 42°C for 1 h and 70°C for 10 min. 1 μl of the reaction mixture was used to amplify cDNA using Taqman® Gene Expression Assays (0.25 μl) (Applied Biosystems) primarily for ACTB (#Hs99999903_m1), GUSB (#Hs99999908_m1), GAPDH (#Hs02758991_g1), IL8 (#Hs00174103_m1), NFKBIA (#Hs00153283_m1), CXCL2 (# Hs00236966_m1), RELA (#Hs01042019_g1) and TaqMan® Fast Universal PCR Master Mix (Applied Biosystems; #4352042).

Techniques: Functional Assay, Derivative Assay, Knockdown

Journal: bioRxiv

Article Title: The proximity-based protein interaction landscape of the transcription factor p65 NF-κB / RELA and its gene-regulatory logics

doi: 10.1101/2024.01.03.574021

Figure Lengend Snippet: (A) Schematic illustrating the strategy to use p65 / RELA ChIPseq data for delineating chromatin recruitment of RELA together with its interactors on the basis of DNA motifs and three possible scenarios of interactions. (B) Windows of 1000 base pairs surrounding experimentally determined p65 / RELA ChIPseq peaks were searched for motifs of RELA and REL using matrices from the JASPAR data base. P values indicated significant enrichment compared to the whole genome. The Venn diagram shows the overlap and inserts show motif compositions. (C) Venn diagrams indicating the overlap of motifs found for RELA or the RELA interactors TFE3, TFEB or GLIS2 in chromosomal regions assigned to p65 / RELA ChIPseq peaks. P values indicated significant enrichment compared to the whole genome. Inserts show motif compositions. (D) All target genes that were significantly up- or downregulated under basal or IL-1α-stimulated conditions as shown in or were collected and were examined for their association with a p65 / RELA ChIPseq peak. The pie charts show the numbers of RELA, TFE3, TFEB and GLIS2 motifs detected in siRNA RELA target genes with an annotated p65 / RELA peak in their promoters or enhancers. (E) Overlap of all genes with a p65 / RELA peak in promoters or enhancers and at least one motif for the indicated transcription factors in IL_1a-stimulated conditions. (F) Genome browser view of the TNFAIP3 locus with p65 / RELA ChIPseq peaks, activated enhancers and promoters (H3K27ac), accessible chromatin (ATACseq) and mRNA production (RNAseq) before and after 1 h of IL-1α stimulation. Data sets were from GSE64224, GSE52470 and GSE134436 and are aligned to HG19 ( ; ). p65 / RELA binding regions of 1000 bp under p65 / RELA peaks and identified TF motifs are indicated by horizontal lines. (G) HeLa cells were left untreated or were starved for 24 h in HBSS. Half of the cells was treated with IL-1α (10 ng / ml) for 1 h before the end of the experiment. ChIP-qPCR was performed with the indicated antibodies or IgG controls and a primer pair covering the TNFAIP3 promoter region (marked with an arrow in ). Floating bar plots show percent input plus the mean of all values from three independent biological replicates performed with two technical replicates. The complete set of data is provided in Supplementary Table 4.

Article Snippet: 1 μg of total RNA was prepared by column purification using the NucleoSpin® RNA Kit (Macherey-Nagel; #740955.250) and transcribed into cDNA using 0.5 μl RevertAid Reverse Transcriptase (Fisher Scientific #EP0441), 4 μl 5x reaction buffer, 0.5 μl Random Hexamer Primer, 0.5 mM dNTP mix (10 mM) in a total volume of 20 μl at 25°C for 10 min, 42°C for 1 h and 70°C for 10 min. 1 μl of the reaction mixture was used to amplify cDNA using Taqman® Gene Expression Assays (0.25 μl) (Applied Biosystems) primarily for ACTB (#Hs99999903_m1), GUSB (#Hs99999908_m1), GAPDH (#Hs02758991_g1), IL8 (#Hs00174103_m1), NFKBIA (#Hs00153283_m1), CXCL2 (# Hs00236966_m1), RELA (#Hs01042019_g1) and TaqMan® Fast Universal PCR Master Mix (Applied Biosystems; #4352042).

Techniques: Binding Assay, ChIP-qPCR

Journal: bioRxiv

Article Title: The proximity-based protein interaction landscape of the transcription factor p65 NF-κB / RELA and its gene-regulatory logics

doi: 10.1101/2024.01.03.574021

Figure Lengend Snippet: Venn diagrams indicating the overlap of RELA motifs with motifs of ZBTB factors that were found by miniTurboID to interact with RELA, in chromosomal regions assigned to p65 / RELA ChIPseq peaks. P values indicated significant enrichment compared to the whole genome. Inserts show motif compositions.

Article Snippet: 1 μg of total RNA was prepared by column purification using the NucleoSpin® RNA Kit (Macherey-Nagel; #740955.250) and transcribed into cDNA using 0.5 μl RevertAid Reverse Transcriptase (Fisher Scientific #EP0441), 4 μl 5x reaction buffer, 0.5 μl Random Hexamer Primer, 0.5 mM dNTP mix (10 mM) in a total volume of 20 μl at 25°C for 10 min, 42°C for 1 h and 70°C for 10 min. 1 μl of the reaction mixture was used to amplify cDNA using Taqman® Gene Expression Assays (0.25 μl) (Applied Biosystems) primarily for ACTB (#Hs99999903_m1), GUSB (#Hs99999908_m1), GAPDH (#Hs02758991_g1), IL8 (#Hs00174103_m1), NFKBIA (#Hs00153283_m1), CXCL2 (# Hs00236966_m1), RELA (#Hs01042019_g1) and TaqMan® Fast Universal PCR Master Mix (Applied Biosystems; #4352042).

Techniques:

Journal: Nature cell biology

Article Title: An androgen receptor switch underlies lineage infidelity in treatment-resistant prostate cancer

doi: 10.1038/s41556-021-00743-5

Figure Lengend Snippet: (a) Heatmap of EZH2 ChIP-seq signal intensity in CRPC 16DCRPC and 42DENZR cell lines (left), with overlaid H3K27Ac and H3K27Me3 histone mark ChIP-seq (right). Each horizontal line represents a 6-kb locus. (b) Representative ChIP-seq tracks surrounding the WNT5A locus in 16DCRPC and 42ENZR cells. Regions of EZH2 co-occupancy with the active H3K27Ac histone mark are highlighted. (c) Relative expression of genes bound by EZH2 alone (EZH2-none) or co-operatively with H3K27Me3 (EZH2-me) and H3K27Ac (EZH2-ac) histone marks in 42DENZR and 42FENZR cell lines. Box plot shows mean and interquartile range. (d) Heatmap of H3K27Me3 and K3K27Ac ChIP-seq signal intensity surrounding AR:EZH2 co-occupied regions in 42DENZR cells. (e) Heatmap indicating AR and EZH2 ChIP-seq signal intensity at AR:EZH2 co-occupied sites (n = 2155) in 42DENZR cells, and EZH2 signal intensity at the corresponding sites in AR-negative cell lines: NCI-H660, DU145 (GEO: GSE135623), and PC-3 (GEO: GSE123204). The shade of green (AR) or blue (EZH2) reflects binding intensity. Each horizontal line represents a 6-kb locus.

Article Snippet: Immunohistochemical staining was performed on deparaffinized FFPE sections using a Ventana Discovery XT automated immunostainer using the following antibodies: AR (clone N-20; 1:50; Santa Cruz sc-816, lot no. G1916), pCDK1-T161 (1:400; Abcam ab47329, lot no. r3260427–5), EZH2 (Clone DR69; 1:50; Cell Signaling 5246S, lot no. 9), pEZH2-T350 (1:75; generated in this study), pEZH2-S21 (1:250; Bethyl IHC-00388, lot no. 6), and SYP (1:500; Abcam ab32127, lot no. {"type":"entrez-nucleotide","attrs":{"text":"GR223336","term_id":"238891844","term_text":"GR223336"}} GR223336 –15).

Techniques: ChIP-sequencing, Expressing, Binding Assay

Journal: Nature cell biology

Article Title: An androgen receptor switch underlies lineage infidelity in treatment-resistant prostate cancer

doi: 10.1038/s41556-021-00743-5

Figure Lengend Snippet: (a) EZH2 was immunoprecipitated in 42ENZR cells, trypsin digested, and analyzed by mass spectrometry. Peptides covering 36% of EZH2 were recovered and analyzed for post-translational modifications. (n = 4 independent replicates). (b) Expression of total and phosphorylated (T350, S21, and T311 residues) EZH2 in the indicated cell lines. Protein abundance was assessed by densitometry and is reported relative to total EZH2. (c) IHC staining of pEZH2-S21 and pEZH2-T350 in serial sections from representative CRPC (n = 39) and NEPC (n = 26) patient tumours (Scale bar, 100 μm). Staining area and intensity was quantified and reported (mean ± SD; two-tailed unpaired t-test). (d) Expression of genes positively regulated by EZH2 when phosphorylated at S21 [defined by Xu et al.] in the indicated cell lines and patient tumours from the Beltran 2016 cohort. Statistical analysis was performed using a two-tailed unpaired t-test. Box plots show mean and interquartile range. ns, not significant. (e) qRT-PCR of NE lineage markers in CRPCcrEZH2 cells expressing myc-tagged EZH2S21A or EZH2S21D mutants, reported relative to empty vector transfected cells. (mean ± SD; two-tailed unpaired t-test, n = 3). Immunoblotting confirmed transgene expression. (f) Proliferation of parental 16DCRPC (control) and CRPCcrEZH2 cells stably expressing EZH2T350A and EZH2T350D phospho-mutants assessed by IncuCyte (mean ± SD, n = 3 replicates). Immunoblotting confirmed transgene expression. (g) qRT-PCR of plasticity and NE markers in VCaP and C4–2 cell lines co-transfected with EZH2 siRNA and siRNA-resistant myc-tagged EZH2WT, EZH2T350A, or EZH2T350D plasmid following treatment with ENZ (10 μM) for 7 days (mean ± SD; two-tailed unpaired t-test, n = 3).

Article Snippet: Immunohistochemical staining was performed on deparaffinized FFPE sections using a Ventana Discovery XT automated immunostainer using the following antibodies: AR (clone N-20; 1:50; Santa Cruz sc-816, lot no. G1916), pCDK1-T161 (1:400; Abcam ab47329, lot no. r3260427–5), EZH2 (Clone DR69; 1:50; Cell Signaling 5246S, lot no. 9), pEZH2-T350 (1:75; generated in this study), pEZH2-S21 (1:250; Bethyl IHC-00388, lot no. 6), and SYP (1:500; Abcam ab32127, lot no. {"type":"entrez-nucleotide","attrs":{"text":"GR223336","term_id":"238891844","term_text":"GR223336"}} GR223336 –15).

Techniques: Immunoprecipitation, Mass Spectrometry, Expressing, Immunohistochemistry, Staining, Two Tailed Test, Quantitative RT-PCR, Plasmid Preparation, Transfection, Western Blot, Stable Transfection

Journal: Nature cell biology

Article Title: An androgen receptor switch underlies lineage infidelity in treatment-resistant prostate cancer

doi: 10.1038/s41556-021-00743-5

Figure Lengend Snippet: a, Abundance of AR, FOXA1, SUZ12 and EED peptides detected using RIME with AR antibodies as bait. Each dot represents an independent replicate, with a solid line denoting the mean. b, SUZ12 immunoprecipitation (IP) followed by immunoblotting for AR and PRC2 subunits. The relative abundance of AR was normalized to SUZ12 pulldown. c, AR–EZH2 PLA and quantification of nuclear PLA signals (red dots) from a single plane (mean ±s.d.; P < 0.0001, two-tailed unpaired t-test; n = 3). Each dot represents the number of PLA signals in a single nucleus. Scale bar, 10 μm. d, Frequency of AR-bound genes with EZH2, SUZ12 and/or EED co-occupancy based on ChIP-seq peak annotation (±50 kb from the nearest TSS) in 42DENZR cells. e, Overlap of genomic regions co-occupied by AR and EZH2 ChIP-seq peaks (AR–EZH2 complex) with ChIP-seq peaks for the H3K27Me3 and H3K27Ac in 42DENZR cells. f, Heat map of AR and EZH2 ChIP-seq signal intensity in 16DCRPC and 42DENZR cells, with corresponding ATAC-seq peak intensity. g, Overlap of AR and EZH2 ChIP-seq peaks in 16DCRPC and 42DENZR cell lines. h, Overlap of AR and EZH2 ChIP-seq peaks in the Ptenf/f;Rb1f/f (DKO) GEMM. i, Enriched reactome pathways with genes co-occupied by AR–EZH2 in 42DENZR cells and the Ptenf/f/Rb1f/f GEMM. The size of each circular data point reflects the degree to which genes in the pathway are enriched based on RNA-seq from 42DENZR compared with 16DCRPC cells. NS, not significant. j, Expression of AR–EZH2 co-bound genes in matched prostate tumours (P1–P3) pre- and post-ENZ therapy (n = 3) from the DARANA trial. Box plot shows mean and interquartile range. Statistical analysis was performed using a paired t-test. k, Venn diagram of overlap in genes downregulated (log2FC < 1) in 42DENZR cells following depletion of AR using CRISPR (crAR) or EZH2 inhibition (10 μm GSK126; 96 h). The heat map depicts relative expression of select AR–EZH2 co-bound genes, reported relative to parental cells. l, Sequential ChIP (Re-ChIP) for selected binding sites in 42DENZR cells treated with vehicle or EZH2 inhibitor (10 μm GSK126, 96 h). Cells were first analysed by chromatin immunoprecipitation with AR antibody and then immunoprecipitated again with an AR or EZH2 antibody, as indicated. Results are reported relative to IgG control (mean ± s.d., n = 2).

Article Snippet: Immunohistochemical staining was performed on deparaffinized FFPE sections using a Ventana Discovery XT automated immunostainer using the following antibodies: AR (clone N-20; 1:50; Santa Cruz sc-816, lot no. G1916), pCDK1-T161 (1:400; Abcam ab47329, lot no. r3260427–5), EZH2 (Clone DR69; 1:50; Cell Signaling 5246S, lot no. 9), pEZH2-T350 (1:75; generated in this study), pEZH2-S21 (1:250; Bethyl IHC-00388, lot no. 6), and SYP (1:500; Abcam ab32127, lot no. {"type":"entrez-nucleotide","attrs":{"text":"GR223336","term_id":"238891844","term_text":"GR223336"}} GR223336 –15).

Techniques: Immunoprecipitation, Western Blot, Two Tailed Test, ChIP-sequencing, RNA Sequencing Assay, Expressing, CRISPR, Inhibition, Binding Assay, Chromatin Immunoprecipitation

Journal: Nature cell biology

Article Title: An androgen receptor switch underlies lineage infidelity in treatment-resistant prostate cancer

doi: 10.1038/s41556-021-00743-5

Figure Lengend Snippet: a, Expression of plasticity and neuroendocrine markers by real-time PCR (rtPCR) and Western blot in 16DCRPC cells with CRISPR-mediated EZH2 knockout (16DCRPC crEZH2) following 7 d ENZ treatment. Cells transfected with a non-silencing scrambled guide RNA (crSCR) served as a control. Data are reported relative to non-transfected cells (mean ± s.d., n = 3). Two-tailed unpaired t-test. b, Tumour growth velocity of CRPC cells with CRISPR-mediated EZH2 knockout transplanted subcutaneously into nude mice, followed by treatment with vehicle (veh) or ENZ (n = 5 mice per group). Box plots show mean and interquartile range. Mann–Whitney test. c, Gene expression analysis (by rtPCR) in 16DCRPC control and crEZH2 xenograft tumours at the experimental end point. Data are reported relative to vehicle-treated mice (mean ± s.d.; *P = 0.05, two-tailed unpaired t-test; n = 3 mice per treatment group). d, Strategy used to establish the 16Dreporter cell line carrying GFP and mCherry fluorescent reporters in the endogenous OCT4 and ASCL1 loci, respectively. Fluorescence-activated cell sorting (FACS) plot shows gating used to isolate the individual cell populations. HL, left homology arm; HR, right homology arm. e, Immunofluorescence images for OCT4-GFP (green) and ASCL1-mCherry (red) in CRPCreporter cells at the indicated time points after ENZ treatment. Single cells were tracked and are denoted with arrows. Scale bar, 100 μm. f, Fold change in transcript abundance of genes unique and common to the OCT4+, ASCL1+ and hybrid (OCT4+ASCL1+) FACS-isolated CRPCreporter cell populations relative to the negative population (log2FC cut-off of 1.5), by RNA-seq. g, MSigDB pathways enriched for common genes (n = 468) upregulated (defined as log2FC > 1.5) across OCT4+, ASCL1+ and hybrid (OCT4+ASCL1+) CRPCreporter populations relative to the negative population. Statistical analysis was performed using a hypergeometric test. h, EZH2 activity score, calculated on the basis of z-score-transformed expression of genes in the ‘Kondo EZH2 targets’ signature from MSigDB, in negative, OCT4+, ASCL1+ and hybrid (OCT4+ASCL1+) CRPCreporter FACS-isolated cell populations. i, Quantification of GFP+ and ASCL1+ fluorescent CRPCreporter cells following treatment with ENZ (10 μM) alone or in combination with EZH2 inhibitor (10 μM GSK126) using the IncuCyte fluorescent object counting algorithm (mean ± s.d., n = 2). Representative images at 8 d after treatment are shown. Scale bar, 50 μm.

Article Snippet: Immunohistochemical staining was performed on deparaffinized FFPE sections using a Ventana Discovery XT automated immunostainer using the following antibodies: AR (clone N-20; 1:50; Santa Cruz sc-816, lot no. G1916), pCDK1-T161 (1:400; Abcam ab47329, lot no. r3260427–5), EZH2 (Clone DR69; 1:50; Cell Signaling 5246S, lot no. 9), pEZH2-T350 (1:75; generated in this study), pEZH2-S21 (1:250; Bethyl IHC-00388, lot no. 6), and SYP (1:500; Abcam ab32127, lot no. {"type":"entrez-nucleotide","attrs":{"text":"GR223336","term_id":"238891844","term_text":"GR223336"}} GR223336 –15).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Western Blot, CRISPR, Knock-Out, Transfection, Two Tailed Test, MANN-WHITNEY, Fluorescence, FACS, Immunofluorescence, Isolation, RNA Sequencing Assay, Activity Assay, Transformation Assay

Journal: Nature cell biology

Article Title: An androgen receptor switch underlies lineage infidelity in treatment-resistant prostate cancer

doi: 10.1038/s41556-021-00743-5

Figure Lengend Snippet: a, Heat map of AR, EZH2 and pEZH2-T350 ChIP-seq binding intensity in 42DENZR cells. Each horizontal line represents a 6-kb locus. b, Frequency of AR ChIP-seq peaks overlapping with EZH2 and pEZH2-T350 ChIP-seq peaks in 42DENZR cells. c, Distribution of AR–EZH2 and AR–pEZH2 co-bound peaks in relation to the TSS. Peaks were mapped into 5-kb bins. d, PLA analysis of the interaction between AR and pEZH2-T350, and quantification of nuclear PLA signals (red dots) from a single plane (mean ± s.d.; P = 3.8 × 10−10, two-tailed unpaired t-test; n = 3). Each dot represents the number of PLA signals in a single nucleus. Scale bar, 10 μm. e, Overlap of genes co-bound to AR–EZH2 occupied by SUZ12- and/or EED, based on ChIP-seq peak annotation in 42DENZR cells. Gene annotation was restricted to ±50 kb from TSS. f, Expression of genes with promoter-bound (defined as ±3 kb from TSS) AR alone or co-occupancy with EZH2 or pEZH2-T350 in 42DENZR and 42FENZR cell lines. Data are mean expression ± s.d., with significance assessed using a two-tailed unpaired t-test. g, Expression of AR–pEZH2 co-bound genes in matched individual patient tumours pre- and post-ENZ therapy from the DARANA trial (n = 3). Box plots show mean and interquartile range. Statistical analysis was performed using a paired t-test. h, Gene ontology signatures from MSigDB enriched for AR–EZH2 and AR–pEZH2 co-bound genes in 42DENZR cells. Statistical analysis was performed using a hypergeometric test. i, Immunohistochemical staining for AR, pEZH2-T350 and SYP (neuroendocrine marker) in serial sections from non-treated (naive) and neoadjuvant ADT/TAX-treated (4.5 months) prostate tumours from the CALGB 90203 clinical trial. Treated tumours were binned on the basis of pEZH2-T350 staining intensity, and matched NanoString-based sequencing was used to assess the expression of plasticity factors in pEZH2-low (n = 8) and pEZH2-high (n = 4) tumours. Box plots show mean and interquartile range of z-score-transformed expression values with significance assessed using a two-tailed unpaired t-test. Scale bar, 100 μm.

Article Snippet: Immunohistochemical staining was performed on deparaffinized FFPE sections using a Ventana Discovery XT automated immunostainer using the following antibodies: AR (clone N-20; 1:50; Santa Cruz sc-816, lot no. G1916), pCDK1-T161 (1:400; Abcam ab47329, lot no. r3260427–5), EZH2 (Clone DR69; 1:50; Cell Signaling 5246S, lot no. 9), pEZH2-T350 (1:75; generated in this study), pEZH2-S21 (1:250; Bethyl IHC-00388, lot no. 6), and SYP (1:500; Abcam ab32127, lot no. {"type":"entrez-nucleotide","attrs":{"text":"GR223336","term_id":"238891844","term_text":"GR223336"}} GR223336 –15).

Techniques: ChIP-sequencing, Binding Assay, Two Tailed Test, Expressing, Immunohistochemical staining, Staining, Marker, Sequencing, Transformation Assay

Journal: Nature cell biology

Article Title: An androgen receptor switch underlies lineage infidelity in treatment-resistant prostate cancer

doi: 10.1038/s41556-021-00743-5

Figure Lengend Snippet: a, Immunoblot of total and phosphorylated EZH2 and CDK1 in the indicated prostate cancer cell lines. HPCS, high-plasticity cell state. b, Immunoblot of EZH2 and pEZH2-T350 following CDK1 inhibition (5 μM RO-3306, 6 h). c, Immunohistochemical staining for pEZH2-T350 and pCDK1-T161 in serial sections from treatment-naive (N, n = 30), CRPC (CR, n = 40) and NEPC (NE, n = 26) clinical samples. Scale bar, 100 μm. Staining intensity was quantified (mean ± s.d.; two-tailed unpaired t-test). d, SUZ12 and EED peptides detected by RIME using EZH2 and pEZH2-T350 antibodies as bait in 42ENZR cells. Each dot represents an independent replicate, with a solid line denoting mean. Significance was defined as ≥4 peptides. e, Myc-tagged wild-type EZH2 (EZH2WT) and T350 phospho-mimicking (EZH2T350D) and phospho-dead (EZH2T350A) mutants were transiently transfected into 16DCRPC cells with endogenous EZH2 deletion for 72 h. Immunoprecipitation was performed using a Myc tag antibody. f, Distribution of pEZH2-T350, SUZ12 and EED ChIP-seq peaks in relation to the nearest TSS. The density of polycomb subunits and H3K27Ac are shown surrounding the WNT5A locus. g, Proportion of EZH2 and pEZH2-T350 ChIP-seq peaks overlapping with H3K27Me3 and H3K27Ac ChIP-seq peaks in 42DENZR cells. The distribution of H3K27Ac alone and co-occupied with pEZH2-T350 (pEZH2-ac) in relation to the TSS is shown. h, Single-sample GSEA (ssGSEA) score of MSigDB pathways in CRPCcrEZH2 cells expressing EZH2T350A or EZH2T350D mutant, and adenocarcinoma (CRPC-Adeno) and NEPC (CRPC-NE) patient specimens from the Beltran 2016 cohort4. The ASC score is shown below each cell line or individual patient. i, rtPCR and immunoblot in 42DENZR cells with EZH2 knockdown, stably expressing siRNA-resistant Myc-tagged EZH2WT or EZH2T350A mutant for 72 h. Data are reported relative to cells transfected with empty vector (EV) (mean ± s.d.; two-tailed unpaired t-test, n = 2). j, Immunohistochemical staining for EZH2 and SYP in serial sections from CRPCcrEZH2 EZH2T350A and EZH2T350D mutant xenografts treated with vehicle or ENZ. Scale bar, 100 μm. SYP staining intensity was quantified; box plots show mean and interquartile range. k, Flow cytometry plots of CD44 and NCAM1 cell surface expression in dissociated tumour cells from EZH2T350A and EZH2T350D mutant xenografts.

Article Snippet: Immunohistochemical staining was performed on deparaffinized FFPE sections using a Ventana Discovery XT automated immunostainer using the following antibodies: AR (clone N-20; 1:50; Santa Cruz sc-816, lot no. G1916), pCDK1-T161 (1:400; Abcam ab47329, lot no. r3260427–5), EZH2 (Clone DR69; 1:50; Cell Signaling 5246S, lot no. 9), pEZH2-T350 (1:75; generated in this study), pEZH2-S21 (1:250; Bethyl IHC-00388, lot no. 6), and SYP (1:500; Abcam ab32127, lot no. {"type":"entrez-nucleotide","attrs":{"text":"GR223336","term_id":"238891844","term_text":"GR223336"}} GR223336 –15).

Techniques: Western Blot, Inhibition, Immunohistochemical staining, Staining, Two Tailed Test, Transfection, Immunoprecipitation, ChIP-sequencing, Expressing, Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Stable Transfection, Plasmid Preparation, Flow Cytometry

Journal: Nature cell biology

Article Title: An androgen receptor switch underlies lineage infidelity in treatment-resistant prostate cancer

doi: 10.1038/s41556-021-00743-5

Figure Lengend Snippet: a, PCA of global transcriptome in the indicated cell lines. 42DENZR cells with AR knockout (AR KO) and inhibited EZH2 activity (10 μM GSK126, 96 h) are shown. b, ASC and NEPC scores in patient tumours from ref. 5 (adenocarcinoma cluster 5, n = 28; AR+NE+, n = 10; AR−NE+, n = 3) and the indicated cell lines. c, GSEA signatures enriched (Fisher’s exact test, P < 0.05) in 42DENZR cells following AR knockout or EZH2 inhibition (10 μM GSK126, 96 h). d, Volcano plot of peptides detected by RIME using EZH2 antibodies as bait in 42DENZR cells treated with DMSO or EZH2 inhibitor (10 μM GSK126, 96 h). Statistical analysis was performed using a two-tailed unpaired t-test (n = 3). e, Immunoprecipitation of EZH2 in 42DENZR cells treated with 10 μM GSK126 for 96 h followed by immunoblotting. f, Immunoblot of SUZ12 in nuclear soluble and chromatin-bound fractions in 42DENZR cells treated with 10 μM GSK126 for 96 h. g, PLA analysis of AR–EZH2 in 42DENZR cells following EZH2 inhibition (10 μM GSK126, 96 h). Nuclear PLA signals from a single plane were quantified (mean ± s.d.; P = 3.1 × 10−16, two-tailed unpaired t-test; n = 3). Scale bar, 10 μm. h, Chromatin immunoprecipitation–PCR (ChIP–PCR) for AR at the AREs within the KLK3 enhancer in 42DENZR cells following treatment with EZH2 inhibitor (10 μM GSK126, 96 h). Results reported relative to IgG control (mean ± s.d.; P = 0.018, two-tailed unpaired t-test; n = 4). F, forward; R, reverse. i, rtPCR in 42DENZR cells treated with EZH2 inhibitor (10 μM GSK126 or GSK343, 96 h) or EED inhibitor (1 μM A-395, 96 h). Data reported relative to vehicle-treated cells (mean ± s.d., two-tailed unpaired t-test; n = 3). Western blot confirmed PRC2 inhibition. j, Confluency measured using IncuCyte (mean ± s.d., n = 2). At 48 h after seeding, cells were treated with EZH2 inhibitor (10 mM GSK126). k, Proliferation of 42DENZR cells treated with ENZ (10 μM) and EZH2 inhibitor (2 μM GSK126) alone or in combination, measured using IncuCyte. EZH2 inhibitor was removed (washout) at 96 h. Data plotted are mean ± s.d. (n = 3), with significance evaluated using a two-tailed unpaired t-test at the end point.

Article Snippet: Immunohistochemical staining was performed on deparaffinized FFPE sections using a Ventana Discovery XT automated immunostainer using the following antibodies: AR (clone N-20; 1:50; Santa Cruz sc-816, lot no. G1916), pCDK1-T161 (1:400; Abcam ab47329, lot no. r3260427–5), EZH2 (Clone DR69; 1:50; Cell Signaling 5246S, lot no. 9), pEZH2-T350 (1:75; generated in this study), pEZH2-S21 (1:250; Bethyl IHC-00388, lot no. 6), and SYP (1:500; Abcam ab32127, lot no. {"type":"entrez-nucleotide","attrs":{"text":"GR223336","term_id":"238891844","term_text":"GR223336"}} GR223336 –15).

Techniques: Knock-Out, Activity Assay, Inhibition, Two Tailed Test, Immunoprecipitation, Western Blot, Chromatin Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction

Journal: Nature cell biology

Article Title: An androgen receptor switch underlies lineage infidelity in treatment-resistant prostate cancer

doi: 10.1038/s41556-021-00743-5

Figure Lengend Snippet: (a) Immunoblot AR, EZH2, and H3K27Me3 (a surrogate marker of EZH2 activity) in 42DENZR cells following CRISPR-mediated AR deletion (crAR) or EZH2 inhibition (10 μM GSK126, 96 hrs). (b) Relative expression (qRT-PCR) of neuroendocrine lineage markers in 16DCRPC and C4–2 cell lines following siRNA-mediated AR silencing for 96 hours. Data are reported relative to cells transfected with a non-silencing scrambled control (mean ± SD, n = 3). A fold change >2 is considered significant. Immunoblotting confirmed AR knockdown.

Article Snippet: Immunohistochemical staining was performed on deparaffinized FFPE sections using a Ventana Discovery XT automated immunostainer using the following antibodies: AR (clone N-20; 1:50; Santa Cruz sc-816, lot no. G1916), pCDK1-T161 (1:400; Abcam ab47329, lot no. r3260427–5), EZH2 (Clone DR69; 1:50; Cell Signaling 5246S, lot no. 9), pEZH2-T350 (1:75; generated in this study), pEZH2-S21 (1:250; Bethyl IHC-00388, lot no. 6), and SYP (1:500; Abcam ab32127, lot no. {"type":"entrez-nucleotide","attrs":{"text":"GR223336","term_id":"238891844","term_text":"GR223336"}} GR223336 –15).

Techniques: Western Blot, Marker, Activity Assay, CRISPR, Inhibition, Expressing, Quantitative RT-PCR, Transfection

Journal: Nature cell biology

Article Title: An androgen receptor switch underlies lineage infidelity in treatment-resistant prostate cancer

doi: 10.1038/s41556-021-00743-5

Figure Lengend Snippet: (a-b) qRT-PCR in 42DENZR (a) and 42FENZR (b) cells following siRNA-mediated EZH2 silencing (siEZH2) for the indicated time, reported relative to non-transfected control cells at day 0 (mean ± SD; two-tailed unpaired t-test, n = 3). NTC, non-targeting control. (c-d) Spheroid formation and ALDH activity in 42DENZR (c) and 42FENZR (d) cells following siRNA-mediated EZH2 silencing (siEZH2; left) or treatment with increasing dose of EZH2 inhibitor (GSK126; right) for 8 days (mean ± SD; two-tailed unpaired t-test, n = 2). (e) qRT-PCR in 42DENZR cells treated with EZH2 inhibitor (10 μM GSK126) for 7 days, followed by removal (washout) for 14 days. Expression is reported relative to cells at day 0 (mean ± SD; two-tailed unpaired t-test, n = 3). Immunoblotting confirmed on-target effect.

Article Snippet: Immunohistochemical staining was performed on deparaffinized FFPE sections using a Ventana Discovery XT automated immunostainer using the following antibodies: AR (clone N-20; 1:50; Santa Cruz sc-816, lot no. G1916), pCDK1-T161 (1:400; Abcam ab47329, lot no. r3260427–5), EZH2 (Clone DR69; 1:50; Cell Signaling 5246S, lot no. 9), pEZH2-T350 (1:75; generated in this study), pEZH2-S21 (1:250; Bethyl IHC-00388, lot no. 6), and SYP (1:500; Abcam ab32127, lot no. {"type":"entrez-nucleotide","attrs":{"text":"GR223336","term_id":"238891844","term_text":"GR223336"}} GR223336 –15).

Techniques: Expressing, Activity Assay, Quantitative RT-PCR, Transfection, Two Tailed Test, Western Blot

Journal: Cell Death & Disease

Article Title: YBX1 orchestrates LDHA-mediated metabolic reprogramming and NF-κB activation to drive clear cell renal cell carcinoma progression

doi: 10.1038/s41419-025-08261-0

Figure Lengend Snippet: A Protein levels of YBX1 in 10 different types of tumors from the CPTAC database. B mRNA levels of YBX1 in 21 different types of tumors from the TCGA database. C–G Enrichment functional analysis of proteins interacting with YBX1 in ccRCC cell line 786-O through Co-IP pull-down and identified by mass spectrometry. C Mass spectrometry identification schematic. D Gene Ontology Biological Process (GO-BP) enrichment. E KEGG pathway enrichment. F UniPort annotation keywords. G Wiki Pathways enrichment analysis. ** P < 0.01, *** P < 0.001, **** P < 0.0001. Unpaired two-sided Student’s t -test in A and B. Data are presented as mean ± SD.

Article Snippet: After removing the supernatant, 50 μL of Antibody Buffer was added to resuspend the beads-bound cells, followed by the addition of 1 μg of YBX1 antibody (Proteintech, USA) and Rabbit IgG (Cell Signaling Technology, USA), and left to stand overnight at 4 °C.

Techniques: Functional Assay, Co-Immunoprecipitation Assay, Mass Spectrometry

Journal: Cell Death & Disease

Article Title: YBX1 orchestrates LDHA-mediated metabolic reprogramming and NF-κB activation to drive clear cell renal cell carcinoma progression

doi: 10.1038/s41419-025-08261-0

Figure Lengend Snippet: A Western blot validation of YBX1 protein expression upon knockdown and overexpression efficiency in ACHN and 786-O cells. B qRT-PCR validation of YBX1 mRNA expression upon knockdown and overexpression efficiency in ACHN and 786-O cells. C Lactate production upon YBX1 knockdown in ACHN and 786-O cells. D ATP production detection upon YBX1 knockdown in ACHN and 786-O cells. E Real-time monitoring of extracellular acidification rate (ECAR) in ACHN and 786-O cells after YBX1 knockdown, measured using a Seahorse Bioscience Analyzer. F Quantitative analysis of glycolytic capacity and glycolytic reserve. G Real-time monitoring of oxygen consumption rate (OCR) in ACHN and 786-O cells after YBX1 knockdown, measured using a Seahorse Bioscience Analyzer. H Quantitative analysis of mitochondrial respiratory capacity and respiratory reserve. I Lactate dehydrogenase (LDH) activity detection upon YBX1 knockdown and overexpression in ACHN and 786-O cells. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Unpaired two-sided Student’s t -test in B – D , F , H and I . Data are presented as mean ± SD.

Article Snippet: After removing the supernatant, 50 μL of Antibody Buffer was added to resuspend the beads-bound cells, followed by the addition of 1 μg of YBX1 antibody (Proteintech, USA) and Rabbit IgG (Cell Signaling Technology, USA), and left to stand overnight at 4 °C.

Techniques: Western Blot, Biomarker Discovery, Expressing, Knockdown, Over Expression, Quantitative RT-PCR, Activity Assay

Journal: Cell Death & Disease

Article Title: YBX1 orchestrates LDHA-mediated metabolic reprogramming and NF-κB activation to drive clear cell renal cell carcinoma progression

doi: 10.1038/s41419-025-08261-0

Figure Lengend Snippet: A , B Effects of YBX1 overexpression on the proliferation of ACHN and 786-O cells. A Colony formation assay. B CCK-8 cell proliferation assay. C, D Effects of YBX1 overexpression on the migration and invasion of ACHN and 786-O cells. C Transwell cell migration assay. D Transwell cell invasion assay. E Detection of bioluminescence intensity of 8 pairs of tumors in nude mice using an in vivo imaging system. F Quantification analysis of bioluminescence intensity at the site of renal orthotopic tumors. G Statistical analysis of in situ tumor weight, expressed as the weight of the left minus the corresponding right kidney of nude mice. H Immunohistochemical staining and quantification to detect protein expression of YBX1, LDHA and Ki-67 in tumor tissues from control and YBX1 knockdown groups in nude mice. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Unpaired two-sided Student’s t -test in A , C , D , F , G and H . Two-way ANOVA with correction for multiple comparisons in B. Data are presented as mean ± SD.

Article Snippet: After removing the supernatant, 50 μL of Antibody Buffer was added to resuspend the beads-bound cells, followed by the addition of 1 μg of YBX1 antibody (Proteintech, USA) and Rabbit IgG (Cell Signaling Technology, USA), and left to stand overnight at 4 °C.

Techniques: Over Expression, Colony Assay, CCK-8 Assay, Proliferation Assay, Migration, Cell Migration Assay, Invasion Assay, In Vivo Imaging, In Situ, Immunohistochemical staining, Staining, Expressing, Control, Knockdown

Journal: Cell Death & Disease

Article Title: YBX1 orchestrates LDHA-mediated metabolic reprogramming and NF-κB activation to drive clear cell renal cell carcinoma progression

doi: 10.1038/s41419-025-08261-0

Figure Lengend Snippet: A, B Protein expression levels of YBX1 ( A ) and LDHA ( B ) in ccRCC from the CPTAC database. C, D Correlation of gene expression between YBX1 and LDHA in ccRCC from the TCGA database ( C ) and in an Asian population ( D ). E Western blot analysis of YBX1 and LDHA protein expression in tumor (T) and paired adjacent non-tumor (P) tissues from 27 ccRCC patients, showing representative images from 4 pairs of tissues. F Quantification of YBX1 and LDHA proteins in ccRCC and adjacent non-cancerous tissues from 27 cases using Image J software, normalized to β-actin. G Correlation of YBX1 and LDHA protein expression in tumor tissues from 27 ccRCC patients. H-M Immunohistochemical staining to assess the expression of YBX1 and LDHA in 63 pairs of ccRCC and adjacent tissues. H, I Representative immunohistochemical staining images and quantification of IHC staining using Image J software. J Correlation of quantified immunohistochemical staining of YBX1 and LDHA in tumor tissues of 63 ccRCC patients. K-M Levels of YBX1 and LDHA in ccRCC patients stratified by tumor size, T stage and Fuhrman grade. N , O Impact of YBX1 expression on overall survival (OS) ( N ) and recurrence free survival (RFS) ( O ) in ccRCC patients from the TCGA database. ** P < 0.01, *** P < 0.001, **** P < 0.0001. Unpaired two-sided Student’s t -test in A , B , K , L and M . Spearman correlation statistics in C , D , G and J . Paired t -test in F and I. Log-rank test in N and O. Data are presented as mean ± SD.

Article Snippet: After removing the supernatant, 50 μL of Antibody Buffer was added to resuspend the beads-bound cells, followed by the addition of 1 μg of YBX1 antibody (Proteintech, USA) and Rabbit IgG (Cell Signaling Technology, USA), and left to stand overnight at 4 °C.

Techniques: Expressing, Gene Expression, Western Blot, Software, Immunohistochemical staining, Staining, Immunohistochemistry

Journal: Cell Death & Disease

Article Title: YBX1 orchestrates LDHA-mediated metabolic reprogramming and NF-κB activation to drive clear cell renal cell carcinoma progression

doi: 10.1038/s41419-025-08261-0

Figure Lengend Snippet: A Identification of LDHA as a potential YBX1-interacting protein by IP/MS analysis. B Protein-protein interaction (PPI) network diagram of YBX1 and LDHA from the GeneMANIA and STRING databases. C Co-IP assay to verify the YBX1-LDHA interaction in ccRCC cell lines. D Immunofluorescence staining to examine the expression of YBX1 and LDHA in ACHN and 786-O cell lines, with confocal microscopy used to observe the co-localization of YBX1 and LDHA. E Molecular docking analysis of the YBX1-LDHA interaction. F Schematic diagram of GFP-tagged YBX1 structural domain peptide fragments. G 786-O cells transfected with GFP-tagged YBX1 or its truncation mutants for 48 h, followed by Co-IP using anti-GFP antibody. H Schematic representation of Flag-tagged LDHA domain protein peptides. I 786-O cells transfected with Flag-tagged LDHA or its truncation mutants for 48 h, followed by Co-IP using anti-Flag antibody. J Assessment of the impact on LDH activity after transfecting YBX1 or its truncated mutants into 786-O cells for 48 h. ** P < 0.01, **** P < 0.0001, ns: no significant difference. One-way ANOVA with correction for multiple comparisons in J. Data are presented as mean ± SD.

Article Snippet: After removing the supernatant, 50 μL of Antibody Buffer was added to resuspend the beads-bound cells, followed by the addition of 1 μg of YBX1 antibody (Proteintech, USA) and Rabbit IgG (Cell Signaling Technology, USA), and left to stand overnight at 4 °C.

Techniques: Protein-Protein interactions, Co-Immunoprecipitation Assay, Immunofluorescence, Staining, Expressing, Confocal Microscopy, Transfection, Activity Assay

Journal: Cell Death & Disease

Article Title: YBX1 orchestrates LDHA-mediated metabolic reprogramming and NF-κB activation to drive clear cell renal cell carcinoma progression

doi: 10.1038/s41419-025-08261-0

Figure Lengend Snippet: A qRT-PCR analysis of LDHA mRNA expression levels in ACHN and 786-O cells following YBX1 knockdown and overexpression. B CUT&Tag peak plots and heatmap showing YBX1 binding intensity in ACHN cells. C Pie chart showing the distribution of YBX1 binding regions. D IGV plot showing the occupancy of YBX1 in the LDHA promoter region. E Motif diagram of YBX1 binding sequences from the JASPAR database. F Molecular docking analysis of YBX1 with four predicted binding sites (BS) in the LDHA promoter sequence. G The LDHA promoter sequence was divided into 3 fragments containing the predicted BS, followed by primer design and ChIP-qPCR to verify YBX1 binding to the LDHA promoter region. H The LDHA promoter sequence was divided into 4 fragments, and the BS-1 site that was mainly bound was mutated, the luciferase-tagged truncated plasmids were designed. After co-transfection of the target and control Renilla plasmids into ccRCC cells, YBX1 binding to the LDHA promoter region was verified using dual-luciferase reporter assays. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns: no significant difference. Unpaired two-sided Student’s t -test in A, G and H. Data are presented as mean ± SD.

Article Snippet: After removing the supernatant, 50 μL of Antibody Buffer was added to resuspend the beads-bound cells, followed by the addition of 1 μg of YBX1 antibody (Proteintech, USA) and Rabbit IgG (Cell Signaling Technology, USA), and left to stand overnight at 4 °C.

Techniques: Quantitative RT-PCR, Expressing, Knockdown, Over Expression, Binding Assay, Sequencing, ChIP-qPCR, Luciferase, Cotransfection, Control

Journal: Cell Death & Disease

Article Title: YBX1 orchestrates LDHA-mediated metabolic reprogramming and NF-κB activation to drive clear cell renal cell carcinoma progression

doi: 10.1038/s41419-025-08261-0

Figure Lengend Snippet: A Western blot analysis of LDHA protein expression levels in ACHN and 786-O cells after YBX1 knockdown and overexpression. B Dual-luciferase reporter assays evaluating the effects of YBX1 and LDHA on glycolysis-related signaling pathways in ccRCC cells co-transfected with target plasmids and control Renilla plasmids. C Western blot analysis of p-p65 (Ser536) and total p65 expression after overexpression of YBX1 and LDHA. D Immunohistochemical staining to detect protein expression of total p65 and p-p65 (Ser536) in tumor tissues from control and YBX1 knockdown groups in nude mice. Representative images and quantification. E, F Western blot ( E ) and qRT-PCR ( F ) analysis of LDHA knockdown efficiency in 786-O cells 72 h post-transfection with si- LDHA . G , H Western blot ( G ) and LDH activity ( H ) analysis of LDHA inhibition in 786-O cells treated with Oxamate (0-80 mM for 48 h). I , J Western blot analysis of p-p65 (Ser536) and total p65 in YBX1-overexpressing cells following LDHA knockdown ( I ) or Oxamate treatment (60 mM for 48 h) ( J ). K-N Lactate production ( K, L ) and cell proliferation ( M , N ) assays in YBX1-overexpressing cells after LDHA knockdown or Oxamate treatment. O Schematic diagram illustrating the YBX1-LDHA-NF-κB mechanism in ccRCC progression. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns: no significant difference. Unpaired two-sided Student’s t -test in B , D , F and H . One-way ANOVA with correction for multiple comparisons in K and L. Two-way ANOVA with correction for multiple comparisons in M and N. Data are presented as mean ± SD.

Article Snippet: After removing the supernatant, 50 μL of Antibody Buffer was added to resuspend the beads-bound cells, followed by the addition of 1 μg of YBX1 antibody (Proteintech, USA) and Rabbit IgG (Cell Signaling Technology, USA), and left to stand overnight at 4 °C.

Techniques: Western Blot, Expressing, Knockdown, Over Expression, Luciferase, Protein-Protein interactions, Transfection, Control, Immunohistochemical staining, Staining, Quantitative RT-PCR, Activity Assay, Inhibition

Journal: bioRxiv

Article Title: Neutralizing Gatad2a-Chd4-Mbd3 Axis within the NuRD Complex Facilitates Deterministic Induction of Naïve Pluripotency

doi: 10.1101/192781

Figure Lengend Snippet: A. MEFs harboring TetO-OKSM and M2rtTA cassettes were transfected with siRNA targeting different canonical NuRD components (indicated in the illustration), 2 and 4 days after reprogramming initiation following DOX administration. Reprogramming was then evaluated by AP staining at day 8. B. Reprograming efficiency following siRNA treatments was evaluated using AP staining, after 8 days of reprogramming (n=3, two-sided Student’s t-test p values are indicated). C. Cell growth curves of MEFs treated with siRNA for the indicated NuRD components (two-sided Student’s t-test p values are indicated). Knockdown (KD) of Gatad2a, unlike KD of Chd4, Mbd3 and Hdac2, does not severely inhibit cell proliferation. D. Representative images of cells treated with siRNA targeting Mbd3 or Gatad2a and exposed to BrdU in order to evaluate proliferation. E. Quantitative evaluation of BrdU incorporation test, which shows normal proliferation in siScramble and siGatad2a, unlike in cells treated with siMbd3 (n=8, two-sided Student’s t-test p values are indicated Student’s t-test). F . Knockdown for different canonical NuRD components does not show a significant elevation in cell death or apoptosis. Viability and apoptosis induction were measured using FACS following Annexin-PI staining. G. Reprogramming efficiency following siRNA treatments targeting different NuRD components, at different time points. KD was performed at two distinct cycles: the early one (Regimen 1, marked in black) started one day prior to DOX induction, and the second one (Regimen 2, marked in grey) started one day post-DOX induction. H. iPSC reprogramming efficiency following different siRNA treatments. was evaluated at day 8. (n=3 per each condition, two-sided Student’s t-test p values are indicated). Gatad2a siRNA improves reprogramming whether administrated prior or post DOX administration, unlike siRNA targeting Mbd3 or Chd4, in which only in Regimen #2 iPSC colony formation efficiency was increased.

Article Snippet: Secondary mouse embryonic fibroblast (MEF) from Mbd3 flox/- cell line (A12 clone: Mbd3 flox/ − cell lines that carries the GOF18-Oct4-GFP transgenic reporter (complete Oct4 enhancer region with distal and proximal enhancer elements) (Addgene plasmid #60527)) and WT* cell line (WT-1 clone that carries the deltaPE-GOF18-Oct4-GFP reporter (Addgene plasmid#52382) were previously described ( ).

Techniques: Transfection, Staining, Knockdown, BrdU Incorporation Assay

Journal: bioRxiv

Article Title: Neutralizing Gatad2a-Chd4-Mbd3 Axis within the NuRD Complex Facilitates Deterministic Induction of Naïve Pluripotency

doi: 10.1101/192781

Figure Lengend Snippet: A . Scheme demonstrating strategy for generating secondary isogenic Gatad2a WT and KO lines, and comparing their reprogramming efficiency side by side. 2i/LIF-KSR conditions were introduced at day 3.5 during the 8-day course. More detailed information is provided in Supplementary regarding different systems used herein. B . Targeting scheme of Gatad2a locus to generate knockouts by CRISPR/Cas9. C . Western blot validation of Gatad2a knockout in iPSC harboring M2rtTA and TetO-OKSM cassettes in comparison to its parental isogenic line. D . Representative images of Gatad2a -/- iPSC derived E13.5 chimera. Red arrow highlights mCherry+ chimera which originates from mCherry labeled iPSCs that were microinjected. E. Bulk iPSC reprogramming as in F , but experiment was terminated after 6 days and iPSC colony formation was evaluated by Alkaline Phosphatase staining (AP+). F . Representative flow cytometry measurements of ΔPEOct4-GFP reactivation dynamics in polyclonal/bulk Gatad2a-WT and Gatad2a-KO isogenic cell lines. Throughout the course of the reprogramming experiment the cells were not passaged to avoid any biases. Reprogramming of secondary MEFs seeded as single cells. G. Representative summaries of single-cell iPSC reprogramming efficiency experiment. Secondary isogenic Gatad2a WT and KO reprogrammable MEFs carrying constitutively expressed mCherry-NLS and naïve pluripotency specific ΔPE-Oct4-GFP reporter were sorted and seeded as single-cell per well. Reprogramming was initiated by DOX administration according to panel A . Reprogramming efficiency was assessed after 8 days based on the number of wells in which mCherry+ cells formed an ΔPE-Oct4-GFP positive colony. Throughout the course of the reprogramming experiment the cells were not passaged to avoid any biases. H. The indicated secondary Gatad2a +/+ and Gatad2a -/- somatic cell types were isolated and subjected to single cell reprogramming and evaluation of iPSC efficiency following 8 days of DOX. Reprogramming efficiency was assessed after 8 days based on the number of wells in which mCherry+ cells formed an ΔPE-Oct4-GFP positive colony. In summary, these results indicate that complete inhibition of Gatad2a (also known as P66a), a NuRD specific subunit, does not compromise somatic cell proliferation as previously seen upon complete Mbd3 protein elimination, and yet disrupts Mbd3/NuRD repressive activity on the pluripotent circuitry and yields 90-100% highly-efficient reprogramming within 8 days as similarly observed previously in Mbd3 hypomorphic Mbd3 flox/- donor somatic cells ( ; ).

Article Snippet: Secondary mouse embryonic fibroblast (MEF) from Mbd3 flox/- cell line (A12 clone: Mbd3 flox/ − cell lines that carries the GOF18-Oct4-GFP transgenic reporter (complete Oct4 enhancer region with distal and proximal enhancer elements) (Addgene plasmid #60527)) and WT* cell line (WT-1 clone that carries the deltaPE-GOF18-Oct4-GFP reporter (Addgene plasmid#52382) were previously described ( ).

Techniques: CRISPR, Western Blot, Biomarker Discovery, Knock-Out, Comparison, Derivative Assay, Labeling, Staining, Flow Cytometry, Isolation, Inhibition, Activity Assay

Journal: bioRxiv

Article Title: Neutralizing Gatad2a-Chd4-Mbd3 Axis within the NuRD Complex Facilitates Deterministic Induction of Naïve Pluripotency

doi: 10.1101/192781

Figure Lengend Snippet: A. Three different clonal sets of isogenic secondary OSKM reprogrammable cells/sets were generated: (i) MEFs were reprogrammed to iPSC following viral infection of FUW-M2rtTA and FUW-OKSM. mCherry constitutive marker and ΔPE-Oct4-GFP markers were then introduced to the iPSC. (ii) R26-M2rtTA +/- m. Col1a-OKSM +/- ES were derived from E3.5 embryos following mating of mice. (iii) ES Kh2 m.Col1a-TetO-2XFlag-Mbd3 were injected to blastocysts, and MEF were harvested at E12.5. MEF were then reprogrammed following viral infection of FUW-OKSM. All described cell lines were then subjected to Gatad2a-KO using CRISPR/Cas9, followed by an injection of both the KO and its isogenic WT to blastocysts. Chimeric fibroblasts were separated from the donor cells by Puromycin selection. B . Summary of CRISPR/Cas9 strategy to generate Gatad2a null cells. sgRNA targeted sequence is indicated. C . Correct targeting efficiency by the strategy described in B to generate mouse Gatad2a KO PSCs as determined both by Western blot analysis and PCR sequencing. D . Representative Western blot analysis for sub cloned lines following targeting Gatad2a with sgRNA. Blue arrows indicate examples of KO clonal lines. E. Transcriptome landscape (RNA-seq), alongside ATAC-seq and H3K27Ac ChIP-seq, of fibroblasts (FSP-1 and Thy1) and pluripotent (Nanog and Sall4) related genes. IGV Data range is indicated at the top right corner of the signal.

Article Snippet: Secondary mouse embryonic fibroblast (MEF) from Mbd3 flox/- cell line (A12 clone: Mbd3 flox/ − cell lines that carries the GOF18-Oct4-GFP transgenic reporter (complete Oct4 enhancer region with distal and proximal enhancer elements) (Addgene plasmid #60527)) and WT* cell line (WT-1 clone that carries the deltaPE-GOF18-Oct4-GFP reporter (Addgene plasmid#52382) were previously described ( ).

Techniques: Generated, Infection, Marker, Derivative Assay, Injection, CRISPR, Selection, Sequencing, Western Blot, Clone Assay, RNA Sequencing, ChIP-sequencing

Journal: bioRxiv

Article Title: Neutralizing Gatad2a-Chd4-Mbd3 Axis within the NuRD Complex Facilitates Deterministic Induction of Naïve Pluripotency

doi: 10.1101/192781

Figure Lengend Snippet: A. Mass spectrometry analysis of NuRD complex components binding efficiency to Mbd3 in Gatad2a-KO and its isogenic Gatad2a-WT line, in MEF cells during reprogramming by OSKM. Only putative NuRD components are presented and can be seen in an equal strength at both platforms, except for Chd4, which does not bind Mbd3 in Gatad2aKO. Flag-Tagged Mbd3 was used to establish a platform for studying Mbd3-binding proteins, by correct targeting of TetO-Mbd3-Flag into the M. Col1a locus. Isogenic Gatad2a-KO were generated from this line with CRISPR/Cas9 and used for the IP and MS analysis indicated above (See ). B. Flag-Mbd3 CoIP in Gatad2a-WT and Gatad2a-KO cells. Experiments were conducted both in MEF cells and ESs. C. Cells during reprogramming were treated with siRNA targeting Gatad2a, and pellets collected after four days. CoIP of Chd4 shows that siGatad2a prevents Mbd3 binding to Gatad2a but also to Chd4. D. Gatad2a-KO MEF with overexpression of Gatad2a (transgenic recovery; abbreviated as Gatad2a-Tg) or Mock were subjected to Chd4-CoIP. Gatad2aoverexpression recovers the binding of Chd4 to Mbd3 and does not affect its binding to Mta2. E. The coiled coil region of Mbd3 is highly conserved between different organisms, and different proteins in the MBD family. The highlighted amino acids are crucial for Gatad2a binding to Mbd2 or Mbd3. F. A scheme of Flag tagged Mbd3, and mutant forms of Mbd3: lacking the Coiled coil region (#CCR-Mbd3) or the methyl-binding domain (#MBD-Mbd3). G. WT-Mbd3 and both mutants were over-expressed in 293T cells and were subjected to Flag-Mbd3 CoIP to examine their protein interactions. While the deletion of MBD prevents the binding of Klf4, only the deletion of the coiled coil region abolished the binding to Chd4 and Gatad2a. H-I. Mbd3 fl/- secondary MEF, harboring #PE-Oct4-GFP reporter, were transfected with two different forms of Flag tagged WTMbd3 and ΔCCR-Mbd3. The cells were then subjected to reprogramming. While WT-Mbd3 significantly reduced reprogramming efficiency ( p Value<0.0001, two-sided Student’s t-test, n=3), ΔCCR-Mbd3 expression was not able to inhibit deterministic reprogramming in the cells, consistent with its inability to interact and recruit Chd4 to the assembled complex.

Article Snippet: Secondary mouse embryonic fibroblast (MEF) from Mbd3 flox/- cell line (A12 clone: Mbd3 flox/ − cell lines that carries the GOF18-Oct4-GFP transgenic reporter (complete Oct4 enhancer region with distal and proximal enhancer elements) (Addgene plasmid #60527)) and WT* cell line (WT-1 clone that carries the deltaPE-GOF18-Oct4-GFP reporter (Addgene plasmid#52382) were previously described ( ).

Techniques: Mass Spectrometry, Binding Assay, Generated, CRISPR, Over Expression, Transgenic Assay, Mutagenesis, Transfection, Expressing

Journal: bioRxiv

Article Title: Neutralizing Gatad2a-Chd4-Mbd3 Axis within the NuRD Complex Facilitates Deterministic Induction of Naïve Pluripotency

doi: 10.1101/192781

Figure Lengend Snippet: A. A scheme of Gatad2a constructs and mutants used. Gatad2a-WT, Gatad2a-Truncated (lacking the CCR2 of Gatad2a) and HA-tagged-CCR1-Gatad2a construct. B. Flag-Mbd3 and different Gatad2a constructs were cotransfected in 293T cells. Flag-Mbd3 binds both forms of Gatad2a (WT and truncated), as shown by Flag-Mbd3 CoIP. C. Dot blot analysis to validate protein expression following HA-Gatad2aCCR1 peptide overexpression. D. Overexpression of Flag-Mbd3 with HA-Gatad2a-CCR1 or control (HA-GFP) in 293T cells, followed by Co-IP for anti-Flag-Mbd3. Over-expression of Gatad2a-CCR1 reduces the binding of Mbd3 to endogenous Gatad2a and Chd4, comparing to the control specimen. Further, CCR can cause a reduction in Mbd3 binding to endogenous Gatad2a and Chd4, without changing other NuRD components (such as Hdac2) binding. E. Overexpression of STEMCCA-OKSM vector, HA-MBD (methyl-binding domain of Mbd3) or HA-GFP in 293T cells, was followed by Co-IP with anti-HA. The results demonstrate that MBD domain can bind to pluripotency factors such as Oct4 and to the NuRD component Hdac2 but cannot bind Gatad2a or Chd4. F. Summarizing scheme for three different approaches for neutralizing Mbd3-Gatad2a-Chd4 axis: (i) by deleting the CCR domain of Mbd3 (ii) KO or KD of Gatad2a protein and (iii) overexpression of an exogenous Gatad2a-CCR competitive peptide that interferes with Gatad2aCCR interaction with Mbd3.

Article Snippet: Secondary mouse embryonic fibroblast (MEF) from Mbd3 flox/- cell line (A12 clone: Mbd3 flox/ − cell lines that carries the GOF18-Oct4-GFP transgenic reporter (complete Oct4 enhancer region with distal and proximal enhancer elements) (Addgene plasmid #60527)) and WT* cell line (WT-1 clone that carries the deltaPE-GOF18-Oct4-GFP reporter (Addgene plasmid#52382) were previously described ( ).

Techniques: Construct, Dot Blot, Expressing, Over Expression, Control, Co-Immunoprecipitation Assay, Binding Assay, Plasmid Preparation

Journal: bioRxiv

Article Title: Neutralizing Gatad2a-Chd4-Mbd3 Axis within the NuRD Complex Facilitates Deterministic Induction of Naïve Pluripotency

doi: 10.1101/192781

Figure Lengend Snippet: A. Rosa26-M2rtTA Col1a:TetO-2XFlag-Mbd3 ES cells were subjected to different differentiation protocols, and cells from 5 distinct states (naïve ESC, EpiLC, EBs, MEF, 4-day OSKM Reprogramming) were subsequently subjected to CoIP with anti- Flag-Mbd3. Lysates were then analyzed by western blot, and reacted with different antibodies against different NuRD components, Pluripotency factors, and other epigenetic proteins. While some of the proteins show constitutive binding to Mbd3 throughout all differentiation states (Mta2, Prmt5)- other proteins show differential binding (Oct4, Klf4, Cdk2ap1) and the latter is not correlated with the proteins level in the cell. B. Rosa26-M2rtTA Col1a:TetO-2XFlag-Mbd3 ES cells were either maintained in ground state naïve conditions or in priming conditions (Fgf2/Activin A). Lysates were subjected to Co-IP with anti-Flag-Mbd3 and examined by Western blot. Oct4 protein expression is significantly reduced after priming, but its binding to Mbd3 can be detected only in the primed pluripotent state. C. Mbd3 expression in ES cells treated with growth media containing different small molecules, after 72 hours of treatment. Unlike other treatments, PKCi Go6983 (5 μM) treatment resulted in a radical decrease in Mbd3 protein expression. D. PKCi Go6983 effect on Mbd3 level is seen after approximately 48 hours, in different concentration ranging from 0.5 to 10 μM. E. WT EpiSCs reversion efficiency to naïve ESCs in different conditions. Anova test P values are indicated. (one representative experiment out of 3 performed is shown). F. Isogenic WT and Gatad2a KO ESCs were expanded on feeder free gelatin coated plates in N2B27 LIF only or LIF/PKCi conditions. Phase images and Oct4-GFP signal maintenance are shown after 8 passages (P8). Oct4-GFP. G. ES cells treated with naïve ground state condition were treated with shRNA targeting Ubc9 or Scramble. Cells were lysed and subsequent CoIP of Chd4 shows a decrease in Gatad2a binding to the protein. H. Cells induced in naïve ground state 2i/LIF conditions and subsequent shRNA targeting either for Ubc9 or scramble negative control. The cells were lysed and fractioned – Cytoplasm, Nucleoplasm and Chromatin fractions, proteins were analyzed by western blot. The NuRD components Mbd3 and Gatad2a, but not Mta2, can be seen mainly in the chromatin fraction, and their expression is significantly reduced following shUbc9 treatment.

Article Snippet: Secondary mouse embryonic fibroblast (MEF) from Mbd3 flox/- cell line (A12 clone: Mbd3 flox/ − cell lines that carries the GOF18-Oct4-GFP transgenic reporter (complete Oct4 enhancer region with distal and proximal enhancer elements) (Addgene plasmid #60527)) and WT* cell line (WT-1 clone that carries the deltaPE-GOF18-Oct4-GFP reporter (Addgene plasmid#52382) were previously described ( ).

Techniques: Western Blot, Binding Assay, Co-Immunoprecipitation Assay, Expressing, Concentration Assay, shRNA, Negative Control

Journal: bioRxiv

Article Title: Neutralizing Gatad2a-Chd4-Mbd3 Axis within the NuRD Complex Facilitates Deterministic Induction of Naïve Pluripotency

doi: 10.1101/192781

Figure Lengend Snippet: A. Western blot showing Mbd3 protein levels in ESCs expanded in the indicated growth conditions. Mbd3 levels were decreased following PKCi (Go6983) treatment in ES cells maintained in different conditions (FBS and KSR based growth media, supplemented with 2i). B. Reduction in Mbd3 protein expression is maintained following long term PKCi (Go6983) treatment, as examined in different concentrations (0.5-5 μM). Media was exchanged ever 48h. C. Western blot analysis for different NuRD components in ESC cells with and without Go6983. Analysis shows that other NuRD components expression level is not affected from PKCi (Go6983) treatment. D. as in A, but a different PKCi was used, termed GFX which does not inhibit atypical PKC pathway. E. Mbd3 protein levels following shRNA treatment of mouse V6.5 ESCs for atypical PKCzeta isoform. F. Mbd3 levels in MEFs following treatment with Go6983 are not affected. G. RT-PCR analysis for Mbd3 transcript abundance 24 and 48h following PKCi Go6983 treatment, do not show significant changes and reduction in Mbd3 transcript level. This suggests that the depletion in Mbd3 protein levels shown in A-C is post-translational. H. Go6983 causes mild depletion in Mbd3 in MEFs following 3 days of OSKM expression. I . Gatad2a +/+ WT MEFs carrying ΔPE -Oct4-GFP reporter and constitutive mCherry markers (secondary system i) were subjected to iPSC reprogramming protocols in in and iPSC efficiency was quantified at day 8. In the last two conditions included in the panel, the MEFs were pre-treated with control and Ubc9 shRNA following with Neomycin selection , and then subjected to DOX mediated iPSC reprogramming. Anova P values are indicated. J. A Schematic representation of Gatad2a known domains and confirmed SUMOylation sites . Importantly, harnessing prediction tools for identification of possible SUMO consensus sites results in 2 additional sites inside the coiled coil regions (Chi-squared test p value<0.05). K. 2-d08 specific small molecule inhibitor for SUMOylation was applied during co-IP experiments for Chd4 at the indicated increasing concentrations known to deplete global SUMOylation levels. Cells were lysed the CoIP of Mbd3 shows a decrease in Gatad2a binding to Chd4, as similarly seen with shRNA depletion of Ubc9 . L. As in with immunoblot for SUMO2/3 on the same exact gel series.

Article Snippet: Secondary mouse embryonic fibroblast (MEF) from Mbd3 flox/- cell line (A12 clone: Mbd3 flox/ − cell lines that carries the GOF18-Oct4-GFP transgenic reporter (complete Oct4 enhancer region with distal and proximal enhancer elements) (Addgene plasmid #60527)) and WT* cell line (WT-1 clone that carries the deltaPE-GOF18-Oct4-GFP reporter (Addgene plasmid#52382) were previously described ( ).

Techniques: Western Blot, Expressing, shRNA, Reverse Transcription Polymerase Chain Reaction, Control, Selection, Co-Immunoprecipitation Assay, Binding Assay

Journal: Nature Communications

Article Title: UBR5 promotes antiviral immunity by disengaging the transcriptional brake on RIG-I like receptors

doi: 10.1038/s41467-024-45141-1

Figure Lengend Snippet: a Immunoblots of UBR5 protein in wild type (WT) and three UBR5 −/− (made with unique guide RNA) in 2fTGH-ISRE-Luc cells. Quantification of the ( b ) intracellular ISRE-Luc activity, c secreted IFN-β protein by ELISA, d cellular IFNB1 mRNA levels in 2fTGH-ISRE-Luc cells transfected with poly (I:C) for 12 h. Quantification of the ( e ) intracellular ISRE-Luc activity, f secreted IFN-β protein, g cellular IFNB1 mRNA, h secreted type I IFNs (Bioassay) in WT, UBR5 −/− (made with gRNA #1) and IFIH1 −/− (gene symbol for MDA5) HEK293T cells transfected with poly (I:C) for 12 h. i Quantification of ISRE-Luc activity in 2fTGH-ISRE-Luc cells transfected with a negative control siRNA (siCtrl) or UBR5 siRNA for 48 h, then poly (I:C) for 12 h. j Measurement of the Luc activity in HEK293T cells transfected with a pcDNA3.1 vector or UBR5 expression plasmid, together with an IFNB1 promoter-driven firefly luciferase (Luc) and an ACTIN promoter-driven renilla Luc plasmid (internal control) for 24 h. The cells were then transfected with poly (I:C) for 12 h. k Quantification of the IFNB1 mRNA in HEK293T cells transfected with a vector or UBR5 expression plasmid for 24 h, then poly (I:C) for 12 h. l Immunoblots of UBR5 protein in mouse primary embryonic fibroblasts (MEF) or bone marrow-derived macrophages. Quantification of ( m ) the Ifnb1 mRNA in MEFs, secreted IFN-β protein in ( n ) MEFs and ( o ) macrophages transfected with poly (I:C). Quantification of the ( p ) Ifnb1 mRNA levels, q secreted IFN-β protein in MEFs transfected with 5-ppp hpRNA (RIG-I agonist). Ubr5 iKO : Ubr5 inducible knockout by Tamoxifen. Data presented in b – d , g – i : mean ± S.E.M, ordinary one-way ANOVA with Dunnett’s test, n = 3 biological independent experiments; for b : **** p < 0.0001, *** p = 0.0003, * p = 0.0305 vs WT; for c : **** p < 0.0001 vs WT; for d : *** p = 0.0003, ** p = 0.0022 vs WT; for g : *** p = 0.0001, **** p < 0.0001 vs WT; for h : **** p < 0.0001 vs WT; for i : **** p < 0.0001, *** p = 0.0005 vs siCtrl. Mult i plicity adjusted p values are p resented. Data presented in e , f , j , k , m – q : mean ± S.E.M, two-tailed student’s t test; for e : *** p = 0.0007, **** p < 0.0001, n = 4 biological independent experiments; for f : *** p = 0.0002, * p = 0.012; for j : ** p = 0.0015, ** p = 0.0018 in sequence; for k : ** p = 0.0014, ** p = 0.0084 in sequence; for m : * p = 0.0224, **** p < 0.0001; for n : ** p = 0.0024, * p = 0.0407; for o : * p = 0.0478, * p = 0.0129, *** p = 0.0002 in sequence; for p : *** p = 0.0005, *** p = 0.0001 in sequence; for q : * p = 0.0263, * p = 0.0138 in sequence; n = 3 biological independent experiments in f , j , k , m – q . Adjusted p values are presented. Source data are provided as a Source Data file.

Article Snippet: The anti-GAPDH (Cat# 60004-1-Ig, 1:2000), UBR5 (Cat# 22782-1-AP, 1:200 for IF) and GFP (Cat # 50430-2-AP, 1:1000) were purchased from Proteintech Group, Inc (Rosemont, IL, USA).

Techniques: Western Blot, Activity Assay, Enzyme-linked Immunosorbent Assay, Transfection, Bioassay, Negative Control, Plasmid Preparation, Expressing, Luciferase, Control, Derivative Assay, Knock-Out, Two Tailed Test, Sequencing

Journal: Nature Communications

Article Title: UBR5 promotes antiviral immunity by disengaging the transcriptional brake on RIG-I like receptors

doi: 10.1038/s41467-024-45141-1

Figure Lengend Snippet: a The immunoblots of UBR5 in various tissues of age- and sex-matched littermates. Black triangle indicates the right band size. The survival curves of littermates infected with ( b ) 100 or ( c ) 1,000 plaque forming units (PFU) of EMCV intraperitoneally. In b , n = 16 for Ubr5 WT and 17 for Ubr5 iKO , p = 0.038 (Log-Rank test); in c , n = 14 for Ubr5 WT and 10 for Ubr5 iKO , p = 0.030 (Log-Rank test). Quantification of ( d ) EMCV RNA in the whole blood cells by qRT-PCR, e viremia and ( f ) viral loads in hearts by a plaque forming assay, in the mice infected with 100 PFU of EMCV. In d – f , n = 6 mice, mean ± S.E.M., two-tailed, unpaired non–parametric Mann–Whitney U test; * p = 0.0411 ( d ), * p = 0.0152 ( e ), * p = 0.0411 ( f ). g , h Quantification of the serum type I IFN and cytokine concentrations by ELISA in the mice infected with 1 × 10 7 PFU of EMCV intravenously. n = 7 mice/group, mean ± S.E.M., two-tailed Student’s t test; *** p = 0.0009 for IFN-β, * p = 0.0221 for IFN-α ( g ); * p = 0.0259 for TNF-α, ** p = 0.0055 for CXCL10, ** p = 0.0034 for MIP−1α, ** p = 0.0049 for MIP−1β ( h ). i The survival curves of age- and sex-matched littermates infected with 1 × 10 7 PFU of VSV intravenously. n = 11 mice/group, p = 0.03 (Log-Rank test). j The arbitrary morbidity score of VSV-infected animals. n = 6 mice/group, mean ± S.E.M., two-tailed Student’s t test; * p = 0.0438, * p = 0.0438 in sequence. Adjusted p values are presented. All the mice used in this study were 8 weeks old. Source data are provided as a Source Data file.

Article Snippet: The anti-GAPDH (Cat# 60004-1-Ig, 1:2000), UBR5 (Cat# 22782-1-AP, 1:200 for IF) and GFP (Cat # 50430-2-AP, 1:1000) were purchased from Proteintech Group, Inc (Rosemont, IL, USA).

Techniques: Western Blot, Infection, Quantitative RT-PCR, Two Tailed Test, MANN-WHITNEY, Enzyme-linked Immunosorbent Assay, Sequencing

Journal: Nature Communications

Article Title: UBR5 promotes antiviral immunity by disengaging the transcriptional brake on RIG-I like receptors

doi: 10.1038/s41467-024-45141-1

Figure Lengend Snippet: The immunoblots of indicated proteins in ( a ) mouse primary MEFs, macrophages and various human cell lines, ( b ) the tissues of age- and sex-matched littermates. The immunoblots of indicated proteins in HEK293T cells ( c ) transfected with poly (I:C)/without (Mock) for 12 h, and ( d ) infected with VSV at a multiplicity of infection (MOI) of 0.5. e The immunoblots of indicated proteins in UBR5 −/− HEK293T cells transfected with a UBR5 expression or vector plasmid for 24 h and then poly (I:C) (+) for 12 h. The bar chart indicates the ratios of MDA5/RIG-I band density to Actin. f The immunoblots of indicated proteins in WT HEK293T cells transfected with a vector, wild-type UBR5, or catalytic mutant C2768A plasmid. The bar chart indicates the ratios of MDA5/RIG-I band density to Actin. n = 2 biologically independent experiments. qRT-PCR quantification of the IFIH1 / DDX58 (gene symbol for MDA5/RIG-I) mRNA levels in ( g ) HEK293T cells, and ( h ) MEFs transfected with poly (I:C). The data are representative of three independent experiments ( a , c , d ) or tissues from two mice ( b ) with similar results. The data shown in ( e ) are from one representative experiment of n = 3 biological independent experiments, mean ± S.E.M, ordinary one-way ANOVA with Dunnett’s test, *** p = 0.0008, ** p = 0.0036 for MDA5; *** p = 0.0009, ** p = 0.0023 for RIG-I. Multiplicity adjusted p values are presented. Data shown in g , h : mean ± S.E.M, two-tailed Student’s t test, n = 3 biologically independent experiments; *** p = 0.0004, ** p = 0.0028 in g ; for h : * p = 0.0493, * p = 0.0458, ** p = 0.0017 in sequence for Ifih1 ; * p = 0.0286, ** p = 0.0086, * p = 0.0472 in sequence for Ddx58 . Adjusted p values are presented. Source data are provided as a Source Data file.

Article Snippet: The anti-GAPDH (Cat# 60004-1-Ig, 1:2000), UBR5 (Cat# 22782-1-AP, 1:200 for IF) and GFP (Cat # 50430-2-AP, 1:1000) were purchased from Proteintech Group, Inc (Rosemont, IL, USA).

Techniques: Western Blot, Transfection, Infection, Expressing, Plasmid Preparation, Mutagenesis, Quantitative RT-PCR, Two Tailed Test, Sequencing

Journal: Nature Communications

Article Title: UBR5 promotes antiviral immunity by disengaging the transcriptional brake on RIG-I like receptors

doi: 10.1038/s41467-024-45141-1

Figure Lengend Snippet: a The immunoblots of indicated proteins in 2fTGH cells transfected with poly (I:C) or infected with VSV-GFP at a MOI of 0.5. b The fluorescent images of VSV-GFP in 2fTGH cells at 12 h p.i . Scale bar: 50 µM. Quantification of the ( c ) IFIH1 / DDX58 (gene symbol for MDA5/RIG-I) and ( d ) IFNB1 mRNA levels by qRT-PCR in 2fTGH cells infected with VSV for 12 h. Data shown in c and d are presented as mean ± S.E.M, two-tailed Student’s t test, n = 3 biologically independent experiments; * p = 0.0492, * p = 0.0189 in sequence for IFIH1 , * p = 0.0286, * p = 0.0483 in sequence for DDX58 in c ; * p = 0.0489 in d . Adjusted p values are presented. e Scatter plot showing the 49 proteins identified by FLAG-UBR5- immunoprecipitated (IP)-mass spectrometer (MS) analysis. The 17 proteins validated at Harmonizome database are labeled. Positive proteins: log 2 (FC of A.P.I) > 1, two-tailed Student’s t test with Benjamini–Hochberg, p < 0.05, FC of A.P.I: fold change of Average Precursor Intensity. f , g FLAG-UBR5 co-immunoprecipitated (IP) with endogenous TRIM28, and vice versa. FLAG-UBR5/TRIM28 or vector plasmid was expressed in HEK293T cells, and immunoprecipitated with an anti-FLAG antibody. The indicated proteins were immunoblotted (IB) with specific antibodies. WCL: whole cell lysate. The immunoblots of indicated proteins in HEK293T cells transfected with ( h ) a negative siRNA (Ctrl) or UBR5 siRNA for 48 h, ( i ) with a UBR5 express i on or vector plasmid for 24 h. The data are representative of two independent experiments with similar results ( a , f – i ). Source data a re provided as a Source Data file.

Article Snippet: The anti-GAPDH (Cat# 60004-1-Ig, 1:2000), UBR5 (Cat# 22782-1-AP, 1:200 for IF) and GFP (Cat # 50430-2-AP, 1:1000) were purchased from Proteintech Group, Inc (Rosemont, IL, USA).

Techniques: Western Blot, Transfection, Infection, Quantitative RT-PCR, Two Tailed Test, Sequencing, Immunoprecipitation, Mass Spectrometry, Labeling, Plasmid Preparation

Journal: Nature Communications

Article Title: UBR5 promotes antiviral immunity by disengaging the transcriptional brake on RIG-I like receptors

doi: 10.1038/s41467-024-45141-1

Figure Lengend Snippet: a WT and UBR5 −/− HEK293T cells were transfected with FLAG-TRIM28, His-UBC9 and HA- or Myc -SUMO plasmids for 24 h, then without (Mock) or with poly (I:C) for 12 h. FLAG-TRIM28 was immunoprecipitated (IP) with an anti-FLAG antibody, and the IP and whole cell lysate (WCL) were immuno-blotted (IB) for the indicated proteins with specific antibodies. b WT and UBR5 −/− HEK293T cells were transfected with FLAG-TRIM28, HA-tagged WT, K48 or K63-only ubiquitin (Ub) for 24 h. The IP and IB was performed as above. c WT and UBR5 −/− HEK293T cells were transfected with FLAG-TRIM28 or vector for 24 h, then with poly (I:C) for 12 h. The IP and IB was carried out as above. The bar chart in a – c indicates the ratios of the indicated protein band density. n = 2 biologically independent experiments. d WT and UBR5 −/− HEK293T cells were infected with VSV at a MOI of 0.5. Endogenous TRIM28 was immunoprecipitated with an anti-TRIM28 antibody. e The bar chart indicates the ratios of the indicated protein band density in d . n = 2 biologically independent experiments. f The method to identify ubiquitinated sites within TRIM28. g The method for generating K507R and K779R mutants of TRIM28. h HEK293T cells were transfected with FLAG-TRIM28 (WT, K507R, K779R), GFP-UBR5, GFP-UBR5-C2758A mutant and HA-K63Ub plasmids for 24 h, then FLAG-TRIM28 was immunoprecipitated with an anti-FLAG antibody, and the IP and WCL were immunoblotted for the indicated proteins with specific antibodies. I – k WT and UBR5 −/− HEK293T cells were infected with VSV at a MOI of 0.5 for 8 h. Chromatin immunoprecipitation (ChIP) was performed with an anti-TRIM28 antibody. i The TRIM28-bound RLR promoter DNA was quantified by qPCR and normalized to its input. Bar: mean ± S.E.M, two-tailed Student’s t test, n = 4 biologically independent samples, *** p = 0.0006, ** p = 0.0094 for IFIH1 ; ** p = 0.0029; * p = 0.0382. Adjusted p values are presented. j ChIP-seq analysis and genomic annotation of TRIM28-bound sites in infected cells. UTR: untranslated regions. k Fold enrichment inTRIM28-bound promoter regions of select ISGs. The P -value was generated in Peak calling statistics using a Poisson distribution with local lambda estimate. Source data are provided as a Source Data file.

Article Snippet: The anti-GAPDH (Cat# 60004-1-Ig, 1:2000), UBR5 (Cat# 22782-1-AP, 1:200 for IF) and GFP (Cat # 50430-2-AP, 1:1000) were purchased from Proteintech Group, Inc (Rosemont, IL, USA).

Techniques: Transfection, Immunoprecipitation, Ubiquitin Proteomics, Plasmid Preparation, Infection, Mutagenesis, Chromatin Immunoprecipitation, Two Tailed Test, ChIP-sequencing, Generated

Journal: Nature Communications

Article Title: UBR5 promotes antiviral immunity by disengaging the transcriptional brake on RIG-I like receptors

doi: 10.1038/s41467-024-45141-1

Figure Lengend Snippet: a , b Volcano plots of differentially expressed genes (DEGs) in UBR5 −/− versus WT cells with/or without poly (I:C)-stimulation. Red and blue represent significant DEGs of upregulated (log 2 FC ≥ 1, p < 0.05) and downregulated (log 2 FC ≤ −1, p < 0.05), res p ectively. DESeq2 was used to perform a comparison of gene expression between defined groups, the Wald test was used to generate log 2 FC and p -values adjusted with the Benjamini–Hochberg. c GSEA plot of a significant gene set associated with RLR pathway and ISGs, p values were calculated by one-way ANOVA with Tukey’s post hoc comparison. d Top KEGG and Reactome pathways enriched from downregulated DEGs in UBR5 −/− versus WT cells upon poly (I:C)-stimulation ( p < 0.05, right-tailed Fisher’s exact t test with Benjamini & Hochberg). e Venn diagram revealing 42 genes overlapping between the DEGs upregulated in TRIM28 −/− and downregulated in UBR5 −/− cells. f Top Reactome pathways enriched from upregulated DEGs in TRIM28 −/− versus WT cells upon poly (I:C)-stimulation. Source data are provided as a Source Data file.

Article Snippet: The anti-GAPDH (Cat# 60004-1-Ig, 1:2000), UBR5 (Cat# 22782-1-AP, 1:200 for IF) and GFP (Cat # 50430-2-AP, 1:1000) were purchased from Proteintech Group, Inc (Rosemont, IL, USA).

Techniques: Comparison, Gene Expression

Journal: bioRxiv

Article Title: SURVIVIN IN SYNERGY WITH BAF/SWI COMPLEX BINDS BIVALENT CHROMATIN REGIONS AND ACTIVATES DNA DAMAGE RESPONSE IN CD4+ T CELLS

doi: 10.1101/2024.03.05.583464

Figure Lengend Snippet: (A) Heatmap of ChIP-seq peaks within and outside the bivalent chromatin regions (BvCR) defined by genomic overlap between the histone H3-marks. (B) Cartoon of BvCR and survivin deposition. (C) Frequency difference of BvCR without and with survivin. Chi-square test p-value is shown. Numbers within bars indicate percentage of BvCR dominant in individual H3 marks. (D) Box plots of survivin peak scores within BvCR dominant by H3K4me3, H3K27me3 and H3K27ac marks. Kolmogorov-Smirnov test p-values are shown. (E) Histogram of fold change in mean H3 peak score in naïve and YM155-treated CD4+ cells within (black) and outside BvCR (colored). (F) Box plot of H3 tag change within H3K4me3 dominant BvCR, after YM155 treatment. Mann-Whitney test p-values are indicated. (G) Frequency of changeable BvCR that shifts in dominant H3 after YM155 treatment. Kolmogorov-Smirnov test p-values are shown.

Article Snippet: For RNA-seq, CD4 + cell cultures were treated with recombinant IFNγ (50 ng/ml; Peprotech, Cranbury, NJ, USA) and survivin inhibitor sepantronium bromide ( ) (10 nM YM155, S1130, Selleck Chemicals, Houston, TX) for 72h.

Techniques: ChIP-sequencing, MANN-WHITNEY

Journal: bioRxiv

Article Title: SURVIVIN IN SYNERGY WITH BAF/SWI COMPLEX BINDS BIVALENT CHROMATIN REGIONS AND ACTIVATES DNA DAMAGE RESPONSE IN CD4+ T CELLS

doi: 10.1101/2024.03.05.583464

Figure Lengend Snippet: (A) Cartoon of analysis strategy. BvCR within genomic regulatory elements ( cis -RE, grey boxes) connected to genes, filtered on the protein-coding genes expressed in CD4 + cells, by RNA-seq. Transcription difference in CD4 + cells treated with IFNg or IFNg+YM155 compared to sham cultures was calculated by DESeq2. Differentially expressed genes (DEG) were defined by a nominal p-value<0.05. (B) Radar plot of Spearman’s rho correlations between H3K4me3 and H3K27me3 tag deposition change in all and survivin-positive BvCR and transcription change in CD4 + cells treated with IFNg or IFNg+YM155. Arrows indicate direction of transcription change. (C) Bubble plot of enrichment in biological processes among CD4 + expressed genes connected to all and survivin-positive BvCR. Bubble size indicates protein number in the process. Color intensity shows false discovery range (FDR). (D) Nodes of the DDR network are colored by dominant H3 mark in BvCR connected to genes within nodes (top map) and by transcription change after IFNγ or YM155 treatment (bottom map). Size of bubble corresponds to percentage of BvCR-connected genes within each node. DDR, DNA damage response. MMR, mismatch repair. RFC, replicator factor C. SSB, single strand break. DSB, double-strand break. HR, homologous recombination. MRN, MRE11-RAD50-NBS1. (E) Confocal image of THP1 nucleus showing presence of survivin (red), nucleus (blue) and BRG1 or H3K4me3 (green). (F) Heatmap of normalized tag deposition of H3 marks, by ChIP-seq, in BvCR connected to DEG treated with IFNγ+YM155. Shaded squares indicate survivin-positive BvCR. Genes connected to multiple BvCR are marked in bold. (G) Heatmap of RNA-seq transcription difference in genes annotated to DNA repair and stress response categories. Transcription difference was calculated by DESeq2 statistics, p-values * < 0.05, ** < 0.01, *** <0.001.

Article Snippet: For RNA-seq, CD4 + cell cultures were treated with recombinant IFNγ (50 ng/ml; Peprotech, Cranbury, NJ, USA) and survivin inhibitor sepantronium bromide ( ) (10 nM YM155, S1130, Selleck Chemicals, Houston, TX) for 72h.

Techniques: RNA Sequencing, Homologous Recombination, ChIP-sequencing

Journal: bioRxiv

Article Title: SURVIVIN IN SYNERGY WITH BAF/SWI COMPLEX BINDS BIVALENT CHROMATIN REGIONS AND ACTIVATES DNA DAMAGE RESPONSE IN CD4+ T CELLS

doi: 10.1101/2024.03.05.583464

Figure Lengend Snippet: (A) Bar plot of motif enrichment in survivin peaks within and outside of BvCR. MEME motifs enriched within BvCR. Venn diagram of TF identified by DNA sequence motif (MEME suite) and location (ChIP-seq) and mass spectrometry (MS). Motif enrichment within BvCR not containing survivin is not shown as no enrichment was observed. (B1) Dot plot of enrichment for human proteins/TF within BvCR, by ChIP-seq based ReMap2022 database. (B2) Box plot of enrichment for BAF/SWI complex proteins in ReMap2022 database. Counts underneath protein names indicates the number of overlaps with BvCR. (C) Coomassie-stained electrophoresis gel depicts survivin-bound proteins precipitated from THP1 cell lysate, separated by molecular weight (MW). Lanes represent two independent experiments (1 and 2, respectively). Bands with BAF/SWI complex proteins identified by mass spectrometry are indicated by boxes. (D) Table of BAF/SWI proteins identified by mass spectrometry. Enrichment score, number of unique peptides, and sequence coverage are calculated by MaxQuant software. (E) Bubble plot of biological processes enriched in proteins colocalized with survivin. Number of proteins in the process is indicated by the bubble size. Fold enrichment is shown by color intensity. (F) Ribbon diagram of the canonical BAF/SWI complex (PDB ID: 6LTJ) depicts the regions with a high probability of survivin binding (red) predicted by functional composition analysis. (G) Ribbon diagram depicting interaction between the human survivin-H3 tail complex (PDB ID: 3UEF) and the canonical BAF/SWI complex (PDB ID: 6LTJ) predicted by protein-protein docking analysis.

Article Snippet: For RNA-seq, CD4 + cell cultures were treated with recombinant IFNγ (50 ng/ml; Peprotech, Cranbury, NJ, USA) and survivin inhibitor sepantronium bromide ( ) (10 nM YM155, S1130, Selleck Chemicals, Houston, TX) for 72h.

Techniques: Sequencing, ChIP-sequencing, Mass Spectrometry, Staining, Electrophoresis, Molecular Weight, Software, Binding Assay, Functional Assay

Journal: bioRxiv

Article Title: SURVIVIN IN SYNERGY WITH BAF/SWI COMPLEX BINDS BIVALENT CHROMATIN REGIONS AND ACTIVATES DNA DAMAGE RESPONSE IN CD4+ T CELLS

doi: 10.1101/2024.03.05.583464

Figure Lengend Snippet: (A) A model of intervention in IFNγ, survivin-BAF complex and BvCR dependent transcription of DNA damage response (DDR) genes by treatment with JAK-inhibitor (JAKi) and survivin inhibitor YM155. Cis-RE, regulatory element. (B) Histogram of 7AAD + CD4 + cell distribution by phases of the cell cycle. Colored areas indicate G1 (blue), S (yellow) and G2 (green) phases. (C) Frequency distribution of 7AAD+ cells by cell cycle phases in CD4 + cells treated with IFNγ (50ng/ml), YM155 (10nM), and JAKi (10µM) compared to sham (DMSO). P-values are obtained by Wilcoxon paired test. (D) Linear plot of gene expression change in CD4 + cells treated with IFNγ (50ng/ml for 48h) measured by qPCR. Expression difference was calculated by Wilcoxon paired test. (E, F) THP1 cells were treated for 24h with YM155 (200nM), JAKi (50 µM), or sham (DMSO), fixed and stained for DNA damage using antibodies against BRG1 (red), γH2AX (green) and nuclear stain (blue). (G) γH2AX foci in nuclei were counted in each nucleus of 90-200 cells per treatment using ImageJ. P-values are obtained by Mann-Whitney U test. (H) Linear plot of gene expression change in CD4 + cells treated with YM155 (10nM) or JAKi (10 μM) for 48h, compared to sham (DMSO), measured by qPCR. Expression difference was calculated by Wilcoxon paired test.

Article Snippet: For RNA-seq, CD4 + cell cultures were treated with recombinant IFNγ (50 ng/ml; Peprotech, Cranbury, NJ, USA) and survivin inhibitor sepantronium bromide ( ) (10 nM YM155, S1130, Selleck Chemicals, Houston, TX) for 72h.

Techniques: Gene Expression, Expressing, Staining, MANN-WHITNEY

Journal: Epigenetics & Chromatin

Article Title: Unraveling the cohesin-chromatin interface: identifying protein interactions that modulate chromosome structure and function

doi: 10.1186/s13072-025-00596-4

Figure Lengend Snippet: Identification of the cohesin interactome with proximity ligation mass spectrometry. A Schematic of the experimental setup for cohesin proximity ligation followed by mass spectrometry. Transgenic NIH-3T3 mouse fibroblast cell lines were generated containing a 3xHA-TurboID or RAD21-3xHA-TurboID transgene with a tetracycline responsive element (TRE). Cell lines were treated with doxycycline for 36 h to promote expression of the transgenes. Additionally, RAD21-3xHA-TurboID cells that were not treated with doxycycline serve as a No TurboID control. Following doxycycline treatment, cells were treated ± biotin for 2 h. Cells were collected, lysed, and nuclear proteins were extracted. A streptavidin enrichment was performed, and a quality control check was done on 10% of the protein sample by western blotting for biotinylated proteins, while the remaining 90% of the streptavidin enriched sample was used for LC–MS/MS. Proteins identified by mass spectrometry were analyzed by two methods to identify proteins enriched relative to controls. Proteins enriched in the RAD21-3xHA-TurboID biotin treated sample relative to the: (1) 3xHA-TurboID biotin treated sample (Tier 1; p < 0.05, FC > 2); or (2) No TurboID biotin treated sample (Tier 2; p < 0.05, FC > 2). Each condition was performed in triplicate. B Co-purification of RAD21-3xHA-TurboID protein with endogenous cohesin subunits. IgG (negative control), anti-SMC3, and anti-HA antibodies were used to purify cohesin complexes in nuclear extracts from RAD21-3xHA-TurboID expressing cells under high stringency conditions. Cohesin subunits were detected by western blotting (SMC3, SMC1, RAD21, and HA). Arrows indicate endogenous RAD21 (black) and transgenic RAD21-3xHA-TurboID proteins (green). Box contains a depiction of core cohesin complex with RAD21-3xHA-TurboID. C Venn diagram showing the overlap of proteins detected in the Tier 1 cohesin interactome, Tier 2 cohesin interactome, and cohesin IP-MS experiments [ – ]. D , E Volcano plots showing proteins detected in the Tier 1 ( D ) and Tier 2 ( E ) cohesin interactomes. Proteins significantly enriched in Tier 1 and Tier 2 shown in red and blue, respectively. Known cohesin subunits and interactors are indicated with green dots and labels

Article Snippet: Primary antibodies used for western blotting include: cohesin subunits SMC1A (Bethyl, A300-080 A), SMC3 (Abcam, ab9263), and RAD21 (Bethyl, A300-080 and Active Motif, 39383); HA (Cell Signaling Technology, 3724S); SWI/SNF subunits SMARCC1 (Cell Signaling Technology 11956S), PBRM1 (Millipore, ABE70), ARID2 (Thermo Fisher Scientific, PA5-35857), ARID1A (Cell Signaling Technology, 12354S), SMARCA4/BRG1 (Abcam, ab110641); ISWI subunit BAZ1B (ABclonal, A9851); NuRD subunits CHD4 (Cell Signaling Technology, 12011S) and MTA1 (Cell Signaling Technology, 5647T); MLL subunits RBBP5 (Bethyl, A300109 A) and WDR5 (Cell Signaling Technology, 13105S); NuA4 subunit EPC1 (ABclonal, A5807); PcG subunit SUZ12 (Cell Signaling Technology, 3737S); mSin3A subunits SAP18 (ABclonal, A43907 ) and SIN3A (ABclonal, A1577); CTCF (Active Motif, 61312); Actin (Abcam, ab8227); and H3 (Abcam, ab1791).

Techniques: Ligation, Mass Spectrometry, Transgenic Assay, Generated, Expressing, Control, Western Blot, Liquid Chromatography with Mass Spectroscopy, Copurification, Negative Control, Protein-Protein interactions

Journal: Epigenetics & Chromatin

Article Title: Unraveling the cohesin-chromatin interface: identifying protein interactions that modulate chromosome structure and function

doi: 10.1186/s13072-025-00596-4

Figure Lengend Snippet: Cohesin co-localizes with SWI/SNF at enhancers, promoters, and CTCF sites. A Genome browser view of the Sox2 gene and super enhancer. ChIP-seq signal for cohesin (RAD21), SWI/SNF (BRG1), H3K4me3 (promoters), H3K27ac (enhancers), and CTCF (insulators) is shown. DNA interactions were detected by Micro-C and are shown at the top with a contact frequency heatmap and arcs for DNA loops (5 kb resolution). RAD21 peaks overlapping BRG1 peaks highlighted in purple, RAD21 peaks not overlapping BRG1 highlighted in green, and BRG1 peaks not overlapping RAD21 highlighted in orange. B Heatmaps showing ChIP-seq signal for cohesin (RAD21), SWI/SNF (BRG1), H3K4me3 (promoters), H3K27ac (enhancers), and CTCF (insulators) at sites where cohesin peaks do not overlap SWI/SNF peaks (top), sites where cohesin peaks overlap SWI/SNF peaks (middle), and sites where SWI/SNF peaks do not overlap cohesin peaks (bottom). Signal is z-score normalized. C UpSet plot of cohesin peaks overlapping CTCF, H3K27ac, H3K4me3, and/or BRG1

Article Snippet: Primary antibodies used for western blotting include: cohesin subunits SMC1A (Bethyl, A300-080 A), SMC3 (Abcam, ab9263), and RAD21 (Bethyl, A300-080 and Active Motif, 39383); HA (Cell Signaling Technology, 3724S); SWI/SNF subunits SMARCC1 (Cell Signaling Technology 11956S), PBRM1 (Millipore, ABE70), ARID2 (Thermo Fisher Scientific, PA5-35857), ARID1A (Cell Signaling Technology, 12354S), SMARCA4/BRG1 (Abcam, ab110641); ISWI subunit BAZ1B (ABclonal, A9851); NuRD subunits CHD4 (Cell Signaling Technology, 12011S) and MTA1 (Cell Signaling Technology, 5647T); MLL subunits RBBP5 (Bethyl, A300109 A) and WDR5 (Cell Signaling Technology, 13105S); NuA4 subunit EPC1 (ABclonal, A5807); PcG subunit SUZ12 (Cell Signaling Technology, 3737S); mSin3A subunits SAP18 (ABclonal, A43907 ) and SIN3A (ABclonal, A1577); CTCF (Active Motif, 61312); Actin (Abcam, ab8227); and H3 (Abcam, ab1791).

Techniques: ChIP-sequencing

Journal: Epigenetics & Chromatin

Article Title: Unraveling the cohesin-chromatin interface: identifying protein interactions that modulate chromosome structure and function

doi: 10.1186/s13072-025-00596-4

Figure Lengend Snippet: Altered cohesin occupancy at CTCF sites and enhancers upon SWI/SNF perturbation . A The SWI/SNF cBAF complex and three small molecules that inhibit or lead to degradation of the BRG1 subunit (dark orange). The ACBI1 PROTAC (red) targets the bromodomain of BRG1, recruiting an E3 ubiquitin ligase for poly-ubiquitination and proteasomal degradation. PFI-3 (purple) targets the bromodomain of the BRG1 subunit. BRM014 (blue) targets the ATPase active site of BRG1. The levels of SWI/SNF subunits shown in light orange were assessed following ACBI1 PROTAC treatment. B Genome browser views of RAD21 peaks that increase (up) or decrease (down) following ACBI1 PROTAC treatment. ChIP-seq signal for RAD21 with DMSO or ACBI1 treatment, BRG1, H3K4me3, H3K27ac, and CTCF is shown. Differential RAD21 peaks are indicated with a gray box. C Violin plots showing differential RAD21 signal in ACBI1 PROTAC treated mESCs relative to DMSO treated mESCs. The RAD21 peaks with significantly increased signal following ACBI1 treatment are shown in red, while peaks with significantly decreased signal are shown in gray. D Violin plots showing BRG1 (orange), H3K27ac (pink), and CTCF (dark blue) ChIP-seq signal at all RAD21 peaks, peaks with increased RAD21 signal (up) following ACBI1 treatment, and peaks with decreased RAD21 signal (down) following ACBI1 treatment. Significance determined by non-parametric Mann–Whitney test (****p-value < 0.0001). E Percentage of differential RAD21 peaks (up on left, down on right) upon ACBI1 treatment that overlap with the anchors of DNA loops detected by Micro-C, and percentage of non-differential RAD21 peaks with similar RAD21 signal intensity shown as a control. F Same as ( C ) for PFI-3 treatment. The RAD21 peaks with significantly increased signal following PFI-3 treatment are shown in purple, while peaks with significantly decreased signal are shown in gray. G Same as ( D ) for all RAD21 peaks and peaks with significantly different RAD21 signal following PFI-3 treatment ( n.s. not significant, *p-value < 0.01). H Same as ( E ) for PFI-3 treatment. I Same as ( B ) for BRM014 treatment. DNA loop anchor and long-range DNA interaction is depicted on top. J Same as ( C ) for BRM014 treatment. The RAD21 peaks with significantly increased signal following BRM014 treatment are shown in blue. K Same as ( D ) for all RAD21 peaks and peaks with increased RAD21 signal (up) following BRM014 treatment (****p-value < 0.0001). L Same as ( E ) for BRM014 treatment

Article Snippet: Primary antibodies used for western blotting include: cohesin subunits SMC1A (Bethyl, A300-080 A), SMC3 (Abcam, ab9263), and RAD21 (Bethyl, A300-080 and Active Motif, 39383); HA (Cell Signaling Technology, 3724S); SWI/SNF subunits SMARCC1 (Cell Signaling Technology 11956S), PBRM1 (Millipore, ABE70), ARID2 (Thermo Fisher Scientific, PA5-35857), ARID1A (Cell Signaling Technology, 12354S), SMARCA4/BRG1 (Abcam, ab110641); ISWI subunit BAZ1B (ABclonal, A9851); NuRD subunits CHD4 (Cell Signaling Technology, 12011S) and MTA1 (Cell Signaling Technology, 5647T); MLL subunits RBBP5 (Bethyl, A300109 A) and WDR5 (Cell Signaling Technology, 13105S); NuA4 subunit EPC1 (ABclonal, A5807); PcG subunit SUZ12 (Cell Signaling Technology, 3737S); mSin3A subunits SAP18 (ABclonal, A43907 ) and SIN3A (ABclonal, A1577); CTCF (Active Motif, 61312); Actin (Abcam, ab8227); and H3 (Abcam, ab1791).

Techniques: Ubiquitin Proteomics, ChIP-sequencing, MANN-WHITNEY, Control

Journal: bioRxiv

Article Title: Two distinct functional axes of positive feedback-enforced PRC2 recruitment in mouse embryonic stem cells

doi: 10.1101/669960

Figure Lengend Snippet: a) Schematic representation of the recruitment of PRC2.1 and PRC2.2. MTF2 binds to DNA, while the EED subunit of core PRC2 (orange) binds to H3K27me3 as part of an allosteric feedback loop. The EZH2 subunit of core PRC2 catalyses H3K27 methylation. The PRC2.2 complex contains JARID2 but not MTF2. Both contain the core PRC2 subunits, however the interactions of the PRC2.1 and PRC2.2-specific subunits with chromatin are different. The arrow from JARID2 to DNA is dashed as DNA binding has been shown in vitro but not in vivo b) PRC2.1 (MTF2) and PRC2.2 (JARID2) co-localize to all EZH2 targets. c-f) Heatmap and rpkm quantification (boxplots) of PRC2 subunits and the catalytic product H3K27me3. EZH2 recruitment is heavily affected by the absence of MTF2, while JARID2 and H3K27me3 absence have minor effects (c). The effect of MTF2 and JARID2 on EZH2 recruitment is reflected on H3K27me3 deposition (d). MTF2 is marginally affected by H3K27me3 removal, but its binding is reduced to approximately half the WT level in the absence of JARID2 (e). JARID2 recruitment is strongly reduced in the absence of either H3K27me3 or MTF2 (f). ChIP profiles are highly reproducible g) Genome browser examples of PRC2 binding to classical Polycomb targets. Box plots represent median and interquartile range (IQR; whiskers, 1.5 IQR).

Article Snippet: ChIP was performed using 3 μl/sample of the following antibodies: MTF2 (Aviva System Biology ARP34292, lot QC49692-42166), H3K27me3 (Millipore 07-449, lot 2717675), EZH2 (Diagenode C15410039, lot 003), JARID2 (Novus Biologicals NB100-2214, Lot E2), RING1B (Abcam, AB3832 lot GR86503-25).

Techniques: Methylation, Binding Assay, In Vitro, In Vivo

Journal: bioRxiv

Article Title: Two distinct functional axes of positive feedback-enforced PRC2 recruitment in mouse embryonic stem cells

doi: 10.1101/669960

Figure Lengend Snippet: a-d) Heatmap of WT ChIP-seq signal on the indicated peak set. H3K27me3-negative JARID2 peaks were excluded from further analysis. e) Venn diagram showing the overlap of peaks called for the ChIP-Seq of each protein independently. f) Mass spectrometry quantification of PRC2 subunits in the different cell lines. Detection of JARID2 and MTF2 in the respective mutants (asterisks) is due to value imputation in Perseus. g) Western blot validation of EED226 depletion of H3K27me3, for the ChIP shown in Figs 1 and 2. h) Scatterplot of peak RPKM showing high reproducibility of ChIP replicates.

Article Snippet: ChIP was performed using 3 μl/sample of the following antibodies: MTF2 (Aviva System Biology ARP34292, lot QC49692-42166), H3K27me3 (Millipore 07-449, lot 2717675), EZH2 (Diagenode C15410039, lot 003), JARID2 (Novus Biologicals NB100-2214, Lot E2), RING1B (Abcam, AB3832 lot GR86503-25).

Techniques: ChIP-sequencing, Mass Spectrometry, Western Blot

Journal: bioRxiv

Article Title: Two distinct functional axes of positive feedback-enforced PRC2 recruitment in mouse embryonic stem cells

doi: 10.1101/669960

Figure Lengend Snippet: a) Clustering of all PRC2 targets using ChIPseq data in multiple PRC2 mutants. Cluster 1-4 are unmethylated CpG islands (strong BioCap) signal, showing bivalent marks in WT (H3K4me3 and H3K27me3). These regions display heavy reduction of EZH2 recruitment in the MTF2 mutant, milder effects of H3K27me3 absence (EED226 treatment), and little or no effect of JARID2 absence. The intensity of MTF2 binding depends on both H3K27me3 and JARID2 but binding is still clearly detectable even in the absence of PRC2 core ( Eed -/- ) indicating a primary binding to DNA, reinforced by other mechanisms, such as JARID2-mediated recruitment, which in turn also depends on both H3K27me3 and MTF2. Cluster 5 and 6 have lower BioCap and H3K4me3 signal, and, while still affected by the absence of MTF2, this has a much less marked effect on recruitment of both EZH2 and JARID2, and on H3K27me3 deposition. b) WT-normalized, input-subtracted RPKM quantification of signal shown in (a). c) Quantification of GCG trinucleotides matching DNA shape requirement for MTF recruitments as defined in . Cluster 1-4 are strongly enriched in shape-matching GCGs, indicating potential for strong DNA-mediated MTF2 recruitment. d) Enrichment of anatomical terms in the genes associated with peaks in the six clusters. Enrichment within PRC2 targets. Cluster 4 show strong enrichment for CNS structures, cluster 5 and 6 for limb and branchial arches tissues and mesenchyme. See for the full overview.

Article Snippet: ChIP was performed using 3 μl/sample of the following antibodies: MTF2 (Aviva System Biology ARP34292, lot QC49692-42166), H3K27me3 (Millipore 07-449, lot 2717675), EZH2 (Diagenode C15410039, lot 003), JARID2 (Novus Biologicals NB100-2214, Lot E2), RING1B (Abcam, AB3832 lot GR86503-25).

Techniques: Mutagenesis, Binding Assay

Journal: bioRxiv

Article Title: Two distinct functional axes of positive feedback-enforced PRC2 recruitment in mouse embryonic stem cells

doi: 10.1101/669960

Figure Lengend Snippet: a) Heatmap showing the cluster specific effect of H3K27me3 depletion on the binding of EZH2. WT and MTF2 GT/GT show mild reduction of EZH2 binding when treated with EED226 inhibitor, while the treatment is highly synergistic with the depletion of JARID2. b) Bootstrapping-based RPKM quantification (methods) of the signal in (a). Each coloured dot represent the median of one round of bootstrapping, grey bar represent 99.9% confidence interval for the mean of bootstrapped values in each condition and cluster. c) Treatment with EED226 further affected MTF2 recruitment in Jarid2 -/- and JARID2 recruitment in Mtf2 GT/GT , with the former leading to recruitment patter closely resembling the Eed -/- line (cf. ), highlighting the recruitment differences between cluster 1-4 and 5-6. d) Bootstrapping-based RPKM quantification (methods) of the signal in (c) similar as in 3b. e) Genome browser view of example Polycomb targets. For each genotype two tracks are overlaid: the darker colour represent EED226 treated samples, the lighter colour untreated cells.

Article Snippet: ChIP was performed using 3 μl/sample of the following antibodies: MTF2 (Aviva System Biology ARP34292, lot QC49692-42166), H3K27me3 (Millipore 07-449, lot 2717675), EZH2 (Diagenode C15410039, lot 003), JARID2 (Novus Biologicals NB100-2214, Lot E2), RING1B (Abcam, AB3832 lot GR86503-25).

Techniques: Binding Assay

Journal: bioRxiv

Article Title: Two distinct functional axes of positive feedback-enforced PRC2 recruitment in mouse embryonic stem cells

doi: 10.1101/669960

Figure Lengend Snippet: a) Heatmap showing EZH2, MTF2 and JARID2 binding in the absence of H3K27me3 in PRC2 and PRC1 mutant lines. In the absence of H3K27me3, JARID2 and RING1A/B mutant phenocopy each other with regard to EZH2 and MTF2 binding, suggesting JARID2 and RING1B act in the same PRC2 recruitment mechanism. JARID2 recruitment is also strongly affected by the absence of RING1A/B, in line with the JARID2-mediated PRC2 recruitment via binding to PRC1-deposited H2AK119ub. b-d) Average plot of the ChIP signal shown in (a), for EZH2 (b) MTF2 (c) and JARID2 (d) centred on called peaks. Lower panels represent the same data with cropped y axis, for better visualization. e) Heatmap showing Ring1b binding in the discussed conditions. Ring1b is only mildly affected by removing H3K27me3 using EED226 (~40%). Binding is further attenuated in MTF2 and JARID2 mutant ESCs. f) Average plot of the ChIP signal shown in (e), centred on called peaks. g) Examples of loci of the data as shown in (e).

Article Snippet: ChIP was performed using 3 μl/sample of the following antibodies: MTF2 (Aviva System Biology ARP34292, lot QC49692-42166), H3K27me3 (Millipore 07-449, lot 2717675), EZH2 (Diagenode C15410039, lot 003), JARID2 (Novus Biologicals NB100-2214, Lot E2), RING1B (Abcam, AB3832 lot GR86503-25).

Techniques: Binding Assay, Mutagenesis

Journal: bioRxiv

Article Title: Two distinct functional axes of positive feedback-enforced PRC2 recruitment in mouse embryonic stem cells

doi: 10.1101/669960

Figure Lengend Snippet: a) On PRC2.1 main targets (clusters 1-4) relatively little MTF2 binding is sufficient to kick start the EED positive feedback loop which heavily relies on JARID2. As primary recruitment is mediated to a large extent via MTF2, such a loop can still exist in the absence JARID2. In the absence of H3K27me3, an alternative route can take over that requires JARID2 binding to H2AK119ub. b) On PRC2.2/PRC1 targets (clusters 5-6), instead, Polycomb binding is initiated by PRC1 that, upon H2AK119ub deposition, is followed by JARID2-containing PRC2.2. These regions also see the presence of MTF2 in physiological conditions, but this is the result of indirect recruitment via the PRC2 core binding to PRC2.2-initiated H3K27me3 deposition.

Article Snippet: ChIP was performed using 3 μl/sample of the following antibodies: MTF2 (Aviva System Biology ARP34292, lot QC49692-42166), H3K27me3 (Millipore 07-449, lot 2717675), EZH2 (Diagenode C15410039, lot 003), JARID2 (Novus Biologicals NB100-2214, Lot E2), RING1B (Abcam, AB3832 lot GR86503-25).

Techniques: Binding Assay

Journal: Nature aging

Article Title: The transcriptional coactivator CBP/p300 is an evolutionarily conserved node that promotes longevity in response to mitochondrial stress

doi: 10.1038/s43587-020-00025-z

Figure Lengend Snippet: a , All putative lysine acetyltransferases (KATs) in C . elegans and their human homologues. The worm KATs were validated/identified by searching the C . elegans protein database for proteins with a conserved acetyltransferase domain, and high amino acid sequences identities of the known human KATs . b , Role of KATs in UPR mt activation in C . elegans . hsp-6p::gfp worms were fed with control (ev) or cco-1 (40%) RNAi in combination with RNAi targeting different KATs (60%). c , d , cbp-1 RNAi attenuates the UPR mt activation induced by cco-1 (c) or mrps-5 (d) RNAi in a dose-dependent manner. RNAi targeting cco-1 or mrps-5 occupied 40%. cbp-1 RNAi occupied 10-60%. Control RNAi was used to supply to a final 100% of RNAi for all conditions. e , Schematic diagram showing the different regions regulated by the two different cbp-1 RNAi, and the two CBP/p300 inhibitors (PF-CBP1 and A-485). KIX, kinase-inducible domain interacting domain; Br, bromodomain; HAT, histone acetyltransferase domain; a.a., amino acids; n.t., nucleotides. f , The alternative cbp-1 RNAi ( cbp-1 _2) also inhibits UPR mt activation. RNAi targeting mrps-5 or spg-7 occupies 40%, cbp-1 RNAi occupies 25%. g , A-485 attenuates UPR mt activation induced by mrps-5 RNAi in a dose-dependent manner. hsp-6p::gfp worms were fed with control or mrps-5 RNAi (40%), in combination with 0-20 μM A-485. h , i , RNAi that specifically targets cbp-2 or cbp-3 failed to abolish UPR mt activation in hsp-6p::gfp worms. Photos (h) and qRT-PCR-results ( n = 4 biologically independent samples) (i) of hsp-6p::gfp worms fed with control, cco-1 (40%), cbp-1 (25%), cbp-2 (Ahringer library) or cbp-3 RNAi. Error bars denote SEM. Statistical analysis was performed by two-tailed unpaired Student’s t -test. j , Schematic diagram showing the protein structure of CBP-1, CBP-2 and CBP-3. The numbers in red indicate the amino acid sequence identities between two proteins in comparison. k - m , cbp-1 RNAi attenuates the UPR ER activation induced by tunicamycin (5 μg/ml) (k), hsp-3 (l) or enpl-1 (m) RNAi in hsp-4p::gfp worms. RNAi targeting hsp-3 or enpl-1 occupies 40%, cbp-1 RNAi occupies 25%, atfs-1 RNAi occupies 60%. n , cbp-1 RNAi does not affect the cytosolic UPR (UPR CYT )/heat shock response activation induced by heat shock. hsp16.2p::gfp reporter worms were fed with different percentages of cbp-1 RNAi as indicated. As positive control, heat shock for 8 h at 31°C could induce the UPR CYT and cbp-1 RNAi did not block this response. Scale bars, 0.3 mm.

Article Snippet: For transfection, plasmids expressing human full-length WT-p300 (Cat. 89094, addgene), and the p300 acetyltransferase activity-defective mutant (Cat. 89095, addgene) , were purchased from addgene and transfected with the Lipofectamine 3000 Reagent (Cat. L3000015, ThermoFisher).

Techniques: Activation Assay, Control, Quantitative RT-PCR, Two Tailed Test, Sequencing, Comparison, Positive Control, Blocking Assay

Journal: Nature aging

Article Title: The transcriptional coactivator CBP/p300 is an evolutionarily conserved node that promotes longevity in response to mitochondrial stress

doi: 10.1038/s43587-020-00025-z

Figure Lengend Snippet: a , b , cbp-1 RNAi attenuates UPR mt activation induced by cco-1 (a) or mrps-5 (b) knockdown in hsp-6p::gfp worms. For RNAi treatment, RNAi targeting cco-1 or mrps-5 occupies 40%, cbp-1 RNAi occupies 25%, atfs-1 RNAi occupies 60%, control RNAi (ev) was used to supply to a final 100% of RNAi for all conditions. DIC, differential interference contrast image. c , CBP/p300 small-molecule inhibitors A-485 and PF-CBP1 attenuate UPR mt activation in worms. hsp-6p::gfp worms were fed with control or the UPR mt -inducing cco-1 (40%) RNAi, in combination with A-485 (10 μM) or PF-CBP1 (80 μM) treatment. d , e , cbp-1 RNAi abolishes UPR mt activation induced by spg-7 , timm-23 , tomm-40 , cts-1 or dlst-1 RNAi (d), and by antimycin A (2.5 μM) or doxycycline (Dox) (30 μg/ml) (e), in hsp-6p::gfp worms. The atfs-1 RNAi was used as a positive control. RNAi targeting tomm-40 , cts-1 or dlst-1 occupies 40%, cbp-1 RNAi occupies 25%, atfs-1 RNAi occupies 60%. f , Volcano plots showing the effects on gene expression of cco-1 RNAi compared to control RNAi (ev) (left), and of cbp-1 (middle) or atfs-1 (right) knockdown in a cco-1 RNAi background. FC, fold change. Genes dependent on both CBP-1 and ATFS-1 for induction after cco-1 RNAi are highlighted in red (common). Genes dependent on CBP-1, but not ATFS-1, for induction are in blue (CBP-1 only). Genes dependent on ATFS-1, but not CBP-1, for induction are highlighted in yellow (ATFS-1 only). g , Venn diagram of CBP-1-dependent UPR mt genes, that are in common with ATFS-1-dependent UPR mt genes in response to cco-1 RNAi, according to the RNA-seq data. h , i , Functional clustering of the 259 (h) and 247 (i) UPR mt genes as indicated in (g). j . Heat-map of the representative UPR mt genes dependent on CBP-1 for induction during cco-1 knockdown. The color-coded heat-map represents gene expression differences in log 2 FC relatively to control RNAi (ev) condition. Genes dependent on both CBP-1 and ATFS-1 are highlighted in red. Genes dependent on CBP-1, but not ATFS-1, are highlighted in blue. Scale bars, 0.3 mm.

Article Snippet: For transfection, plasmids expressing human full-length WT-p300 (Cat. 89094, addgene), and the p300 acetyltransferase activity-defective mutant (Cat. 89095, addgene) , were purchased from addgene and transfected with the Lipofectamine 3000 Reagent (Cat. L3000015, ThermoFisher).

Techniques: Activation Assay, Knockdown, Control, Positive Control, Gene Expression, RNA Sequencing, Functional Assay

Journal: Nature aging

Article Title: The transcriptional coactivator CBP/p300 is an evolutionarily conserved node that promotes longevity in response to mitochondrial stress

doi: 10.1038/s43587-020-00025-z

Figure Lengend Snippet: a , H3K18 and H3K27 acetylation increases in a CBP-1-dependent manner during UPR mt activation induced by cco-1 or mrps-5 RNAi. Western blots of hsp-6p::gfp worms fed with control, cbp-1 , atfs-1 , cco-1 or mrps-5 RNAi. RNAi targeting cbp-1 occupies 25%, atfs-1 , cco-1 or mrps-5 occupies 50%, control RNAi was used to supply to a final 100% of RNAi for all conditions. b , The increase of H3K18Ac and H3K27Ac upon cco-1 RNAi is strongly blocked by chemical inhibition of CBP/p300 with A-485. Western blots of hsp-6p::gfp worms fed with control or cco-1 (50%) RNAi, in combination with DMSO or A-485 (10 μM) treatment. c , Western blots of hsp-6p::gfp worms showing increased H3K18Ac and H3K27Ac during UPR mt activation induced by RNAi targeting different mitochondrial genes as indicated. d , Summary of the H3K27Ac and H3K18Ac ChIP-seq results in worms fed with control or cco-1 RNAi. e - g , Treatment of cco-1 RNAi increases H3K18Ac and H3K27Ac at the genomic loci of representative UPR mt genes including hsp-6 , hsp-60 and timm-23 . Genome tracks showing the ChIP-seq analysis for H3K27Ac and H3K18Ac over the genomic loci of hsp-6 (e), hsp-60 (f) and timm-23 (g) in worms fed with control (ev) or cco-1 RNAi. The two tracks were shown with the same total count range between basal and mitochondrial stress condition for each gene. h , i , Functional clustering of the 4639 (h) and 2283 (i) genes as indicated in (d). j - l , cco-1 RNAi-induced increase of H3K18Ac and H3K27Ac at the loci of representative UPR mt genes is completely blocked by cbp-1 RNAi. ChIP-qPCR analysis of hsp-6 (j), hsp-60 (k) and timm-23 (l) in worms fed with RNAi targeting cco-1 and/or cbp-1 ( n = 4 biologically independent samples). RNAi targeting cco-1 occupies 50%, cbp-1 occupies 25%. ChIP was performed by using antibodies to H3K18Ac or H3K27Ac. Error bars denote SEM. Statistical analysis was performed by ANOVA followed by Tukey post-hoc test. For uncropped gel source data, see .

Article Snippet: For transfection, plasmids expressing human full-length WT-p300 (Cat. 89094, addgene), and the p300 acetyltransferase activity-defective mutant (Cat. 89095, addgene) , were purchased from addgene and transfected with the Lipofectamine 3000 Reagent (Cat. L3000015, ThermoFisher).

Techniques: Activation Assay, Western Blot, Control, Inhibition, ChIP-sequencing, Functional Assay, ChIP-qPCR

Journal: Nature aging

Article Title: The transcriptional coactivator CBP/p300 is an evolutionarily conserved node that promotes longevity in response to mitochondrial stress

doi: 10.1038/s43587-020-00025-z

Figure Lengend Snippet: a , Pearson’s correlation co-expression heat-map for CBP , p300, Kdm6b , Phf8 and UPR mt genes in spleen, pituitary, adrenal and eye of the BXD mouse genetic reference population 43, 52. Positive and negative correlations are indicated in red and blue, respectively. The intensity of the colors corresponds to correlation coefficients. b , Positive correlations between lifespan and CBP transcript levels in spleen, pituitary and adrenal of BXD mice (Pearson’s r , two-sided). c , Positive correlations between lifespan and p300 transcript levels in spleen, pituitary and eye of BXD mice (Pearson’s r , two-sided). d , Circos plot of the expression correlations between CBP/p300 transcripts and the UPR mt gene transcripts in the brain (cerebellar hemisphere), hypothalamus, liver, heart (left ventricle), stomach, pancreas, kidney and small intestine of human samples derived from the Genotype-Tissue Expression (GTEx) database (v8) . Red bar ring: the Pearson’s correlation coefficients ( r ) between UPR mt gene transcripts and CBP (light red) or p300 (dark red). Blue bar ring: the Spearman Rank correlation coefficients ( Rho ) between UPR mt gene transcripts and CBP (light blue) or p300 (dark blue). All correlations were found to be positive.

Article Snippet: For transfection, plasmids expressing human full-length WT-p300 (Cat. 89094, addgene), and the p300 acetyltransferase activity-defective mutant (Cat. 89095, addgene) , were purchased from addgene and transfected with the Lipofectamine 3000 Reagent (Cat. L3000015, ThermoFisher).

Techniques: Expressing, Derivative Assay

Journal: Nature aging

Article Title: The transcriptional coactivator CBP/p300 is an evolutionarily conserved node that promotes longevity in response to mitochondrial stress

doi: 10.1038/s43587-020-00025-z

Figure Lengend Snippet: a , Pearson’s correlation co-expression heat-map for CBP / p300, Kdm6b / Phf8 and UPR mt genes in hippocampus and hypothalamus of the BXD mouse genetic reference population , . Positive and negative correlations are indicated in red and blue, respectively. The intensity of the colors corresponds to correlation coefficients. b , Pearson’s correlation co-expression heat-map for CBP / p300, Kdm6b / Phf8 and UPR mt genes in the brain (whole brain) and prefrontal cortex of the LXS mouse genetic reference population . c , Positive correlations between lifespan and CBP or p300 transcript levels in hippocampus and hypothalamus of BXD mice (Pearson’s r , two-sided). Each dot indicates an independent BXD strain.

Article Snippet: For transfection, plasmids expressing human full-length WT-p300 (Cat. 89094, addgene), and the p300 acetyltransferase activity-defective mutant (Cat. 89095, addgene) , were purchased from addgene and transfected with the Lipofectamine 3000 Reagent (Cat. L3000015, ThermoFisher).

Techniques: Expressing

Journal: Nature aging

Article Title: The transcriptional coactivator CBP/p300 is an evolutionarily conserved node that promotes longevity in response to mitochondrial stress

doi: 10.1038/s43587-020-00025-z

Figure Lengend Snippet: a , Knockout of CBP/p300 attenuates Dox-induced transcription of key UPR mt genes in MEFs. qRT-PCR-results of wild-type (WT) and CBP/p300 -/- MEFs treated with or without Dox (30 μg/ml) for 24 h ( n = 4 biologically independent samples). b , Diagram of the UPR mt genes that are dependent (blue) or independent (grey) on CBP/p300 for induction upon Dox treatment in MEFs, according to the RNA-seq data. c , Functional clustering of the 197 genes as indicated in (b). d , e , Transcripts ( n = 4 biologically independent samples) (d) and protein levels (e) of CBP/p300 -/- MEFs reconstituted with empty vector, WT-p300 or the D1399Y acetyltransferase activity-defective mutant of p300, after control or Dox (30 μg/ml) treatment for 24 h. f , Western blots showing time-dependent changes of H3K18Ac and H3K27Ac in WT and CBP/p300 -/- MEFs treated with Dox (30 μg/ml) for 0-12 h. g , CBP/p300 is essential for Dox-induced H3K18Ac and H3K27Ac at the promoters of UPR mt genes (e.g. Hspd1 and Hspa9 ). ChIP-qPCR analysis of Hspd1 and Hspa9 in wild-type and CBP/p300 -/- MEFs treated with or without Dox (30 μg/ml) for 3 h ( n = 4 biologically independent samples). ChIP was performed by using antibodies to H3K18Ac or H3K27Ac. h , CBP/p300 acetyltransferase activity inhibitor A-485 strongly suppresses Dox-induced transcription of key UPR mt genes in HepG2 cells. qRT-PCR-results of HepG2 cells pretreated with DMSO or A-485 (5 μM) for 1 h, and then co-treated with or without Dox (30 μg/ml) for 24 h ( n = 4 biologically independent samples). i , Functional clustering of the 299 genes up-regulated upon Dox treatment (30 μg/ml, 24 h) and the CBP/p300 activity-dependency in human HepG2 cells, according to the RNA-seq data with A-485. CBP/p300 activity-dependent genes are indicated in red. j , qRT-PCR-results of HepG2 cells overexpressing empty vector, WT-p300 or the acetyltransferase activity-defective mutant D1399Y-p300 ( n = 4 biologically independent samples). Error bars denote SEM. Statistical analysis was performed by ANOVA followed by Tukey post-hoc test. For uncropped gel source data, see .

Article Snippet: For transfection, plasmids expressing human full-length WT-p300 (Cat. 89094, addgene), and the p300 acetyltransferase activity-defective mutant (Cat. 89095, addgene) , were purchased from addgene and transfected with the Lipofectamine 3000 Reagent (Cat. L3000015, ThermoFisher).

Techniques: Knock-Out, Quantitative RT-PCR, RNA Sequencing, Functional Assay, Plasmid Preparation, Activity Assay, Mutagenesis, Control, Western Blot, ChIP-qPCR

Journal: Nature aging

Article Title: The transcriptional coactivator CBP/p300 is an evolutionarily conserved node that promotes longevity in response to mitochondrial stress

doi: 10.1038/s43587-020-00025-z

Figure Lengend Snippet: a , Multidimensional scaling (MDS) plot of the RNA-seq profiles of wild-type (WT) and CBP/p300 -/- MEFs treated with or without Dox (30 μg/ml) for 24 h. Note the decreased distance between Dox-treated and un-treated condition in the CBP/p300 -/- background compared to that in WT background. b , CBP/p300 -/- MEFs are insensitive to the treatment of mitochondrial stress inducer Dox. Volcano plots showing the effect of Dox treatment in wild-type (WT) (left) or CBP/p300 -/- (right) MEFs on gene expression. FC, fold change. Genes that were up-regulated (log 2 FC > 0.5, adjusted P < 0.05) during Dox treatment were highlighted in red. Genes that were down-regulated (log 2 FC < -0.5, adjusted P < 0.05) were highlighted in blue. c , Heat-map of the representative UPR mt genes dependent on CBP/p300 for induction in response to Dox treatment in WT and CBP/p300 -/- (KO) MEFs, according to the RNA-seq data. The heat-map was shown in log 2 FC values. d , MDS plot of the RNA-seq profiles of HepG2 cells treated with or without CBP/p300 acetyltransferase activity inhibitor A-485 (5 μM) and/or Dox (30 μg/ml) for 24 h. e , The CBP/p300 catalytic inhibitor A-485 attenuates the effect of Dox on gene expression in HepG2 cells. Volcano plots showing the effect of Dox on gene expression of HepG2 cells in control (DMSO) (left) or A-485 (right) treatment background. FC, fold change. Genes that were up-regulated (log 2 FC > 0.5, adjusted P < 0.05) during Dox treatment were highlighted in red. Genes that were down-regulated (log 2 FC < -0.5, adjusted P < 0.05) were highlighted in blue. f , Diagram of the UPR mt genes that are dependent (orange) or independent (grey) on CBP/p300 activity in response to Dox treatment in HepG2 cells, according to the RNA-seq data with A-485.

Article Snippet: For transfection, plasmids expressing human full-length WT-p300 (Cat. 89094, addgene), and the p300 acetyltransferase activity-defective mutant (Cat. 89095, addgene) , were purchased from addgene and transfected with the Lipofectamine 3000 Reagent (Cat. L3000015, ThermoFisher).

Techniques: RNA Sequencing, Gene Expression, Activity Assay, Control

Journal: Nature aging

Article Title: The transcriptional coactivator CBP/p300 is an evolutionarily conserved node that promotes longevity in response to mitochondrial stress

doi: 10.1038/s43587-020-00025-z

Figure Lengend Snippet: When the mitochondria are stressed in response to various intracellular or extracellular stimuli, CBP-1 or the mammalian CBP/p300 act downstream of demethylases JMJD-3.1/JMJD-1.2 or mammalian KDM6B/PHF8, switching the transcription-repressive histone methylation marks (e.g. H3K27Me3) to the transcription-active acetylation marks (e.g. H3K27Ac), and thereby relays the mitochondrial stress signal to the transcriptional induction of diverse UPR mt genes in C . elegans as well as in mammals. Many of these UPR mt effectors play positive roles in the recovery of mitochondrial function, improved immune response and proteostasis, and lifespan extension.

Article Snippet: For transfection, plasmids expressing human full-length WT-p300 (Cat. 89094, addgene), and the p300 acetyltransferase activity-defective mutant (Cat. 89095, addgene) , were purchased from addgene and transfected with the Lipofectamine 3000 Reagent (Cat. L3000015, ThermoFisher).

Techniques: Methylation

Journal: Nature aging

Article Title: The transcriptional coactivator CBP/p300 is an evolutionarily conserved node that promotes longevity in response to mitochondrial stress

doi: 10.1038/s43587-020-00025-z

Figure Lengend Snippet: a , ATFS-1 was acetylated by CBP-1 in vivo . Flag-tagged ATFS-1 was expressed with or without CBP-1 acetyltransferase domain (CBP-1-HAT) in HEK293T cells, immunoprecipitated with anti-Flag antibody and analyzed by western blots. TCL, total cell lysate. b , ATFS-1 was acetylated by CBP-1 in vitro . Bacterially expressed GST tagged CBP-1-HAT was incubated with Flag-ATFS-1 with or without acetyl-CoA (Ac-CoA) and immunoblotted as indicated. c , Schematic diagram showing the protein structure of ATFS-1 and the acetylated sites identified by mass spectrometry. MTS, mitochondrial targeting sequence; SRR, Serine-rich region; NLS, Nuclear localization signal; LZD, Leucine zipper domain. d , HEK293T cells transfected as indicated were treated with or without TSA (class I/II HDAC inhibitor), or NAM (class III HDAC inhibitor) 8 h before harvesting, and analyzed by western blots. For uncropped gel source data, see .

Article Snippet: For transfection, plasmids expressing human full-length WT-p300 (Cat. 89094, addgene), and the p300 acetyltransferase activity-defective mutant (Cat. 89095, addgene) , were purchased from addgene and transfected with the Lipofectamine 3000 Reagent (Cat. L3000015, ThermoFisher).

Techniques: In Vivo, Immunoprecipitation, Western Blot, In Vitro, Incubation, Mass Spectrometry, Sequencing, Transfection

Journal: Cell Death & Disease

Article Title: HNF4α-HKDC1 axis orchestrates a metabolic rewiring to promote migration and metastasis in advanced gastric cancer

doi: 10.1038/s41419-026-08627-y

Figure Lengend Snippet: A A heatmap depicted the mRNA expression levels of glycolytic genes (as mentioned in Fig. 5E, G in high-expression and low-expression HNF4α groups within the TCGA GC cohort. The median value of HNF4α was utilized as the cutoff threshold to categorize GC patients into high-expression and low-expression HNF4α groups. B A scatter plot illustrated the correlation between HNF4α and HKDC1 mRNA expression in TCGA GC samples ( n = 408, Spearman’s correlation test). C Another scatter plot displayed the correlation between HNF4α and HKDC1 mRNA expression in CCLE GC cell lines ( n = 71, Spearman’s correlation test). D A boxplot presented HKDC1 mRNA expression in GC tissues and the corresponding normal gastric tissues in TCGA and Genotype-Tissue Expression (GTEx) databases ( n = 408 for tumor and n = 211 for normal) using GEPIA ( http://gepia.cancer-pku.cn/ ). E Assessment of the prognostic values of overall survival in GC patients based on the mRNA expression level of HKDC1 via Kaplan–Meier plotter by GEPIA (cutoff: high HKDC1 60%, low HKDC1 40%). F The expression profiles of HKDC1 across the ten identified cell types derived from the single-cell data referenced in Fig. and Supplementary Fig. were depicted. G A scatter plot illustrated the correlation between HNF4α and HKDC1 mRNA expression in single-cell RNAseq datasets, as determined by Spearman’s correlation test. H , I The expression level of HKDC1 was upregulated in HGC-27 and OCUM-1 cells by HNF4α7 overexpression and down-regulated in AGS, NUGC-4, and KATO-III cells by HNF4α knockdown, as analyzed by real-time PCR. J The expression level of HKDC1 was upregulated in HGC-27 and OCUM-1 cells by HNF4α7 overexpression and down-regulated in AGS cells by HNF4α knockdown, as analyzed by WB. Data were presented as the mean ± SEM. The difference in significance was analyzed by unpaired two-tailed Student’s t -test ( H ) or one-way ANOVA ( I ).

Article Snippet: Rabbit anti-HKDC1 antibody (Cat. #25874-1-AP, 1:1000 for WB) was obtained from Proteintech, Wuhan, China.

Techniques: Expressing, Derivative Assay, Single Cell, RNA sequencing, Over Expression, Knockdown, Real-time Polymerase Chain Reaction, Two Tailed Test

Journal: Cell Death & Disease

Article Title: HNF4α-HKDC1 axis orchestrates a metabolic rewiring to promote migration and metastasis in advanced gastric cancer

doi: 10.1038/s41419-026-08627-y

Figure Lengend Snippet: A – D HKDC1 knockdown significantly suppressed the peritoneal metastasis of GC cells. shCtrl and shHKDC1 sublines of AGS cells were subjected to intraperitoneal injection into nude mice (seven mice per group). Forty days later, images ( A ) and quantification ( B ) of luciferase expression, and representative images ( C ) and number ( D ) of metastatic nodules in the abdominal cavity of nude mice for each group were presented. E The diminished migratory capacity of AGS cells resulting from HNF4α knockdown was restored by HKDC1 overexpression, as demonstrated by the wound healing assay (scale bar: 100 μm). Representative images (upper panel) and quantification of wound distance (lower panel) were presented ( n = 9 biologically independent samples). F , G HKDC1 knockdown attenuated the effects of HNF4α7 overexpression on the migration of HGC-27 cells by the 3D tumor spheroid migration assay. Representative images ( F , scale bar: 50 μm) and quantification of wound distance ( G ) were shown ( n = 4 biologically independent samples). H Metabolite levels of organic acid compounds (including glycolysis/TCA cycle Intermediates) before and after HNF4α or HKDC1 knockdown in AGS cells were assayed by targeted mass spectrometry ( n = 3 biologically independent samples). I Metabolite levels of organic acid compounds (including glycolysis/TCA cycle Intermediates) of peritoneal metastases in each mouse of Fig. 6A were assayed by targeted mass spectrometry (seven mice per group). J – L The effects of HNF4α knockdown on lactate production ( J ), ROS levels ( K ), and ATP production ( L ) were restored by HKDC1 overexpression in AGS cells ( n = 3 biologically independent samples). M , N The diminished transwell migration ability of AGS cells caused by HNF4α or HKDC1 knockdown was rescued by treatment with lactate (5 mM) or α-KG (5 mM). Representative images ( M ) and quantification ( N ) were shown ( n = 3 biologically independent samples). Scale bar: 50 μm. Data were presented as the mean ± SEM. The difference significance was analyzed by unpaired two-tailed Student’s t -test ( B , D , I ) or one-way ANOVA ( E , G , H , J – L , N ).

Article Snippet: Rabbit anti-HKDC1 antibody (Cat. #25874-1-AP, 1:1000 for WB) was obtained from Proteintech, Wuhan, China.

Techniques: Knockdown, Injection, Luciferase, Expressing, Over Expression, Wound Healing Assay, Migration, Mass Spectrometry, Two Tailed Test

Journal: Cell Death & Disease

Article Title: HNF4α-HKDC1 axis orchestrates a metabolic rewiring to promote migration and metastasis in advanced gastric cancer

doi: 10.1038/s41419-026-08627-y

Figure Lengend Snippet: A Overexpression of HNF4α7, but not HNF4α7(ΔDBD), resulted in an elevation of HKDC1 mRNA expression in HGC-27 and OCUM-1 cells. B Integration of HNF4α ChIP-seq data in seven HNF4α-positive GC cell lines (IM95, Ist1, KATO-III, NUGC4, OCUM-1, SNU16, and YCC3) and histone ChIP-seq data (H3K27ac, H3K4me3, and H3K4me1) in IM95 cell line from the GEO database ( GSE114018 and GSE75898 ) was performed at human HKDC1 gene locus ( NR_120648.1 ). The arrow indicated the direction of transcription. The blue frames denoted potential HNF4α binding regions, namely E1, E2, E3, E4 and E5. C HNF4α promoted the transcription of the potential HNF4α binding region E4. HEK293T cells transiently expressing or HGC-27 cells stably expressing pLV vector or pLV-HNF4α7 were cotransfected with pRL-TK and pGL6-TA constructs containing E1, E2, E3, E4, or E5, as indicated. Twenty-four hours later, cells were harvested, and a dual-luciferase reporter assay was conducted. D Two potential HNF4α response elements (HREs), namely S1 ( + 4323 ~ +4334 bp) and S2 ( + 4365 ~ +4376 bp), were predicted in the potential HNF4α binding region E4 within the human HKDC1 gene locus. The sequence (+4323 ~ +4381 bp, named S3), containing S1 and S2, was employed in subsequent experiments. E Overexpression of HNF4α7 promoted the transcription of S3, but not its mutant with mutations in both S1 and S2 mentioned in ( D ). HEK293T cells transiently expressing or HGC-27 cells stably expressing pLV vector or pLV-HNF4α7 were cotransfected with pRL-TK and pGL6-TA constructs containing S3 or its mutant, as indicated. F – H The binding of HNF4α to S3 within the human HKDC1 gene locus was detected by EMSA ( F ) and ChIP ( H ) assay. For EMSA, recombinant GST-HNF4α7 or its mutant GST-HNF4α7(ΔDBD) protein was incubated with biotin-labeled oligonucleotides of S3. Coomassie blue staining of the purified recombinant GST-HNF4α7 and GST-HNF4α7(ΔDBD) proteins is indicated in ( G ). For ChIP, HGC-27 cells (with HNF4α7 overexpressed beforehand) or AGS cells were harvested, and the ChIP assay was carried out. All data were shown as the mean ± SEM. The difference significance was analyzed by one-way ANOVA ( A ) or unpaired two-tailed Student’s t -test ( C , E ).

Article Snippet: Rabbit anti-HKDC1 antibody (Cat. #25874-1-AP, 1:1000 for WB) was obtained from Proteintech, Wuhan, China.

Techniques: Over Expression, Expressing, ChIP-sequencing, Binding Assay, Stable Transfection, Plasmid Preparation, Construct, Luciferase, Reporter Assay, Sequencing, Mutagenesis, Recombinant, Incubation, Labeling, Staining, Purification, Two Tailed Test

Journal: Cell Death & Disease

Article Title: HNF4α-HKDC1 axis orchestrates a metabolic rewiring to promote migration and metastasis in advanced gastric cancer

doi: 10.1038/s41419-026-08627-y

Figure Lengend Snippet: A , B The suppressive effects of MMF on the mRNA expression level of HKDC1 and the migration of AGS cells by real-time PCR ( A ) and wound healing assay ( B ). Representative images ( B , upper panel) and quantification of wound distance ( B , lower panel) were presented ( n = 7 biologically independent samples). Scale bar: 100 μm. C – H MMF significantly suppressed the peritoneal metastasis of HNF4α-overexpressed HGC-27 cells, but not that of HGC-27 cells with minimal HNF4α expression in nude mice. One of the HGC-27 pLV-Vector and pLV-HNF4α7 cell sublines (all stably expressing luciferase, 1 × 10 7 cells per mouse) was intraperitoneally injected into the nude mice. Ten days later, the mice were randomly divided into two groups ( n = 7 per group), and treated with vehicle (0.9% NaCl) or MMF (40 mg/kg) once daily for 26 days by oral administration. Presented were images ( C ) and quantification ( D ) of luciferase expression, representative images ( E ) and number ( F ) of metastatic nodules in the abdominal cavity, and body weight curve ( G ) of nude mice for each group. All macroscopic metastatic lesions discernible by visual inspection were included in the quantitative analysis ( E , F ). The expression levels of HKDC1 and HNF4α in each group were examined by WB ( H ). I A proposed a working model for HNF4α-HKDC1 in the metabolism and metastasis of GC, which was antagonized by MPA. Data were presented as the mean ± SEM. The significance of differences was analyzed by one-way ANOVA ( A , D , F , G ) or unpaired two-tailed Student’s t -test ( B ).

Article Snippet: Rabbit anti-HKDC1 antibody (Cat. #25874-1-AP, 1:1000 for WB) was obtained from Proteintech, Wuhan, China.

Techniques: Expressing, Migration, Real-time Polymerase Chain Reaction, Wound Healing Assay, Plasmid Preparation, Stable Transfection, Luciferase, Injection, Process/Product Development, Two Tailed Test

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Histone demethylase KDM9 activates the CCND1/AKT pathway to promote the malignant progression of gliomas through interaction with BRD2

doi: 10.1007/s00018-025-05900-9

Figure Lengend Snippet: KDM9 regulates CCND1 transcription through interaction with BRD2. ( A ) Protein silver staining of IP via KDM9 antibody enrichment in LN229 cells. ( B ) Transcription factors in the protein mass spectrometry data are shown. Exogenous and endogenous IP experiments were performed to analyze the interaction between KDM9 and BRD4 ( C ) or BRD2 ( D ). ( E ) The IF assay was used to detect the expression and co-localization of KDM9 and BRD2 in LN229 cells. ( F ) After the transfection of pcDNA3.1-BRD2-His into LN229-shKDM9 cells for 48 h, ChIP-qPCR was performed to detect the enrichment of H4K20me2 at the CCND1 P4 promoter region (with IgG as negative control). ( G ) After the transfection of wild-type or mutant KDM9 plasmids into U251 cell lines for 48 h, luciferase reporter assays were conducted to detect CCND1 transcription activity (Left). After the transfection of pcDNA3.1-BRD2-His into LN229-shKDM9 cells for 48 h, luciferase reporter assays were performed to detect CCND1 transcription activity (Right). ( H ) After the transfection of shBRD2 plasmids into U251-KDM9-WT cell lines for 48 h, luciferase reporter assays were conducted to detect CCND1 transcription activity. ( I ) After the transfection of pcDNA3.1-BRD2-His into shKDM9 glioma cells for 48 h, a Western blot experiment was conducted to detect the protein expression of KDM9, BRD2, CCND1, AKT/P-AKT, mTOR/P-mTOR, and CD133. Data are shown as the mean ± SD. * P < 0.05, ** P < 0.01, and *** P < 0.001

Article Snippet: CD133 (18470–1-AP, 1:1000), Histone H4 (16047–1-AP, 1:500), CCND1 (60186–1-Ig, 1:2000), SMAD2 (12570–1-AP, 1:2000), P-AKT (66444–1-IG, 1:1000), AKT (60230–2-IG, 1:1000), Flag-Tag (66008–4-Ig, 1:5000), BRD2 (22236–1-AP, 1:1000), and BRD4 (28486–1-AP, 1:1000) were purchased from Proteintech (Chicago, IL).

Techniques: Silver Staining, Mass Spectrometry, Expressing, Transfection, ChIP-qPCR, Negative Control, Mutagenesis, Luciferase, Activity Assay, Western Blot

Journal: Neuron

Article Title: ARNT2 tunes activity-dependent gene expression through NCoR2-mediated repression and NPAS4-mediated activation

doi: 10.1016/j.neuron.2019.02.007

Figure Lengend Snippet: A. ChIP-seq signal for ARNT2 at presumptive NPAS4 sites in unstimulated cortical neuron cultures (left) and CA1 hippocampal tissue derived from standard housed mice (right) sorted by descending binding strength. Each ARNT2 binding site is represented as a single horizontal line (orange) centered at the peak summit with flanking 2kb. Color denotes ChIP-seq signal intensity (displayed in log2(counts per million + 1)). Peaks are split between distal enhancers bound by NPAS4 (n= 6,352) and TSS-proximal promoters bound by NPAS4 (n = 2,614).

Article Snippet: To prepare antibody bead complexes, Protein A Dynabeads (Life Technologies) were incubated with antibodies [NPAS4 (20 μg/45–50 million cells), ARNT2 (20 μg/35–50 million cells), rabbit IgG (Santa Cruz H270 (sc-66931); 20 μg/45–50 million cells)] for 30 min at room temperature followed by washing in NE1 buffer (20 mM HEPES pH 7.9, 10 mM KCl, 1 mM MgCl 2 , 0.1% Triton X100, 1 mM DTT, 250 mM NaCl) to remove unbound antibody.

Techniques: ChIP-sequencing, Derivative Assay, Binding Assay

Journal: Neuron

Article Title: ARNT2 tunes activity-dependent gene expression through NCoR2-mediated repression and NPAS4-mediated activation

doi: 10.1016/j.neuron.2019.02.007

Figure Lengend Snippet: A. Heatmaps of normalized ChIP-seq signal for unstimulated (0 hr KCl) and depolarized (2 hr KCl) cortical neuron cultures (left eight columns) at presumptive NPAS4 enhancers. Rightmost two columns show ARNT2 binding in adult hippocampal tissue in standard housing (Strd) or following neuronal stimulation via the injection of kainate (KA). Each binding site is represented as a single horizontal line centered at the peak summit and intensity of color correlates with sequencing signal for the indicated factor. Peaks are ordered by decreasing NPAS4 peak intensity.

Article Snippet: To prepare antibody bead complexes, Protein A Dynabeads (Life Technologies) were incubated with antibodies [NPAS4 (20 μg/45–50 million cells), ARNT2 (20 μg/35–50 million cells), rabbit IgG (Santa Cruz H270 (sc-66931); 20 μg/45–50 million cells)] for 30 min at room temperature followed by washing in NE1 buffer (20 mM HEPES pH 7.9, 10 mM KCl, 1 mM MgCl 2 , 0.1% Triton X100, 1 mM DTT, 250 mM NaCl) to remove unbound antibody.

Techniques: ChIP-sequencing, Binding Assay, Injection, Sequencing

Journal: Neuron

Article Title: ARNT2 tunes activity-dependent gene expression through NCoR2-mediated repression and NPAS4-mediated activation

doi: 10.1016/j.neuron.2019.02.007

Figure Lengend Snippet: A. −log10(FDR) versus log2 fold change in peptides obtained by immunoprecipitation followed by mass spectrometry (IP-MS) with an anti-ARNT2 antibody in stimulated primary cortical neurons relative to peptides immunoprecipitated with an IgG control. Blue points indicate proteins with FDR <0.1 and log2FC > 0. Purple points indicate members of the NCoR2 co-repressor complex.

Article Snippet: To prepare antibody bead complexes, Protein A Dynabeads (Life Technologies) were incubated with antibodies [NPAS4 (20 μg/45–50 million cells), ARNT2 (20 μg/35–50 million cells), rabbit IgG (Santa Cruz H270 (sc-66931); 20 μg/45–50 million cells)] for 30 min at room temperature followed by washing in NE1 buffer (20 mM HEPES pH 7.9, 10 mM KCl, 1 mM MgCl 2 , 0.1% Triton X100, 1 mM DTT, 250 mM NaCl) to remove unbound antibody.

Techniques: Immunoprecipitation, Mass Spectrometry, Protein-Protein interactions, Control

Journal: Neuron

Article Title: ARNT2 tunes activity-dependent gene expression through NCoR2-mediated repression and NPAS4-mediated activation

doi: 10.1016/j.neuron.2019.02.007

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: To prepare antibody bead complexes, Protein A Dynabeads (Life Technologies) were incubated with antibodies [NPAS4 (20 μg/45–50 million cells), ARNT2 (20 μg/35–50 million cells), rabbit IgG (Santa Cruz H270 (sc-66931); 20 μg/45–50 million cells)] for 30 min at room temperature followed by washing in NE1 buffer (20 mM HEPES pH 7.9, 10 mM KCl, 1 mM MgCl 2 , 0.1% Triton X100, 1 mM DTT, 250 mM NaCl) to remove unbound antibody.

Techniques: Virus, shRNA, Recombinant, Reporter Assay, Luciferase, Binding Assay, Modification, Software