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: Cell Reports Medicine

Article Title: Disrupting AGR2/IGF1 paracrine and reciprocal signaling for pancreatic cancer therapy

doi: 10.1016/j.xcrm.2024.101927

Figure Lengend Snippet:

Article Snippet: Pevonedistat (MLN4924) , Selleck , Cat# S7109; CAS: 905579-51-3.

Techniques: Virus, Recombinant, Control, Enzyme-linked Immunosorbent Assay, Isolation, Membrane, Protein Extraction, Chromatin Immunoprecipitation, Bicinchoninic Acid Protein Assay, Sircol Collagen Assay, Luciferase, RNA Sequencing Assay, Sequencing, Expressing, Real-time Polymerase Chain Reaction, shRNA, Plasmid Preparation, Software, Flow Cytometry

Journal: iScience

Article Title: Microtubule-associated proteins MAP7 and MAP7D1 promote DNA double-strand break repair in the G1 cell cycle phase

doi: 10.1016/j.isci.2023.106107

Figure Lengend Snippet: Knock-down of MAP7D1 leads to arrest in G1, while downregulation of MAP7 and MAP7D1 in G1 cells leads to defects in DNA repair (A) Representative images of cell cycle analysis of MCF7 (top) and RPE1 cells (bottom) treated with siLuc, siMAP7 or siMAP7D1 siRNA. (B) Quantification of FACS cell cycle data from MCF7 (right) and RPE1 (left) cells. Data from three independent biological replicates are shown in the bar graph from which on average 30,000 cells for MCF7 and 10,000 cells for RPE1 were acquired. Results are presented as mean ± SEM of values. Statistical differences were determined from nested t-test: p < 0.05, ∗; p < 0.01, ∗∗, ns, not significant. (C) Quantification of the immunofluorescence staining of 53BP1 foci in MCF7 cells untreated or treated with 10Gy γ-irradiation. Cells were treated with siRNA targeting luciferase, MAP7 or MAP7D1 and arrested in the G1 cell cycle phase using 250 nM palbociclib. Data from three independent biological replicates are shown in the dot plot from which at least 300 cells were quantified. Results are presented as mean ± SEM of values. Statistical differences were determined from nested t-test: p < 0.05, ∗; p < 0.01, ∗∗; p < 0.001, ∗∗∗. (D) Representative images of alkaline comet assay with MCF7 cells arrested in G1 cell cycle phase using 250 nM palbociclib. The cells were downregulated with siRNA targeting luciferase, MAP7 or MAP7D1 and treated or untreated with 10 Gy of γ-irradiation. - Scale bar, 10 μm. (E) Quantification of alkaline comet assay experiments with MCF7 cells from (D). Data from three independent biological replicates are shown in the dot plot from which at least 150 cells were quantified. Results are presented as mean ± SEM of values. Statistical differences were determined from nested t-test: p < 0.05, ∗; p < 0.01, ∗∗. (F and G) Western blot analysis of MCF7 cell fractionation. MCF7 cells were treated with siRNA and treated or untreated with 10 Gy of γ-irradiation and collected 1 h after the treatment. (H) Immunoprecipitation of endogenous MAP7 and MAP7D1 with 53BP1 from HEK293T whole cell extract (both MAP7 and MAP7D1 are recognized by MAP7 antibody). (I) Immunoprecipitation of full length MAP7 (GFP-FL) wild type and MAP7 mutated on Arg144/145 to Ala with deletion between aa151-300 (GFP- Δ 2) with endogenous 53BP1 from HEK293T whole cell extract.

Article Snippet: Cells were treated with 10Gy γ-IR, 0.1 ng/ml nocodazole (Sigma-Aldrich), 0.01μM–10μM paclitaxel (TOKU-E), 250 nM palbociclib (Selleck), 100 or 500 units of recombinant CK2 (NEB), 5μM or 15μM silmitaserib (CK2 inhibitor, Cayman Chemical) and 10J/m 2 UVC.

Techniques: Knockdown, Cell Cycle Assay, Immunofluorescence, Staining, Irradiation, Luciferase, Alkaline Single Cell Gel Electrophoresis, Western Blot, Cell Fractionation, Immunoprecipitation

Journal: iScience

Article Title: Microtubule-associated proteins MAP7 and MAP7D1 promote DNA double-strand break repair in the G1 cell cycle phase

doi: 10.1016/j.isci.2023.106107

Figure Lengend Snippet:

Article Snippet: Cells were treated with 10Gy γ-IR, 0.1 ng/ml nocodazole (Sigma-Aldrich), 0.01μM–10μM paclitaxel (TOKU-E), 250 nM palbociclib (Selleck), 100 or 500 units of recombinant CK2 (NEB), 5μM or 15μM silmitaserib (CK2 inhibitor, Cayman Chemical) and 10J/m 2 UVC.

Techniques: Virus, Subcloning, Recombinant, Sequencing, Protease Inhibitor, Single Cell Gel Electrophoresis, Mutagenesis, Mass Spectrometry, Construct, Software

Journal: iScience

Article Title: The HRD1-SEL1L ubiquitin ligase regulates stress granule homeostasis in couple with distinctive signaling branches of ER stress

doi: 10.1016/j.isci.2024.110196

Figure Lengend Snippet:

Article Snippet: GSK2606414 , MedChemExpress , Cat# HY-18072.

Techniques: Recombinant, cDNA Synthesis, Bicinchoninic Acid Protein Assay, Software

Journal: The EMBO Journal

Article Title: Mouse models to investigate in situ cell fate decisions induced by p53

doi: 10.1038/s44318-024-00189-z

Figure Lengend Snippet: ( A ) Thymocytes were extracted from Puma-tdTomato KI/+ mice and Trp53 −/− ;Puma-tdTomato KI/+ mice, and treated for 48 h in vitro with either DMSO (vehicle control), 10 µM nutlin-3a, 1 µg/mL etoposide, 1 µg/mL ionomycin, 1 µM dexamethasone or 10 ng/mL PMA. The percentages of live thymocytes (annexin-V/DAPI double negative) were determined by flow cytometric analysis. p values were calculated using a two-way ANOVA using Sidak’s correction for multiple tests. p value* ≤0.05, *** ≤0.001, **** ≤0.0001. Data were presented as mean ± SD. n = 3–7 for mice of each genotype and treatment. ( B ) MDFs from Puma-tdTomato KI/+ and Trp53 −/− ;Puma - tdTomato KI/+ mice were treated for 72 h with either DMSO (vehicle control), 10 µM nutlin-3a, 1 µg/mL etoposide, 250 nM taxol, 10 µM palbociclib or 5 µM abemaciclib. Cell cycle distribution was determined by flow cytometric analysis after staining with Hoechst 33342 at the start of treatment (0 h), and at 24, 48 and 72 h of drug treatment. p values were calculated using a two-way ANOVA using Sidak’s correction for multiple tests and displayed in Appendix Table . Data were presented as mean ± SD. n = 3 mice for each genotype and treatment. ( C ) Representative flow cytometry histograms of cell cycle distribution of MDFs from Trp53 −/− ;Puma - tdTomato KI/+ mice following in vitro treatment for 24 h with DMSO (vehicle control; in red), 10 µM nutlin-3a or 1 µg/mL etoposide (overlaid in blue). Cell cycle distribution was determined by flow cytometric analysis after staining with Hoechst 33342. The data shown were representative of n = 3 for mice of each genotype and treatment. ( D ) qRT-PCR analysis examining the levels of Puma mRNA in MDFs from Puma - tdTomato KI/+ and Puma +/− mice treated with either DMSO (vehicle control) or 10 µM nutlin-3a for 24 h. Data were normalised using the ΔΔCT method, using Hmbs as a housekeeping gene. Data were plotted as fold-change compared to the average of the wt DMSO-treated samples. Data were presented as mean ± SD. n = 3 mice of each genotype and treatment. The p values were calculated using a one-way ANOVA using Tukey’s correction for multiple tests. p value** ≤0.01, **** ≤0.0001. ( E ) Western blot analysis for TRP53 and PUMA protein levels in MDFs from Puma - tdTomato KI/+ and Puma +/ − mice 24 h after treatment with either DMSO (vehicle control) or 10 µM nutlin-3a. Probing for HSP70 and β-ACTIN were used as loading controls. Created with BioRender.com.

Article Snippet: palbociclib , MedChemExpress , HY-50767.

Techniques: In Vitro, Control, Staining, Flow Cytometry, Quantitative RT-PCR, Western Blot

Journal: The EMBO Journal

Article Title: Mouse models to investigate in situ cell fate decisions induced by p53

doi: 10.1038/s44318-024-00189-z

Figure Lengend Snippet: ( A ) Flow cytometry histograms showing the levels of tdTomato in untreated Puma-tdTomato KI/+ MDFs compared to untreated MDFs from a Puma +/− mouse. Data shown were representative of n = 3–5 for each genotype. ( B ) Flow cytometry histograms showing the levels of tdTomato in MDFs from a Puma-tdTomato KI/+ mouse and MDFs from a Trp53 −/− ; Puma-tdTomato KI/+ mouse that had been treated for 24 h with DMSO (vehicle control), 10 µM nutlin-3a, 1 µg/mL etoposide, 250 nM taxol, 5 µM abemaciclib or 10 µM palbociclib. Representative histograms from n = 5 independent cell cultures for cells of each genotype are shown. ( C ) Flow cytometry histograms showing the levels of tdTomato in untreated Puma-tdTomato KI/+ thymocytes compared to untreated thymocytes from a Puma +/− mouse. Data shown were representative of n = 3–5 mice for each genotype. ( D ) Flow cytometry histograms showing the levels of tdTomato in thymocytes from a Puma-tdTomato KI/+ mouse and a Trp53 −/− ; Puma-tdTomato KI/+ mouse that had been treated for 24 h with DMSO (vehicle control), 10 µM nutlin-3a, 1 µg/mL etoposide, 1 µg/mL ionomycin, 1 µM dexamethasone or 10 ng/mL PMA in the presence of the broad-spectrum caspase inhibitor QVD-oPH. Representative histograms from n = 3 independent cell cultures for each genotype and treatment are shown. ( E ) Summary plots of tdTomato expression in murine dermal fibroblasts displayed as fold-change relative to the level at 0 h. Cells were treated with DMSO (vehicle control), 10 µM nutlin-3a, 1 µg/mL etoposide, 250 nM taxol or 5 µM abemaciclib for 24 h before drug-containing medium was removed and replaced with non-drug-containing medium. p values were calculated using a linear model using Sidak’s correction for multiple tests. Data displayed as mean ± SD from n = 3 cultures derived from independent mice of each genotype. p value ** ≤0.01, **** ≤0.0001. .

Article Snippet: palbociclib , MedChemExpress , HY-50767.

Techniques: Flow Cytometry, Control, Expressing, Derivative Assay

Journal: The EMBO Journal

Article Title: Mouse models to investigate in situ cell fate decisions induced by p53

doi: 10.1038/s44318-024-00189-z

Figure Lengend Snippet: Reagents and tools table

Article Snippet: palbociclib , MedChemExpress , HY-50767.

Techniques: Recombinant, Affinity Purification, Sequencing, Protease Inhibitor, Modification, Software, Multiplex Assay