Journal: eLife

Article Title: Macrophage inflammation resolution requires CPEB4-directed offsetting of mRNA degradation

doi: 10.7554/eLife.75873

Figure Lengend Snippet: ( A ) Bone marrow-derived macrophages (BMDMs) were obtained from wildtype or Cpeb4MKO mice. Immunoblot analysis of CPEB4 during lipopolysaccharide (LPS) stimulation. Vinculin served as loading control. ( B ) Percentage of mice of the indicated phenotype born from matings between Cpeb4 +/+ or Cpeb4 lox/lox females and a corresponding male carrying the Lyz2 Cre gene (n > 47 for each genotype). ( C ) Total animal body weight (n > 10). ( D ) Normalized organ weight (n = 6). ( E ) Complete blood counts from wildtype or Cpeb4MKO mice (n = 6). WBC, white blood cells; LYM, lymphocytes; MID, monocytes; GRA, granulocytes; PLT, platelets; RBC, red blood cells.

Article Snippet: On day 8, BMDMs were primed with LPS (10 ng/ml, E. coli 0111:B4, Santa Cruz SC-3535) for the indicated time points.

Techniques: Derivative Assay, Western Blot, Control

Journal: eLife

Article Title: Macrophage inflammation resolution requires CPEB4-directed offsetting of mRNA degradation

doi: 10.7554/eLife.75873

Figure Lengend Snippet: ( A–C ) Lipopolysaccharide (LPS)-stimulated wildtype (WT) bone marrow-derived macrophages (BMDMs). ( A ) Cpeb1–4 levels were measured by RT-qPCR (n = 6). ( B, left) CPEB4 immunoblot, using α-tubulin as loading control. (Right) CPEB4 quantification normalized to α-tubulin (n = 3; data shown in ). ( C , left) CPEB4 immunoblot in protein extracts treated with λphosphatase when indicated. (Right) Quantification of P-CPEB4 signal (n = 3). ( D–H ) LPS-stimulated WT and Cpeb4 –/– BMDMs. ( D ) Number of differentially expressed genes (p<0.01) between genotypes. mRNA levels were quantified by RNAseq (n = 4). Statistics: DESeq2 R package. ( E ) Z-score signature of the indicated pathways. mRNA levels were quantified by RNAseq (n = 4). Statistics: rotation gene set enrichment analysis. ( F , left) HIF1a and CPEB4 immunoblot, vinculin served as loading control. (Right) Normalized quantification, signal intensity was normalized to vinculin and fold change to WT at 9 hr after LPS induction was calculated (n = 3; data shown in ). ( G, H ) Hif1a and Il10 levels measured by RT-qPCR (n = 6). ( A, G ) Tbp was used to normalize. ( B, C, E, F ) Data are represented as mean ± SEM. ( F–H ) Statistics: two-way ANOVA. ( D, E ) See also . Figure 2—source data 1. Blots corresponding to and . Figure 2—source data 2. Blots corresponding to . Figure 2—source data 3. Blots corresponding to and .

Article Snippet: On day 8, BMDMs were primed with LPS (10 ng/ml, E. coli 0111:B4, Santa Cruz SC-3535) for the indicated time points.

Techniques: Derivative Assay, Quantitative RT-PCR, Western Blot, Control

Journal: eLife

Article Title: Macrophage inflammation resolution requires CPEB4-directed offsetting of mRNA degradation

doi: 10.7554/eLife.75873

Figure Lengend Snippet: ( A ) Immunoblot analysis of CPEB4 in control or LPS-treated macrophages. α-Tubulin served as loading control. ( B ) Immunoblot analysis of CPEB4 in LPS-stimulated bone marrow-derived macrophages (BMDMs) obtained from wildtype or Cpeb4 –/– mice. Vinculin served as loading control.

Article Snippet: On day 8, BMDMs were primed with LPS (10 ng/ml, E. coli 0111:B4, Santa Cruz SC-3535) for the indicated time points.

Techniques: Western Blot, Control, Derivative Assay

Journal: eLife

Article Title: Macrophage inflammation resolution requires CPEB4-directed offsetting of mRNA degradation

doi: 10.7554/eLife.75873

Figure Lengend Snippet: ( A ) Immunoblot analysis of HIF1a in LPS-stimulated bone marrow-derived macrophages (BMDMs) obtained from wildtype or Cpeb4 –/– mice. Vinculin served as loading control. Quantification is shown in . ( B ) Gene set analysis of Hallmark pathways in LPS-stimulated WT and Cpeb4 –/– BMDMs. Differential mRNA expression was measured by RNAseq (n = 4). Statistics: DESeq2 R package. See also .

Article Snippet: On day 8, BMDMs were primed with LPS (10 ng/ml, E. coli 0111:B4, Santa Cruz SC-3535) for the indicated time points.

Techniques: Western Blot, Derivative Assay, Control, Expressing

Journal: eLife

Article Title: Macrophage inflammation resolution requires CPEB4-directed offsetting of mRNA degradation

doi: 10.7554/eLife.75873

Figure Lengend Snippet: ( A ) Bone marrow-derived macrophages (BMDMs) were stimulated with lipopolysaccharide (LPS) and mRNA levels were measured by RT-qPCR, normalizing to Tbp (n = 6). Cpeb4 mRNA values are also shown in . ( B ) Schematic representation of the Cpeb4 3’-UTR showing AU-rich element (ARE) domains.

Article Snippet: On day 8, BMDMs were primed with LPS (10 ng/ml, E. coli 0111:B4, Santa Cruz SC-3535) for the indicated time points.

Techniques: Derivative Assay, Quantitative RT-PCR

Journal: eLife

Article Title: Macrophage inflammation resolution requires CPEB4-directed offsetting of mRNA degradation

doi: 10.7554/eLife.75873

Figure Lengend Snippet: ( A ) Cpeb4 levels in wildtype (WT) and p38αMKO bone marrow-derived macrophages (BMDMs) stimulated with lipopolysaccharide (LPS) (n = 3). ( B ) Cpeb4 levels in LPS-stimulated BMDMs treated with the p38α inhibitor PH-797804 (or DMSO as control) (n = 4). ( C ) WT or p38αMKO BMDMs were stimulated with LPS for 1 hr; Cpeb4 mRNA stability was measured after treating with actinomycin D (ActD). Statistics: paired t -test (60 min time point; n = 3). See also . ( D ) Cpeb4 mRNA levels in HuR RNA-immunoprecipitates (IP) performed in WT or p38αMKO BMDMs, after LPS stimulation as indicated. IgG IPs served as control. IP/input enrichment is shown, normalized to WT IP LPS (n = 2). See also . ( E ) Immunoblot of TTP protein in WT BMDMs treated with LPS for 0–9 hr. Vinculin served as loading control (n = 2). ( F ) Cpeb4 and Tnf decay rates in WT and TTPMKO BMDMs stimulated for 6 hr with LPS (data from ). Data represents the mean of three biological replicates. ( G, H ) U2OS cells were treated with tetracycline to induce the expression of a constitutively active MKK6, which induces p38α MAPK activation . ( G ) Cpeb4 levels upon p38α activation (+MKK6) or inhibition with SB203580 or PH-797804 (n = 3). ( H ) Cpeb4 levels in control or HuR-depleted U2OS cells, where p38α MAPK signaling has been activated (+MKK6) or inhibited (SB) (n = 2). See also . ( A–D, G, H ) mRNA levels were quantified by RT-qPCR. Gapdh ( A, B, C, G ) was used to normalize. ( A, B, D, G, H ) Data are represented as mean ± SEM. ( A, B ) Statistics: two-way ANOVA. ( C ) Statistics: paired t -test. ( G, H ) Statistics: one-way ANOVA, selected pvadj are shown. Figure 3—source data 1. Blots corresponding to .

Article Snippet: On day 8, BMDMs were primed with LPS (10 ng/ml, E. coli 0111:B4, Santa Cruz SC-3535) for the indicated time points.

Techniques: Derivative Assay, Control, Western Blot, Expressing, Activation Assay, Inhibition, Quantitative RT-PCR

Journal: eLife

Article Title: Macrophage inflammation resolution requires CPEB4-directed offsetting of mRNA degradation

doi: 10.7554/eLife.75873

Figure Lengend Snippet: ( A ) WT or p38αMKO BMDMs were stimulated with lipopolysaccharide (LPS) for 1 hr; and mRNA stability was measured after treating with actinomycin D (ActD). mRNA levels were quantified by RT-qPCR. Gapdh was used to normalize.

Article Snippet: On day 8, BMDMs were primed with LPS (10 ng/ml, E. coli 0111:B4, Santa Cruz SC-3535) for the indicated time points.

Techniques: Quantitative RT-PCR

Journal: eLife

Article Title: Macrophage inflammation resolution requires CPEB4-directed offsetting of mRNA degradation

doi: 10.7554/eLife.75873

Figure Lengend Snippet: ( A ) HuR IP was performed in wildtype (WT) and p38αMKO BMDMs stimulated with lipopolysaccharide (LPS) for 3 hr when indicated. IgG IP was used as control.

Article Snippet: On day 8, BMDMs were primed with LPS (10 ng/ml, E. coli 0111:B4, Santa Cruz SC-3535) for the indicated time points.

Techniques: Control

Journal: eLife

Article Title: Macrophage inflammation resolution requires CPEB4-directed offsetting of mRNA degradation

doi: 10.7554/eLife.75873

Figure Lengend Snippet: ( A ) TTP PAR-iCLIP was performed in bone marrow-derived macrophages (BMDMs) treated with lipopolysaccharide (LPS) for 6 hr. Coverage plots represent the number of crosslink sites (CL) detected in each position of Cpeb4 mRNA. For binding sites located in the Cpeb4 3′-UTR, distance to the transcription start site (TSS) and their scores is indicated (data from ).

Article Snippet: On day 8, BMDMs were primed with LPS (10 ng/ml, E. coli 0111:B4, Santa Cruz SC-3535) for the indicated time points.

Techniques: Derivative Assay, Binding Assay

Journal: eLife

Article Title: Macrophage inflammation resolution requires CPEB4-directed offsetting of mRNA degradation

doi: 10.7554/eLife.75873

Figure Lengend Snippet: ( A–D ) CPEB4 RNA-Immunoprecipitation (IP) and sequencing was performed using total lysates (input) from wildtype (WT) or Cpeb4 –/– bone marrow-derived macrophages (BMDMs) that had been treated or not with LPS for 9 hr (n = 1). ( A ) CPEB4 immunoblot, using vinculin as a loading control. ( B ) Examples of read coverage of input or IP of selected mRNAs. Peak enrichments between WT and Cpeb4 –/– IPs are shown in blue. ( C ) Cytoplasmic polyadenylation element (CPE) and CPE G-containing transcripts according to ) in input and CPEB4 IPs. The script from was modified to consider TTTTGT as a CPE motif. Statistics: Fisher’s exact test. ( D ) Read coverage of IPs of selected mRNAs. ( E ) CPEB4 IP and RT-qPCR were performed for WT or Cpeb4 –/– BMDMs stimulated with LPS for 9 hr. IP/input enrichment is shown (n = 3). ( F ) Socs1 mRNA levels in LPS-stimulated WT and Cpeb4 –/– BMDMs. mRNA levels were measured by RT-qPCR normalizing to Tbp (n = 6). ( G ) Immunoblot of SOCS1 in WT and Cpeb4 –/– BMDMs treated with LPS. Vinculin served as loading control. Quantification is shown (FC to WT, after 9 hr of LPS) (n = 3). ( H ) Differential expression between WT and Cpeb4 –/– BMDMs treated with LPS measured by RNAseq (n = 4). Statistics: DESeq2 R package. ( I ) mRNA stability was measured by treating with actinomycin D (ActD) WT and Cpeb4 –/– BMDMs stimulated with LPS for the indicated times. Gene expression was analyzed by RT-qPCR, normalized to Gapdh/Tbp (n = 4). ( J ) RAW 264.7 macrophages were transfected with a Firefly luciferase reporter under the control of the cyclin B1 3′-UTR, containing either WT (CPE+) or mutated (CPE–) CPE motifs. The same plasmid contained Renilla luciferase reporter as a control. Macrophages were stimulated with LPS for 3 hr, at which point ActD was added. mRNA levels were measured by RT-qPCR. ( B, D ) Integrated Genomic Viewer (IGV) images. ( E–G ) Data are represented as mean ± SEM. ( F, G, I, J ) Statistics: two-way ANOVA. See also . Figure 4—source data 1. Blots corresponding to . Figure 4—source data 2. Blots corresponding to .

Article Snippet: On day 8, BMDMs were primed with LPS (10 ng/ml, E. coli 0111:B4, Santa Cruz SC-3535) for the indicated time points.

Techniques: RNA Immunoprecipitation, Sequencing, Derivative Assay, Western Blot, Control, Modification, Quantitative RT-PCR, Quantitative Proteomics, Gene Expression, Transfection, Luciferase, Plasmid Preparation

Journal: eLife

Article Title: Macrophage inflammation resolution requires CPEB4-directed offsetting of mRNA degradation

doi: 10.7554/eLife.75873

Figure Lengend Snippet: ( A, B ) CPEB4 RNA-Immunoprecipitation (IP) and sequencing was performed in total lysates (Input) from wildtype and Cpeb4 KO bone marrow-derived macrophages (BMDMs), untreated or stimulated with LPS for 9 hr (n = 1). ( A ) CPE-A- or CPE-G-containing transcripts in Inputs and CPEB4 IPs. Statistics with Fisher’s exact test. ( B ) Top 10 Gene Ontology KEGG categories enriched in CPEB4 target mRNAs in wildtype BMDMs stimulated with LPS for 9 hr. Mus musculus transcriptome was used as background. Statistics: Benjamini–Hochberg adjusted p-value is shown. See also .

Article Snippet: On day 8, BMDMs were primed with LPS (10 ng/ml, E. coli 0111:B4, Santa Cruz SC-3535) for the indicated time points.

Techniques: RNA Immunoprecipitation, Sequencing, Derivative Assay

Journal: eLife

Article Title: Macrophage inflammation resolution requires CPEB4-directed offsetting of mRNA degradation

doi: 10.7554/eLife.75873

Figure Lengend Snippet: ( A, B ) Differential expression between wildtype (WT) and bone marrow-derived macrophages (BMDMs) treated with LPS measured by RNAseq (n = 4). Statistics: DESeq2cR package ( C ) Dusp1 mRNA levels were measured by RT-qPCR, normalizing to Tbp .

Article Snippet: On day 8, BMDMs were primed with LPS (10 ng/ml, E. coli 0111:B4, Santa Cruz SC-3535) for the indicated time points.

Techniques: Quantitative Proteomics, Derivative Assay, Quantitative RT-PCR

Journal: eLife

Article Title: Macrophage inflammation resolution requires CPEB4-directed offsetting of mRNA degradation

doi: 10.7554/eLife.75873

Figure Lengend Snippet: ( A ) ARE-containing transcripts in the input and CPEB4 immunoprecipitations (IPs) from . Statistics: Fisher’s exact test. ( B ) Percentage of CPEB4 targets in lipopolysaccharide (LPS)-stimulated bone marrow-derived macrophages (BMDMs) transcriptome and TTP targets in LPS-stimulated BMDMs . ( C ) Genome-wide correlation between ARE and CPE motifs in 3′-UTRs. The black line shows the linear regression trend line. R 2 = 0.6364. ( D ) mRNA levels in wildtype (WT) and Cpeb4 –/– BMDMs were measured by RT-qPCR, normalizing to Tbp (n = 6). Statistics: two-way ANOVA. Socs1 data are also shown in . ( E ) mRNA expression in WT or TTPMKO BMDMs treated with LPS. Statistics: DESeq2 software, qval is shown (data from ). ( F ) Common TTP and CPEB4 target mRNAs were classified according to the ARE:CPE score as ARE-dominant (ARE-d; red; 30 mRNAs) or CPE-dominant (CPE-d; blue; 61 mRNAs) (see also ). ( G ) CPEB4 and TTP target mRNAs were plotted according to the number of AREs and CPEs in the 3′-UTR. (Left) The dashed line separates ARE-d and CPE-d mRNAs. (Right) mRNAs were classified according to only the number of AREs in their 3'-UTR. The dashed line separates ARE high (>4 AREs; yellow; 43 mRNAs) from ARE low (≤4 AREs; navy; 56 mRNAs) mRNAs. ( H, I ) WT BMDMs were stimulated with LPS and mRNA levels were quantified by RNAseq (n = 4). ( H ) Common CPEB4 and TTP target mRNAs were classified as AREd/CPEd (left) or ARE high /ARE low (right). For each mRNA, the levels after 9 hr of LPS treatment were normalized by its expression at 6 hr LPS. ( I ) 1521 CPE- and ARE-containing mRNAs were classified as sustained >0.5 (1319 mRNAs) or downregulated <0.5 (202 mRNAs) according to their expression after 9 hr of LPS treatment, after normalizing to the peak of expression throughout the LPS response. For each mRNA, the ARE:CPE score was calculated. ( J, K ) RAW 264.7 macrophages were transfected with a Firefly luciferase reporter under the control of a chimeric 3′-UTR combining Ier3 and cyclin B1 AREs and CPEs motifs, respectively. Inactivating specific CPE or ARE motifs, six different 3′-UTRs with distinct ARE:CPE scores were generated. The same plasmid contained Renilla luciferase reporter as a control. ( J ) Scheme of the six constructs used for the dual luciferase reporter assay. Inactivated motifs are shown in gray. ( K ) RAW 264.7 macrophages were stimulated with LPS for 6 hr and Firefly/Renilla levels were measured by RT-qPCR. Values were normalized to the 0AREs/0CPEs construct. Statistics: one-way ANOVA Friedman test. All significant differences are shown except 5AREs/0CPEs vs. 0AREs/3CPEs (**) and 2AREs/3CPEs (*). ( D, H, K ) Data are represented as mean ± SEM. ( H, I ) Statistics: Mann–Whitney t -test. See also .

Article Snippet: On day 8, BMDMs were primed with LPS (10 ng/ml, E. coli 0111:B4, Santa Cruz SC-3535) for the indicated time points.

Techniques: Derivative Assay, Genome Wide, Quantitative RT-PCR, Expressing, Software, Transfection, Luciferase, Control, Generated, Plasmid Preparation, Construct, Reporter Assay, MANN-WHITNEY

Journal: eLife

Article Title: Macrophage inflammation resolution requires CPEB4-directed offsetting of mRNA degradation

doi: 10.7554/eLife.75873

Figure Lengend Snippet: ( A ) ARE/CPE score definition. ARE and CPE motifs used to calculate the score are specified. ( B–E ) Wildtype bone marrow-derived macrophages (BMDMs) were treated with lipopolysaccharide (LPS) and mRNA levels were quantified by RNAseq (n = 4). ( B–D ) Common TTP and CPEB4 target mRNAs were considered. ( B ) ARE-dominant (AREd, red) and CPE-dominant (CPEd, blue) mRNA levels after 3 hr of LPS stimulation, normalized for its expression at 1 hr. Statistics: Mann–Whitney t -test. ( C ) Mean expression profile of CPEd and AREd mRNAs in LPS-stimulated BMDMs. For each mRNA, values were normalized to its peak of expression. Statistics: two-way ANOVA. ( D ) Mean mRNA expression profile of ARE high and ARE low mRNAs in LPS-stimulated BMDMs. For each mRNA, values were normalized to its peak of expression. ( E ) 1521 CPE- and ARE-containing mRNAs were classified as sustained >0.5 or downregulated <0.5, on the basis of their expression after 9 hr of LPS treatment, normalized by their peak of expression during the LPS response. For each mRNA, the number of AREs in its 3’-UTR was calculated. ( B ) Data are represented as mean ± SEM. ( C, D ) Data are represented as mean ± SD. See also .

Article Snippet: On day 8, BMDMs were primed with LPS (10 ng/ml, E. coli 0111:B4, Santa Cruz SC-3535) for the indicated time points.

Techniques: Derivative Assay, Expressing, MANN-WHITNEY

Journal: eLife

Article Title: Macrophage inflammation resolution requires CPEB4-directed offsetting of mRNA degradation

doi: 10.7554/eLife.75873

Figure Lengend Snippet: ( A ) Lipopolysaccharide (LPS) stimulates the MAPK signaling cascades downstream of TLR4. p38α controls TTP phosphorylation, causing a shift in the competitive binding equilibrium between Hu-antigen R (HuR) and tristetraprolin (TTP) towards HuR, which stabilizes AU-rich element (ARE)-containing mRNAs. The p38α/HuR/TTP axis also regulates Cpeb4 mRNA stability and promotes CPEB4 expression during the late phase of the LPS response. CPEB4 then accumulates in its active state, which involves phosphorylation by ERK1/2 MAPK signaling. During the resolutive phase of the LPS response, CPEB4 and TTP compete to stabilize/destabilize mRNAs containing cytoplasmic polyadenylation elements (CPEs) and AREs. The equilibrium between these positive and negative cis -acting elements in the mRNA 3′-UTRs generates customized temporal expression profiles. CPEB4 stabilizes CPE-dominant mRNAs, which are enriched in transcripts encoding negative regulators of MAPKs that contribute to inflammation resolution. ( B ) Immunoblot of the indicated proteins in wildtype (WT) BMDMs treated with LPS for 0–9 hr. Vinculin and Ponceau staining served as loading control. Figure 6—source data 1. Blots corresponding to .

Article Snippet: On day 8, BMDMs were primed with LPS (10 ng/ml, E. coli 0111:B4, Santa Cruz SC-3535) for the indicated time points.

Techniques: Phospho-proteomics, Binding Assay, Expressing, Western Blot, Staining, Control

Journal: Cell reports

Article Title: CUX1 regulates human hematopoietic stem cell chromatin accessibility via the BAF complex

doi: 10.1016/j.celrep.2024.114227

Figure Lengend Snippet: (A) Co-immunoprecipitation for CUX1 in K562 cells was followed by mass spectrometry (n=2 biological replicates). The heatmap indicates BAF members ranked by the mean label-free quantification fold enrichment compared to IgG controls. (B) Representative co-immunoprecipitation followed by immunoblot in K562 (n=2 biological replicates). (C) K562 CUX1 and SMARCA4 ChIP-seq overlap (n=2 biological replicates, IDR<0.05). (D) Enriched motifs84 at CUX1 and SMARCA4 co-occupied sites. (E) Overlap of SMARCA4 peaks (n=2 biological replicates, IDR<0.05) in gHPRT and gCUX1 K562 cells. (F) Heatmaps showing overlap between CUX1-dependent or CUX1-independent SMARCA4 sites with CUX1. The values are normalized ChIP-seq reads (RPKM). The direct model represents CUX1 recruitment of SMARCA4. The indirect model represents SMARCA4 sites bound but not recruited by CUX1. Example genome snapshots for each category are shown (G).85 (H) Distance to the nearest transcription start site (TSS) of CUX1-recruited and non-CUX1-recruited SMARCA4 sites and hematopoietic TF occupancy (I).

Article Snippet: 12 μg of CUX1 antibody (B-10 Santa Cruz sc-514008) and mouse IgG (Santa Cruz sc-2025) antibody were added to the lysate and incubated overnight on a rocker at 4°C.

Techniques: Immunoprecipitation, Mass Spectrometry, Quantitative Proteomics, Western Blot, ChIP-sequencing

Journal: Cell reports

Article Title: CUX1 regulates human hematopoietic stem cell chromatin accessibility via the BAF complex

doi: 10.1016/j.celrep.2024.114227

Figure Lengend Snippet: (A) Volcano plot comparing ATAC-seq signal in gCUX1 vs. gHPRT K562 cells (n=2 biological replicates). Significance calculated by csaw.41 Top enriched motifs for the significant down sites are shown. (B) H3K27ac ChIP-seq reads (n=2 biological replicates) at significantly down, up and non-significant ATAC sites. (C) CUX1 and SMARCA4 occupancy at down (n=933) vs. CUX1-independent ATAC sites (n=14,256). ATAC-seq signal from gHPRT and gCUX1 cells for CUX1 and SMARCA4 co-occupied sites (D), CUX1-recruited SMARCA4 sites (E), and SMARCA4 sites bound but not recruited by CUX1 (F). Significance for (B-F) calculated by two-sided Wilcoxon rank-sum test.

Article Snippet: 12 μg of CUX1 antibody (B-10 Santa Cruz sc-514008) and mouse IgG (Santa Cruz sc-2025) antibody were added to the lysate and incubated overnight on a rocker at 4°C.

Techniques: ChIP-sequencing

Journal: Cell reports

Article Title: CUX1 regulates human hematopoietic stem cell chromatin accessibility via the BAF complex

doi: 10.1016/j.celrep.2024.114227

Figure Lengend Snippet: (A) Overlap of CUX1 and SMARCA4 CUT&RUN peaks in primary human CD34+ HSPCs (n=2 biological replicates, IDR<0.05). (B) Genome-wide correlation of CUX1 and SMARCA4 CUT&RUN signals with histone marks from Roadmap Epigenomics.44 All pairwise correlations have p<0.001. (C) CUX1 and SMARCA4 peaks absolute distance (log2 transformed) to the nearest TSS. The dash line indicates 2 Kb. (D) Top GO terms for TSS-proximal and -distal CUX1/SMARCA4 co-bound sites (Bonferroni corrected p-value<0.05).45,46 (E) Volcano plot of ATAC-seq changes in gCUX1 and gHPRT CD34+ HSPCs (n=2 biological replicates). Significance calculated by csaw.41 Top motifs for the down sites are shown. (F) Normalized ATAC reads at genome-wide CUX1-bound enhancers (n=3,902) and a randomly sampled, size-matched list of enhancers not bound by CUX1 (top). Normalized ATAC reads at CUX1-bound enhancers (n=3,902) comparing the control gHPRT and gCUX1 conditions (bottom). (G) Normalized CUT&RUN reads of CUX1 and SMARCA4 in CD34+ HSPC at down vs. CUX1-independent ATAC sites. Significance for (F) and (G) is by two-sided Wilcoxon rank-sum test.

Article Snippet: 12 μg of CUX1 antibody (B-10 Santa Cruz sc-514008) and mouse IgG (Santa Cruz sc-2025) antibody were added to the lysate and incubated overnight on a rocker at 4°C.

Techniques: Genome Wide, Transformation Assay, Control

Journal: Cell reports

Article Title: CUX1 regulates human hematopoietic stem cell chromatin accessibility via the BAF complex

doi: 10.1016/j.celrep.2024.114227

Figure Lengend Snippet: (A) ATAC-seq accessibility for gHPRT and gCUX1 CD34+ HSPCs at distal 3D chromatin contact points looped to CUX1-bound promoters from published CD34+ HSPC Hi-C data.59 (B) IGV snapshot of CUX1 binding at the promoter of KIT and the reduced accessibility of multiple enhancers looped to the promoter. Enhancer and promotor annotations are from Roadmap Epigenomics.44 (C) Integration of CD34+ HSPC ATAC-seq and RNA-seq (n=2 biological replicates).13 Scatterplot shows the RNA log2FC vs. ATAC-seq log2FC for 406 DEGs (FDR<0.1, |log2FC|>0.75). Enriched GO terms related to HSPC lineage commitment are shown.86 (D) Log2FC of ATAC-seq signal comparing CD34+ HSPC gCUX1 vs. gHPRT cells at the hematopoietic cell-type specific enhancers from the VISION database87 (9,657 myeloid enhancers, 11,653 erythroid enhancers, 15,323 lymphoid enhancers), and 10,000 randomly sampled non-cell type specific enhancers. (E) Performance score of cell fate prediction using the published murine HSPC scRNA-seq.68 From left to right: positive control is the top 2,000 genes with the highest cell-cell variation; negative controls are a randomly sampled gene set (n=1,000) and curated list of mouse TFs (n=1,636);88 CUX1-bound genes from human CD34+ HSPC CUT&RUN (n=6,758); overlap of CUX1-bound and differentially-expressed upon CUX1 knockdown in CD34+ HSPC (n=923). Equivalent gene sets were tested for PU.1 and RUNX1 as benchmarks (n = 336 and 325).69–72 For all gene sets larger than 1,000, 50 bootstraps were performed to sample for 1,000 genes. Logistic regression and deep neural network were used to construct the classifier. Significance for (A), (D), (E) is by two-sided Wilcoxon rank sum test.

Article Snippet: 12 μg of CUX1 antibody (B-10 Santa Cruz sc-514008) and mouse IgG (Santa Cruz sc-2025) antibody were added to the lysate and incubated overnight on a rocker at 4°C.

Techniques: Hi-C, Binding Assay, RNA Sequencing, Positive Control, Knockdown, Construct

Journal: Cell reports

Article Title: CUX1 regulates human hematopoietic stem cell chromatin accessibility via the BAF complex

doi: 10.1016/j.celrep.2024.114227

Figure Lengend Snippet: Key resources table

Article Snippet: 12 μg of CUX1 antibody (B-10 Santa Cruz sc-514008) and mouse IgG (Santa Cruz sc-2025) antibody were added to the lysate and incubated overnight on a rocker at 4°C.

Techniques: Purification, Mass Spectrometry, Software

Journal: Cell reports

Article Title: Insulin Potentiates JAK/STAT Signaling to Broadly Inhibit Flavivirus Replication in Insect Vectors

doi: 10.1016/j.celrep.2019.10.029

Figure Lengend Snippet:

Article Snippet: For ERK inhibition experiments, culture media was supplemented with 10 μM U0126 in DMSO (Cell Signaling 9903) ( ; ) 24 hours prior to and during infection.

Techniques: Virus, Recombinant, Control, Mutagenesis, Software, Genome Wide

Journal: Foods

Article Title: Development of a DNA Metabarcoding Method for the Identification of Bivalve Species in Seafood Products

doi: 10.3390/foods10112618

Figure Lengend Snippet: Multi-species sequence alignment of the mitochondrial 16S rDNA barcoding region for bivalve species. Colored bars indicate the binding sites of the primer sets for scallops (blue), oysters (green), and mussels (red, CLC Genomics Workbench software version 10.1.1, Qiagen, Hilden, Germany).

Article Snippet: Reference sequences for commonly consumed bivalve species and some exotic seafood species, that are permitted for consumption in Austria (“Codex Alimentarius Austriacus” chapter B35, [ ]), were downloaded from the NCBI databases ( ) by using CLC Genomics Workbench software (version 10.1.1, Qiagen, Hilden, Germany).

Techniques: Sequencing, Binding Assay, Software

Journal: bioRxiv

Article Title: The NAT10 acetyltransferase modulates DNA damage-related factors and global 3D-genome architecture

doi: 10.1101/2025.03.05.641614

Figure Lengend Snippet: (A) The NAT10 acetyltransferase (green) occupies the nucleolus visualized by the antibody against fibrillarin (red). Nuclear DNA was stained by DAPI (blue). Bars represent 0.8 µm. (B) NAT10 interaction with 18S rRNA is documented with pTM = 0.51. (C) AlphaFold 3 model of NAT10 dimerization is shown (pTM = 0.54). Structures are colored using AlphaFold 3 pLDDT confidence score.

Article Snippet: Following this, the membranes were blocked with 5% non-fat dry milk for 2 hours and were incubated overnight at 4 °C with specific primary antibodies: anti-NAT10 (#sc-271770, Santa Cruz Biotechnology Inc., USA), anti-p53 (#sc-126), anti-XPC (#sc-30156); anti-XPA (#sc-28353), anti-DDB2 (#sc-25368), and anti-GAPDH (#60004-1, Proteintech, Germany).

Techniques: Staining

Journal: bioRxiv

Article Title: The NAT10 acetyltransferase modulates DNA damage-related factors and global 3D-genome architecture

doi: 10.1101/2025.03.05.641614

Figure Lengend Snippet: Data from AlphaFold 3, predicting (A) NAT10 dimerization and the degree of interaction between (B) NAT10-DDB2, (C) NAT10-p53, and (D) DDB2-p53, were imported to ChimeraX 1.9 software showing individual proteins. NAT10 is highlighted by orange, DDB2 is green, p53 is blue colour. The source data are shown at https://www.ibp.cz/en/research/departments/cellular-biology-and-epigenetics/open-data . This raw data from AlphaFold 3 can be uploaded to ChimeraX 1.9 for more detailed analysis.

Article Snippet: Following this, the membranes were blocked with 5% non-fat dry milk for 2 hours and were incubated overnight at 4 °C with specific primary antibodies: anti-NAT10 (#sc-271770, Santa Cruz Biotechnology Inc., USA), anti-p53 (#sc-126), anti-XPC (#sc-30156); anti-XPA (#sc-28353), anti-DDB2 (#sc-25368), and anti-GAPDH (#60004-1, Proteintech, Germany).

Techniques: Software

Journal: bioRxiv

Article Title: The NAT10 acetyltransferase modulates DNA damage-related factors and global 3D-genome architecture

doi: 10.1101/2025.03.05.641614

Figure Lengend Snippet: (A) The following proteins were studied in non-irradiated and UVC-irradiated NAT10 wt and dn cells: NAT10, p53, XPA, XPC, and DDB2. In detail, panel (A) shows western blot analysis of proteins NAT10, p53, XPA, XPC, and DDB2 in non-irradiated and UVC-irradiated cells without and with a depletion of the NAT10 acetyltransferase. Data revealed lower p53, XPA, XPC, and DDB2 protein levels in NAT10 dn cells, with further reductions of XPA, XPC, and DDB2 protein levels upon UVC exposure. UVC irradiation also decreased the pool of p53, XPA, and XPC in NAT10 wt cells. Panels (B-D) show the quantification of western blot data from panel (A) using ImageJ software (NIH freeware, USA). Non-irradiated cells and cells irradiated by UVC light were analyzed. (E) TP53 deficiency led to a decrease in the pool of NAT10 and DDB2 proteins. Panel (F) shows the quantification of western blot data from panel (E) using ImageJ software (NIH freeware, USA). The protein levels were normalized to GAPDH (reference and loading control). The asterisks in panels B-D and F represent statistically significant differences with either a p-value ≤ 0.05 (*) or a p-value ≤ 0.001 (***). AlphaFold 3 models of ( G) NAT10 interaction with p53 (pTM = 0.53), ( H ) NAT10 and DDB2 (pTM = 0.5), and ( I ) DDB2 and p53 protein (pTM = 0.47). Structures are colored with AlphaFold 3 pLDDT confidence score.

Article Snippet: Following this, the membranes were blocked with 5% non-fat dry milk for 2 hours and were incubated overnight at 4 °C with specific primary antibodies: anti-NAT10 (#sc-271770, Santa Cruz Biotechnology Inc., USA), anti-p53 (#sc-126), anti-XPC (#sc-30156); anti-XPA (#sc-28353), anti-DDB2 (#sc-25368), and anti-GAPDH (#60004-1, Proteintech, Germany).

Techniques: Irradiation, Western Blot, Software, Control

Journal: bioRxiv

Article Title: The NAT10 acetyltransferase modulates DNA damage-related factors and global 3D-genome architecture

doi: 10.1101/2025.03.05.641614

Figure Lengend Snippet: Panels (A, B) show AlphaFold 3 multimer prediction of human NAT10 interaction with (A) NAT10 and p53 and DDB2 proteins (pTM = 0.48) or (B) NAT10 and p53 and DDB1-DDB2 protein complex complex (pTM = 0.53).

Article Snippet: Following this, the membranes were blocked with 5% non-fat dry milk for 2 hours and were incubated overnight at 4 °C with specific primary antibodies: anti-NAT10 (#sc-271770, Santa Cruz Biotechnology Inc., USA), anti-p53 (#sc-126), anti-XPC (#sc-30156); anti-XPA (#sc-28353), anti-DDB2 (#sc-25368), and anti-GAPDH (#60004-1, Proteintech, Germany).

Techniques:

Journal: bioRxiv

Article Title: The NAT10 acetyltransferase modulates DNA damage-related factors and global 3D-genome architecture

doi: 10.1101/2025.03.05.641614

Figure Lengend Snippet: Interaction properties between p53-DDB2 and NAT10-DDB2 in human NAT10 wt and NAT10 dn cells were studied using PLA. (A) The panel shows an example of PLA methodology, adapted from BioRender software. In (B) NAT10 wt and (C) NAT10 dn cells, after fixation, the PLA was performed using antibodies to detect the p53-DDB2 or NAT10-DDB2 complexes in both non-irradiated and UVC-irradiated NAT10 wt and NAT10 dn cells. Red dots indicate amplified interaction signals, with cell nuclei counterstained with DAPI, used for visualization of DNA content. NAT10-DDB2 in NAT10 dn acted as a control, showing no external impact on PLA outcomes. This section quantifies PLA signals (dots per nucleus) for (D) the p53-DDB2 complex in non-irradiated and UVC-irradiated NAT10 wt cells, (E) p53-DDB2 in non-irradiated and UVC-irradiated NAT10 dn cells. (F) The UVC irradiation significantly increased NAT10-DDB2 PLA signals in NAT10 wt cells, suggesting enhanced interaction. Non-irradiated cells and cells irradiated by UVC light were analyzed. We used ImageJ software (NIH freeware, USA) to analyze the number of PLA signals. The statistical analysis was performed using GraphPad Prism 9 software (USA) and the nonparametric Mann-Whitney U -test. The asterisk in panel F represents statistically significant differences with a p-value ≤ 0.05 (*).

Article Snippet: Following this, the membranes were blocked with 5% non-fat dry milk for 2 hours and were incubated overnight at 4 °C with specific primary antibodies: anti-NAT10 (#sc-271770, Santa Cruz Biotechnology Inc., USA), anti-p53 (#sc-126), anti-XPC (#sc-30156); anti-XPA (#sc-28353), anti-DDB2 (#sc-25368), and anti-GAPDH (#60004-1, Proteintech, Germany).

Techniques: Software, Irradiation, Amplification, Control, MANN-WHITNEY

Journal: bioRxiv

Article Title: The NAT10 acetyltransferase modulates DNA damage-related factors and global 3D-genome architecture

doi: 10.1101/2025.03.05.641614

Figure Lengend Snippet: (A, B) Animation documents chromatin density inside the cell nucleus (see graphical illustration created by BioRender software). Panel (A) shows cell nucleolus (Nu) and nuclear lamina in dark blue, and panel (B) shows localization of heterochromatin (green) decorating the periphery of cell nucleus and nucleolus, while euchromatin (pale blue) is shown as de-condensed treads inside the cell nucleus. An analysis of the DDB2 nuclear distribution pattern was done using immunohistochemistry in (C) NAT10 wt and NAT10 dn cells. (D) The NAT10 protein (red) was accumulated into tiny, well-visible foci in DAPI-dense (considered as heterochromatin), DAPI-poor (euchromatin, more decondensed) genomic region as well as inside compartment of nucleoli. Panel (E) shows representative images of the DDB2 protein recruitment to UVA-microirradiated chromatin in NAT10 wt and NAT10 dn cells. Microirradiation using a 405 nm laser line was performed, followed by immunofluorescent staining for DDB2 and DNA damage marker γH2A.X. DAPI was used for nuclear counterstaining in blue fluorescence. The scale bar represents 7.5 μm.

Article Snippet: Following this, the membranes were blocked with 5% non-fat dry milk for 2 hours and were incubated overnight at 4 °C with specific primary antibodies: anti-NAT10 (#sc-271770, Santa Cruz Biotechnology Inc., USA), anti-p53 (#sc-126), anti-XPC (#sc-30156); anti-XPA (#sc-28353), anti-DDB2 (#sc-25368), and anti-GAPDH (#60004-1, Proteintech, Germany).

Techniques: Software, Immunohistochemistry, Staining, Marker, Fluorescence

Journal: bioRxiv

Article Title: The NAT10 acetyltransferase modulates DNA damage-related factors and global 3D-genome architecture

doi: 10.1101/2025.03.05.641614

Figure Lengend Snippet: The results of the differential loop analysis are shown for the following comparisons: (A) NAT10 wild-type non-irradiated cells versus NAT10 double null non-irradiated cells; (B) NAT10 wild-type non-irradiated cells versus NAT10 wt UVC-irradiated cells; (C) NAT10 double null non-irradiated cells versus NAT10 double null UVC-irradiated cells. The contact maps show the differential loop analysis for common (unspecific loops) (left), and unique (conditional specific) loops (the middle showing an increased number of loops and the right panels showing a reduced number of loops) in the comparison between two experimental conditions as shown in (A) , (B) , and (C) . Labeling ES means an enrichment score. The color scale indicates ES, with red regions representing higher loop enrichment and blue regions representing loops with reduced interaction properties.

Article Snippet: Following this, the membranes were blocked with 5% non-fat dry milk for 2 hours and were incubated overnight at 4 °C with specific primary antibodies: anti-NAT10 (#sc-271770, Santa Cruz Biotechnology Inc., USA), anti-p53 (#sc-126), anti-XPC (#sc-30156); anti-XPA (#sc-28353), anti-DDB2 (#sc-25368), and anti-GAPDH (#60004-1, Proteintech, Germany).

Techniques: Irradiation, Comparison, Labeling

Journal: bioRxiv

Article Title: The NAT10 acetyltransferase modulates DNA damage-related factors and global 3D-genome architecture

doi: 10.1101/2025.03.05.641614

Figure Lengend Snippet: Changes in the 3D-organization (see contact maps and A/B compartments) of human acrocentric chromosome 13 (HSA13) were studied in non-irradiated and UVC-irradiated NAT10 wt and NAT10 dn HeLa cells. Genome-wide contact maps are shown in panels (A, B) for acrocentric chromosome HSA13 in 360 kb per bin. Red-blue panels show difference maps of acrocentric chromosome 13 visualized using the HiGlass software studied in non-irradiated NAT10 wt and NAT10 wt/UVC-irradiated cells or NAT10 wt and NAT10 dn cells. The missing data in panels A and B are shown in white color. Hi-C data were also analyzed as an average over (C) all acrocentric chromosomes and compared with the average of (D) all human chromosomes and (E) all chromosomes without acrocentric ones. Panel (F) shows compartment switching regions (B-to-A) (A in red shows active and open, positive, chromatin, and B in blue shows condensed and inactive, negative, chromatin) of human chromosome 13 analyzed in non-irradiated and UVC-irradiated NAT10 wt and NAT10 dn HeLa cells. The A/B compartments were identified according to eigenvector analysis, at a medium resolution of 250,000 base pairs. Decomposition from normalized correlation maps was applied.

Article Snippet: Following this, the membranes were blocked with 5% non-fat dry milk for 2 hours and were incubated overnight at 4 °C with specific primary antibodies: anti-NAT10 (#sc-271770, Santa Cruz Biotechnology Inc., USA), anti-p53 (#sc-126), anti-XPC (#sc-30156); anti-XPA (#sc-28353), anti-DDB2 (#sc-25368), and anti-GAPDH (#60004-1, Proteintech, Germany).

Techniques: Irradiation, Genome Wide, Software, Hi-C

Journal: bioRxiv

Article Title: The NAT10 acetyltransferase modulates DNA damage-related factors and global 3D-genome architecture

doi: 10.1101/2025.03.05.641614

Figure Lengend Snippet: Changes in 3D-genome nuclear architecture of human acrocentric chromosomes 13 and 14 caused by NAT10 deficiency and UVC irradiation in HeLa cells. Genome-wide contact maps are shown for acrocentric chromosomes (A) HSA13 and (C) HSA14 in 1 Mb per bin. Panels (B) and (D) show regions of Hi-C matrices corresponding to individual acrocentric chromosomes that were visualized using the HiGlass software at a resolution of 360 Kb per bin (left panel in B and D). The visualization shows difference maps between the control (NAT10 wt or NAT10 dn) and comparative datasets (NAT10 wt / UVC; NAT10 dn or NAT10 dn / UVC) (red-blue right panels in B and D). The missing data in panels B and D are shown in white color. The following panels show global changes in average genomic interactions over all genomic distances of HSA13 (E) and HSA14 (F) studied in non-irradiated and UVC-irradiated NAT10 dn and NAT10 wt cells.

Article Snippet: Following this, the membranes were blocked with 5% non-fat dry milk for 2 hours and were incubated overnight at 4 °C with specific primary antibodies: anti-NAT10 (#sc-271770, Santa Cruz Biotechnology Inc., USA), anti-p53 (#sc-126), anti-XPC (#sc-30156); anti-XPA (#sc-28353), anti-DDB2 (#sc-25368), and anti-GAPDH (#60004-1, Proteintech, Germany).

Techniques: Irradiation, Genome Wide, Hi-C, Software, Control

Journal: bioRxiv

Article Title: The NAT10 acetyltransferase modulates DNA damage-related factors and global 3D-genome architecture

doi: 10.1101/2025.03.05.641614

Figure Lengend Snippet: Changes in 3D-genome nuclear architecture of human acrocentric chromosome 22 caused by NAT10 deficiency and UVC irradiation in HeLa cells. Panel (A) shows contact maps of HSA22 analyzed in resolution 1 Mb per bin, studied in non-irradiated and UVC-irradiated NAT10 wt and NAT10 dn HeLa cells. Panel (B) documents regions of Hi-C matrices corresponding to individual acrocentric chromosome 22 visualized using the HiGlass software at a resolution of 360 Kb per bin (left panels in B and D). The visualization shows differences between the control (NAT10 wt or NAT10 dn) and comparative datasets (NAT10 wt / UVC; NAT10 dn or NAT10 dn / UVC) (red-blue right panels in B). The missing data in panel B are shown in white color. Panels (C) show global changes in average genomic interactions and overall genomic distances of HSA22 studied in non-irradiated and UVC-irradiated NAT10 dn and NAT10 wt cells. Hi-C data were also analyzed as an average over (D) all acrocentric chromosomes and compared with the average of (E) all human chromosomes and (F) all chromosomes without acrocentric ones.

Article Snippet: Following this, the membranes were blocked with 5% non-fat dry milk for 2 hours and were incubated overnight at 4 °C with specific primary antibodies: anti-NAT10 (#sc-271770, Santa Cruz Biotechnology Inc., USA), anti-p53 (#sc-126), anti-XPC (#sc-30156); anti-XPA (#sc-28353), anti-DDB2 (#sc-25368), and anti-GAPDH (#60004-1, Proteintech, Germany).

Techniques: Irradiation, Hi-C, Software, Control

Journal: bioRxiv

Article Title: The NAT10 acetyltransferase modulates DNA damage-related factors and global 3D-genome architecture

doi: 10.1101/2025.03.05.641614

Figure Lengend Snippet: Volcano plots illustrate the results of the differential analysis of chromatin loops (cLoops). These plots depict the relationship between fold change and statistical significance (p-value = 0.01, red line) of individual loops under two experimental conditions. The x-axis represents the logarithmic values of the fold change, indicating the degree of change in interactions between the two conditions. The y-axis shows the negative logarithmic value of the p-value (-log10(p-value)), representing the measure of statistical significance. Differentially enriched chromatin loops are highlighted in green (highly enriched) and purple indicates a reduced number of loops. Insignificantly changed loops are shown in grey. The number of chromatin loop interactions was studied in (A) NAT10 dn non-irradiated cells when compared with NAT10 wt counterpart, (B) NAT10 wt UVC-irradiated cells compared with NAT10 wt non-irradiated cells, and (C) NAT10 dn UVC-irradiated cells compared with NAT10 dn non-irradiated cells.

Article Snippet: Following this, the membranes were blocked with 5% non-fat dry milk for 2 hours and were incubated overnight at 4 °C with specific primary antibodies: anti-NAT10 (#sc-271770, Santa Cruz Biotechnology Inc., USA), anti-p53 (#sc-126), anti-XPC (#sc-30156); anti-XPA (#sc-28353), anti-DDB2 (#sc-25368), and anti-GAPDH (#60004-1, Proteintech, Germany).

Techniques: Irradiation

Journal: bioRxiv

Article Title: The NAT10 acetyltransferase modulates DNA damage-related factors and global 3D-genome architecture

doi: 10.1101/2025.03.05.641614

Figure Lengend Snippet: Changes in TADs (shown in resolution 320 kB per bin) of HSA13 were studied in non-irradiated and UVC-irradiated NAT10 wt and NAT10 dn HeLa cells. Hi-C contact maps are shown with respective TAD annotations for all experimental events (upper orange panel in A-F). A difference map (bottom red panels) highlighting regions where NAT10 wt has more interaction than NAT10 wt/UVC (A) , where NAT10 wt/UVC has more interaction than NAT10 wt (B) , where NAT10 wt has more interaction than NAT10 dn (C) , where NAT10 dn has more interaction than NAT10 wt (D) , where NAT10 dn has more interaction than NAT10 dn/UVC (E) , and where NAT10 dn/UVC has more interaction than NAT10 dn (F) .

Article Snippet: Following this, the membranes were blocked with 5% non-fat dry milk for 2 hours and were incubated overnight at 4 °C with specific primary antibodies: anti-NAT10 (#sc-271770, Santa Cruz Biotechnology Inc., USA), anti-p53 (#sc-126), anti-XPC (#sc-30156); anti-XPA (#sc-28353), anti-DDB2 (#sc-25368), and anti-GAPDH (#60004-1, Proteintech, Germany).

Techniques: Irradiation, Hi-C

Journal: bioRxiv

Article Title: The NAT10 acetyltransferase modulates DNA damage-related factors and global 3D-genome architecture

doi: 10.1101/2025.03.05.641614

Figure Lengend Snippet: TADs are shown in resolution 40 kb per bin for human acrocentric chromosome 13. TADs were studied in non-irradiated and UVC-irradiated NAT10 wt and dn HeLa cells.

Article Snippet: Following this, the membranes were blocked with 5% non-fat dry milk for 2 hours and were incubated overnight at 4 °C with specific primary antibodies: anti-NAT10 (#sc-271770, Santa Cruz Biotechnology Inc., USA), anti-p53 (#sc-126), anti-XPC (#sc-30156); anti-XPA (#sc-28353), anti-DDB2 (#sc-25368), and anti-GAPDH (#60004-1, Proteintech, Germany).

Techniques: Irradiation

Journal: bioRxiv

Article Title: The NAT10 acetyltransferase modulates DNA damage-related factors and global 3D-genome architecture

doi: 10.1101/2025.03.05.641614

Figure Lengend Snippet: The levels of selected histone markers, characterized for (A) euchromatin or (B) heterochromatin, were studied in non-irradiated and UVC-irradiated NAT10 wt and NAT10 dn cells. Analyses were performed using western blots. (C) In non-irradiated and UVC-irradiated NAT10 wt and dn cells, the level of the following histone markers was analyzed: H4ac, H3K9ac, H3K9me1/me2/me3, H3K36me3, and H3K79me1/me2/me3. (D-F) Western blot data were quantified by the ImageJ software. Statistically significant differences at p ≤ 0.05 are shown by asterisk (*). Using immunofluorescence and confocal microscopy, the following histone markers were analyzed in NAT10 wt and NAT10 dn cells: (G) H3K9ac, (H) H3K9me3, (I) H3K36me3, (J) H3K79me3, (K) H3K79me2, and (L) H3K79me3. AlphaFold 3 predicted a low degree of interaction between the NAT10 protein and human core histone H3.3C (pTM = 0.57). Structures are colored with AlphaFold 3 pLDDT confidence score.

Article Snippet: Following this, the membranes were blocked with 5% non-fat dry milk for 2 hours and were incubated overnight at 4 °C with specific primary antibodies: anti-NAT10 (#sc-271770, Santa Cruz Biotechnology Inc., USA), anti-p53 (#sc-126), anti-XPC (#sc-30156); anti-XPA (#sc-28353), anti-DDB2 (#sc-25368), and anti-GAPDH (#60004-1, Proteintech, Germany).

Techniques: Irradiation, Western Blot, Software, Immunofluorescence, Confocal Microscopy

Journal: bioRxiv

Article Title: The NAT10 acetyltransferase modulates DNA damage-related factors and global 3D-genome architecture

doi: 10.1101/2025.03.05.641614

Figure Lengend Snippet: Results from AlphaFold 3 predictions of (A) NAT10 interaction with MDM2 (pTM = 0.49) and (B) MDM2 DNA repair-related protein interaction with p53 (pTM = 0.35). (C) Interaction properties of NAT10-MDM2-p53 trimer (pTM = 0.45) For source data, see https://www.ibp.cz/en/research/departments/cellular-biology-and-epigenetics/open-data .

Article Snippet: Following this, the membranes were blocked with 5% non-fat dry milk for 2 hours and were incubated overnight at 4 °C with specific primary antibodies: anti-NAT10 (#sc-271770, Santa Cruz Biotechnology Inc., USA), anti-p53 (#sc-126), anti-XPC (#sc-30156); anti-XPA (#sc-28353), anti-DDB2 (#sc-25368), and anti-GAPDH (#60004-1, Proteintech, Germany).

Techniques:

Journal: bioRxiv

Article Title: The NAT10 acetyltransferase modulates DNA damage-related factors and global 3D-genome architecture

doi: 10.1101/2025.03.05.641614

Figure Lengend Snippet: Changes in 3D-genome nuclear architecture of human acrocentric chromosomes 15 and 21 caused by NAT10 deficiency and UVC irradiation in HeLa cells. Genome-wide contact maps are shown for acrocentric chromosomes (A) HSA15 and (C) HSA21 in 1 Mb per bin. Panels (B) and (D) show regions of Hi-C matrices corresponding to HSA15 and HSA21 that were visualized using the HiGlass software at a resolution of 360 Kb per bin (left panels in B and D). The visualization shows difference maps between the control (NAT10 wt or NAT10 dn) and comparative datasets (NAT10 wt / UVC; NAT10 dn or NAT10 dn / UVC) (red-blue right panels in B and D). The missing data in panels B and D are shown in white color. The following panels show global changes in average genomic interactions over all genomic distances of HSA15 (E) and HSA21 (F) studied in non-irradiated and UVC-irradiated NAT10 dn and NAT10 wt cells.

Article Snippet: Following this, the membranes were blocked with 5% non-fat dry milk for 2 hours and were incubated overnight at 4 °C with specific primary antibodies: anti-NAT10 (#sc-271770, Santa Cruz Biotechnology Inc., USA), anti-p53 (#sc-126), anti-XPC (#sc-30156); anti-XPA (#sc-28353), anti-DDB2 (#sc-25368), and anti-GAPDH (#60004-1, Proteintech, Germany).

Techniques: Irradiation, Genome Wide, Hi-C, Software, Control