Journal: G3: Genes|Genomes|Genetics

Article Title: A long reads-based de-novo assembly of the genome of the Arlee homozygous line reveals chromosomal rearrangements in rainbow trout

doi: 10.1093/g3journal/jkab052

Figure Lengend Snippet: Statistical data summary of the USDA_OmykA_1.1 (Arlee) rainbow trout genome assembly

Article Snippet: The Hi-C proximity ligation sequence data were used to generate chromosome scale scaffolds from the Bionano scaffolds assembly.

Techniques:

Journal: eLife

Article Title: Association with TFIIIC limits MYCN localisation in hubs of active promoters and chromatin accumulation of non-phosphorylated RNA polymerase II

doi: 10.7554/eLife.94407

Figure Lengend Snippet: ( A ) Immunoblots showing levels of FLAG-tagged MYCN (amino acids [aa] 2–137) and the six subunits of the TFIIIC complex after a pull-down assay using anti-FLAG affinity columns. Multiple columns labelled ‘Wash’ represent the sequential washings (n=2). ( B ) Size exclusion chromatography graph of MYCN (aa 1–137)/TauA (τA) (black trace) or MYCN alone (red trace). The blue box marks the fractions used for panels C and D (n=2). ( C ) Coomassie staining of fractions of the MYCN (aa 1–137)/τA complex (fractions marked with blue box in panel B). ( D ) Immunoblot of fractions of the MYCN (aa 1–137)/τA complex (fractions marked with blue box in panel B). ( E ) Growth curve (measured as % confluence) of SH-EP-MYCN-ER cells expressing doxycycline (Dox)-inducible shRNA targeting TFIIIC2 , TFIIIC3, or TFIIIC5 under the indicated conditions. Data show mean ± standard deviation (SD) (n=3). Figure 1—source data 1. Raw unedited gels for . Figure 1—source data 2. Uncropped and labelled gels for . Figure 1—source data 3. Raw unedited Coomassie images for . Figure 1—source data 4. Uncropped and labelled Coomassie images for . Figure 1—source data 5. Raw data for graphs shown in .

Article Snippet: Antibody , TFIIIC5 (rabbit polyclonal) , Bethyl Laboratories , Cat# A301-242A RRID: AB_890669 , WB (1:1000) Seq (10–15 µg) PLA (1:1000).

Techniques: Western Blot, Pull Down Assay, Size-exclusion Chromatography, Staining, Expressing, shRNA, Standard Deviation

Journal: eLife

Article Title: Association with TFIIIC limits MYCN localisation in hubs of active promoters and chromatin accumulation of non-phosphorylated RNA polymerase II

doi: 10.7554/eLife.94407

Figure Lengend Snippet: ( A ) SDS-PAGE gel of recombinant purified MYCN (amino acids [aa] 1–137) expressed in Escherichia coli cells. ( B ) SDS-PAGE gel of recombinant purified TauA (τA) subcomplex which was expressed in Spodoptera frugiperda (Sf9) cells. ( C ) Deconvoluted spectra of native mass spectrometry for the τA/3x FLAG-MYCN complex. Masses are shown in red, identities of complexes are marked in blue. ( D ) Boxplot of the 30 possibleτA/3x FLAG-MYCN complex molecular weights based on the masses observed using intact mass spectrometry. The red line indicates the mass observed for the complex by native mass spectrometry (204,763 Da). ( E ) Immunoblot showing levels of TFIIIC2, TFIIIC3, or TFIIIC5 and MYC in SH-EP-MYCN-ER cells expressing doxycycline (Dox)-inducible shRNAs targeting the different TFIIIC subunits (n=3). Where indicated cells were treated with Dox (1 µg/ml, 48 hr) and/or 4-hydroxytamoxifen (4-OHT) (200 nM, 4 hr), respectively. EtOH was used as control. In the following panels addition of 4-OHT is indicated by ‘+MYCN’ and Dox treatment by ‘– TFIIIC’. ( F ) Immunoblot of MYC and MYCN in SH-EP-MYCN-ER cells after induction of MYCN with 4-OHT (200 nM, 4 hr). VCL was used as a loading control. Figure 1—figure supplement 1—source data 1. Raw unedited Coomassie images for . Figure 1—figure supplement 1—source data 2. Uncropped and labelled Coomassie images for . Figure 1—figure supplement 1—source data 3. Raw data for graphs shown in . Figure 1—figure supplement 1—source data 4. Raw unedited gels for . Figure 1—figure supplement 1—source data 5. Uncropped and labelled gels for .

Article Snippet: Antibody , TFIIIC5 (rabbit polyclonal) , Bethyl Laboratories , Cat# A301-242A RRID: AB_890669 , WB (1:1000) Seq (10–15 µg) PLA (1:1000).

Techniques: SDS Page, Recombinant, Purification, Mass Spectrometry, Western Blot, Expressing, Control

Journal: eLife

Article Title: Association with TFIIIC limits MYCN localisation in hubs of active promoters and chromatin accumulation of non-phosphorylated RNA polymerase II

doi: 10.7554/eLife.94407

Figure Lengend Snippet: ( A ) Browser tracks for non-phosphorylated RNAPII (top) and RNAPII pSer2 (bottom) ChIP-Rx at the indicated gene loci. SH-EP-MYCN-ER cells were treated with doxycycline (Dox) (1 µg/ml, 48 hr) and/or 4-hydroxytamoxifen (4-OHT), respectively. EtOH was used as control. ( B ) Average density plot of ChIP-Rx signal for non-phosphorylated RNAPII. Data show mean (line) ± standard error of the mean (SEM indicated by the shade) of different gene sets based on an RNA-sequencing (RNA-seq) of SH-EP-MYCN-ER cells ± 4-OHT. The y-axis shows the number of spike-in normalised reads and it is centred to the TSS ± 2 kb. N=number of genes in the gene set defined in the Methods (n=2). ( C ) Density plot of ChIP-Rx signal for RNAPII pSer2 as described for panel B. The signal is centred to the transcription end site (TES) ± 2 kb (n=2). ( D ) Average bin dot plot showing fold change for RNAPII pSer2 ChIP-Rx reads over TES ± 2 kb and RNA-seq of SH-EP-MYCN-ER for the same genes ± MYCN + TFIIIC5 (blue) or + MYCN ± TFIIIC5 (red). The plot shows 20 bins representing a total of 13,239 and 12,330 genes for ± MYCN + TFIIIC5 and + MYCN ± TFIIIC5 datasets, respectively (n=3 for RNA-seq, n=2 pSer2 RNAPII ChIP-Rx). ( E ) Average bin dot plot for RNA-seq of SH-EP-MYCN-ER showing log2 mRNA expression normalised by control per bin. Cells were treated with 1 µg/ml Dox (‘– TFIIIC5’, 48 hr) and/or 4-OHT (‘+MYCN’, 4 hr) or EtOH as control. Expression was normalised by its control. Each bin represents 150 genes of a total of 14,085 genes. Dotted line marks the relative expression at 0 (n=3). ( F ) Density plot of ChIP-Rx signal for TFIIIC5. Data show mean (line) ± SEM (shade) for 14,722 genes. The signal is centred to the TSS ± 2 kb (n=2).

Article Snippet: Antibody , TFIIIC5 (rabbit polyclonal) , Bethyl Laboratories , Cat# A301-242A RRID: AB_890669 , WB (1:1000) Seq (10–15 µg) PLA (1:1000).

Techniques: Control, RNA Sequencing Assay, Expressing

Journal: eLife

Article Title: Association with TFIIIC limits MYCN localisation in hubs of active promoters and chromatin accumulation of non-phosphorylated RNA polymerase II

doi: 10.7554/eLife.94407

Figure Lengend Snippet: ( A ) Metagene plot of ChIP-Rx signal for non-phosphorylated RNAPII. Data show mean (line) ± standard error of the mean (SEM indicated by the shade) of different gene sets based on an RNA-sequencing (RNA-seq) of SH-EP-MYCN-ER cells ± 4-hydroxytamoxifen (4-OHT). ( B ) Metagene plot of ChIP-Rx signal for RNAPII pSer2. Data are presented as described in (A). ( C ) Average density plot of ChIP-Rx signal for non-phosphorylated RNAPII in SH-EP cells treated with 4-OHT. Data show mean (line) ± SEM (indicated by the shade) of different gene sets based on an RNA-seq of SH-EP-MYCN-ER cells ±4 OHT. The signal is centred to the TSS ± 2 kb (n=2). ( D ) Average bin dot plot for RNA-seq of SH-EP-MYCN-ER showing mRNA expression normalised by control per bin. Cells were co-treated with 1 µg/ml doxycycline (Dox) (‘– TFIIIC3’, 48 hr) and/or 4-OHT (‘+ MYCN’, 4 hr) as EtOH as control. Expression was normalised by its control. Each bin represents 100 genes of a total of 12,091 genes. Dotted line marks the relative expression at 1 (n = 3). ( E ) Browser tracks for TFIIIC5 ChIP-Rx at the indicated gene loci. SH-EP-MYCN-ER cells were treated with 5,6-dichlorobenzimidazole-1-β-D-ribofuranoside (DRB) and/or 4-OHT, respectively. EtOH was used as control.

Article Snippet: Antibody , TFIIIC5 (rabbit polyclonal) , Bethyl Laboratories , Cat# A301-242A RRID: AB_890669 , WB (1:1000) Seq (10–15 µg) PLA (1:1000).

Techniques: RNA Sequencing Assay, Expressing, Control

Journal: eLife

Article Title: Association with TFIIIC limits MYCN localisation in hubs of active promoters and chromatin accumulation of non-phosphorylated RNA polymerase II

doi: 10.7554/eLife.94407

Figure Lengend Snippet: ( A ) Diagram showing the workflow of p hosphorylated l inker HiChIP (pLHiChIP). The workflow corresponds to the original HiChIP protocol. For a detailed description, see Methods. ( B ) MYCN and MYC ChIP in SH-EP-MYCN-ER cells before and after activation of MYCN with 4-hydroxytamoxifen (4-OHT) (200 nM, 24 hr). Shown is the mean and points of technical triplicates normalised to an intergenic region (n=2). ( C ) (Top) Diagram illustrating the difference between valid and invalid pairs after mapping of s pike-in p hosphorylated l inker HiChIP (spLHiChIP) data. Valid pairs harbour non-contiguous elements of chromosomal DNA, whereas invalid pairs harbour contiguous stretches of chromosomal DNA. (Bottom) Bar graph showing percentage of valid and invalid reads in our dataset (right) compared to a published HiC protocol (left). ( D ) Table showing quality controls for MYCN and TFIIIC5 spLHiChIP compared to the original Oct4 HiChIP. ( E ) Representative example of pLHiChIP track for MYCN (red) and a phosphorylated linker Hi-C (pLHi-C) track (blue) showing the strongly increased PETs number of the MYCN interactions after MYCN immunoprecipitation relative to input (pLHi-C). Data are superimposed with a browser view of an MYCN ChIP-sequencing (ChIP-seq). ( F ) Table showing interactions involving tRNA genes in MYCN pLHiChIP. ( G ) Table showing the top three terms, their p-values, and the false discovery rate (FDR) from enrichment analysis for MSigDB C5 collection of all MYCN network. Figure 3—figure supplement 1—source data 1. Raw data for data shown in .

Article Snippet: Antibody , TFIIIC5 (rabbit polyclonal) , Bethyl Laboratories , Cat# A301-242A RRID: AB_890669 , WB (1:1000) Seq (10–15 µg) PLA (1:1000).

Techniques: HiChIP, Activation Assay, Hi-C, Immunoprecipitation, ChIP-sequencing

Journal: eLife

Article Title: Association with TFIIIC limits MYCN localisation in hubs of active promoters and chromatin accumulation of non-phosphorylated RNA polymerase II

doi: 10.7554/eLife.94407

Figure Lengend Snippet: ( A ) Top: Representative browser track of MYCN three-dimensional chromatin interactions. Height shows the number of p aired e nd t ags (PETs) indicating the interaction intensity and the width of the line shows the start and end positions of each anchor. Middle and bottom: Browser tracks showing the number of reads of MYCN and total RNAPII ChIP-Rx, respectively. Unless stated, all experiments were performed in SH-EP-MYCN-ER cells treated with 4-hydroxytamoxifen (4-OHT) (200 nM, 4 hr). The ruler at the bottom shows the genomic coordinates (n=3 independent biological replicates for MYCN p hosphorylated l inker HiChIP [pLHiChIP]; n=2 for RNAPII ChIP-Rx). ( B ) Bar chart listing functional annotations of all binary MYCN interactions (N=4591; N indicates total number). ( C ) Boxplots showing relative binding of the indicated proteins (RNAPII, MYCN, TFIIIC5) to promoter regions or expression levels of the corresponding genes (mRNA by RNA-sequencing [RNA-seq]; 4sU by 4sU-seq). Red boxes: Genes bound by MYCN and part of MYCN-hubs. Blue boxes: Genes bound by MYCN that are not part of MYCN-hubs. Each pair was normalised to the median of the corresponding ‘blue’ gene set. p-Values were obtained by pairwise comparisons using Student’s t - test (n=2 for TFIIIC5 and RNAPII ChIP-Rx). ( D ) Boxplot showing the number of promoters in each cluster, with each red dot representing one cluster. ( E ) Network reconstruction of the three biggest clusters based on MYCN pLHiChIP interactions. Each anchor is represented by a node (‘triangle’) and the lines show interactions between the anchors. The colours are indicating the different functional annotation.

Article Snippet: Antibody , TFIIIC5 (rabbit polyclonal) , Bethyl Laboratories , Cat# A301-242A RRID: AB_890669 , WB (1:1000) Seq (10–15 µg) PLA (1:1000).

Techniques: HiChIP, Functional Assay, Binding Assay, Expressing, RNA Sequencing Assay

Journal: eLife

Article Title: Association with TFIIIC limits MYCN localisation in hubs of active promoters and chromatin accumulation of non-phosphorylated RNA polymerase II

doi: 10.7554/eLife.94407

Figure Lengend Snippet: ( A ) Representative example of p hosphorylated l inker HiChIP (pLHiChIP) track for MYCN (red) and TFIIIC5 (blue) interactions (conventions as in ) (n=2). ( B ) Bar chart listing the total number of functional annotations for all TFIIIC5 binary interactions (N=3499). ( C ) Venn diagram showing the number of interactions shared between MYCN and TFIIIC5. The diagram at the left shows the types of overlaps between connections. ( D ) Bar chart listing the interaction functional annotations for MYCN anchors not overlapping with TFIIIC5 anchors (‘MYCN only’) as well as TFIIIC5 anchors without overlapping MYCN anchors (‘TFIIIC5 only’) and their joint anchors. ( E ) Representative example of MYCN s pike-in p hosphorylated l inker HiChIP (spLHiChIP) track for MYCN interactions in the presence (blue) or absence (red) of TFIIIC5 (n=2). ( F ) Bar graph showing the fold change of all MYCN spLHiChIP interactions comparing ‘+TFIIIC5’ and ‘– TFIIIC5’ in SH-EP-MYCN-ER cells expressing a doxycycline (Dox)-inducible shRNA targeting TFIIIC5 . n1,2 indicates two independent biological replicates. ( G ) Representative example of TFIIIC5 spLHiChIP track without (blue) or with (red) induction of MYCN for SH-EP-MYCN-ER cells (conventions as in ). ( H ) Bar graph showing the number of TFIIIC5 interactions normalised by the relative binding of TFIIIC5 ChIP-Rx signals for the same coordinates. Coordinates defined as TSS ± 2 kb of 14,722 genes.

Article Snippet: Antibody , TFIIIC5 (rabbit polyclonal) , Bethyl Laboratories , Cat# A301-242A RRID: AB_890669 , WB (1:1000) Seq (10–15 µg) PLA (1:1000).

Techniques: HiChIP, Functional Assay, Expressing, shRNA, Binding Assay

Journal: eLife

Article Title: Association with TFIIIC limits MYCN localisation in hubs of active promoters and chromatin accumulation of non-phosphorylated RNA polymerase II

doi: 10.7554/eLife.94407

Figure Lengend Snippet: ( A ) Heatmap showing analysis of sequence motifs characteristic for interacting boxes on both anchors of the interactions. Colour reflects the number of interactions. ( B ) Table summarising numbers of MYCN interactions in the presence and absence of TFIIIC5 in SH-EP-MYCN-ER cells and the TFIIIC5-dependent change. Data merged of two independent biological replicates. ( C ) Table displaying total numbers of TFIIIC5 interactions in SH-EP-MYCN-ER±MYCN + 5,6-dichlorobenzimidazole-1-β-D-ribofuranoside (DRB).

Article Snippet: Antibody , TFIIIC5 (rabbit polyclonal) , Bethyl Laboratories , Cat# A301-242A RRID: AB_890669 , WB (1:1000) Seq (10–15 µg) PLA (1:1000).

Techniques: Sequencing

Journal: eLife

Article Title: Association with TFIIIC limits MYCN localisation in hubs of active promoters and chromatin accumulation of non-phosphorylated RNA polymerase II

doi: 10.7554/eLife.94407

Figure Lengend Snippet: ( A ) Volcano plot depicting changes in intron retention upon TFIIIC3 knock-down in the absence (left) and presence (right) of MYCN. Triangle reflects the effect of the splicing error after TFIIIC knock-down. ( B ) Volcano plot depicting changes in exon skipping upon TFIIIC3 knock-down in the absence (left) and presence (right) of MYCN. Triangle reflects the effect of the splicing error after TFIIIC knock-down. ( C ) Controls for proximity ligation assays (PLAs) shown in . Immunofluorescence show specificity of the antibody. Single antibodies PLAs were performed in parallel to each PLA. Mean of signal in the nucleus and cytoplasm was calculated and compared to mean of the signal per nucleus of the PLA. ( D ) Boxplots showing the number of PLA signals between RNAPII and TFIIIC5. SH-EP-MYCN-ER cells were treated with 1 µg/ml doxycycline (Dox) (‘– TFIIIC5’, 48 hr) and/or 4-hydroxytamoxifen (4-OHT) (‘+MYCN’). EtOH was used as control. For clarity purposes, 500 cells pooled from different replicates were plotted. p-Values were calculated comparing the PLA signal of all cells using unpaired Wilcoxon rank sum test. The grey dotted line indicates the median in the control condition (n=3). ( E ) BRCA1 ChIP in SH-EP-MYCN-ER cells expressing a Dox-inducible shRNA targeting TFIIIC5 treated with 4-OHT. Data show fold change of BRCA1 binding after induction of MYCN in the presence (blue) or absence (red) of TFIIIC5 (n=2). Figure 5—figure supplement 1—source data 1. Raw data for plots shown in .

Article Snippet: Antibody , TFIIIC5 (rabbit polyclonal) , Bethyl Laboratories , Cat# A301-242A RRID: AB_890669 , WB (1:1000) Seq (10–15 µg) PLA (1:1000).

Techniques: Knockdown, Ligation, Immunofluorescence, Control, Expressing, shRNA, Binding Assay

Journal: eLife

Article Title: Association with TFIIIC limits MYCN localisation in hubs of active promoters and chromatin accumulation of non-phosphorylated RNA polymerase II

doi: 10.7554/eLife.94407

Figure Lengend Snippet: ( A ) Boxplots showing the number of proximity ligation assay (PLA) signals between RNA polymerase II (RNAPII) and NELFE, PP2A, PNUTS, or XRN2. SH-EP-MYCN-ER cells were treated with 1 µg/ml doxycycline (Dox) (‘– TFIIIC5’, 48 hr) and/or 4-hydroxytamoxifen (4-OHT) (‘+MYCN’). EtOH was used as control. For clarity purposes, 500 cells pooled from different replicates were plotted. p-Values were calculated comparing the PLA signal of all cells using unpaired Wilcoxon rank sum test. The grey dotted line indicates the median in the control condition (n=3). ( B ) Density plot of CUT&RUN for EXOSC5 binding (N=14,704 genes) in SH-EP-MYCN-ER cells expressing a Dox-inducible shRNA targeting TFIIIC5 treated with 4-OHT. Data show mean ± SEM (shade). ( C ) BRCA1 ChIP in SH-EP-MYCN-ER cells expressing a Dox-inducible shRNA targeting TFIIIC5 treated with 4-OHT (4 hr). Shown is the mean of technical triplicates of one representative experiment with identical results (n=2). Figure 5—source data 1. Raw data for plots shown in .

Article Snippet: Antibody , TFIIIC5 (rabbit polyclonal) , Bethyl Laboratories , Cat# A301-242A RRID: AB_890669 , WB (1:1000) Seq (10–15 µg) PLA (1:1000).

Techniques: Proximity Ligation Assay, Control, Binding Assay, Expressing, shRNA

Journal: eLife

Article Title: Association with TFIIIC limits MYCN localisation in hubs of active promoters and chromatin accumulation of non-phosphorylated RNA polymerase II

doi: 10.7554/eLife.94407

Figure Lengend Snippet:

Article Snippet: Antibody , TFIIIC5 (rabbit polyclonal) , Bethyl Laboratories , Cat# A301-242A RRID: AB_890669 , WB (1:1000) Seq (10–15 µg) PLA (1:1000).

Techniques: Recombinant, Expressing, Plasmid Preparation, Variant Assay, Sequencing, shRNA, In Situ, Affinity Purification, Fluorescence, Isolation, Multiplex Assay, Transfection, Magnetic Beads, Software, Imaging

Journal: bioRxiv

Article Title: HIC1 interacts with FOXP3 multi protein complex: a novel mechanism to regulate human regulatory T cell differentiation and function

doi: 10.1101/2023.05.15.540505

Figure Lengend Snippet: Naïve CD4 + CD25 - T cells were isolated from human umbilical cord blood and polarized to Treg lineage for 72 h. The cultured, Treg cells were lysed and HIC1 protein was immunoprecipitated (IP) using HIC1 specific antibody. Western blot (WB) analysis of HIC-1 immunoprecipitation (IP) with antibody specific to C-terminus region of HIC1, in 72 h polarized Treg cells. For HIC1 targeting antibody, an IgG control antibody was used. The figure is a representative WB of three experiments. Input, IgG control-IP and HIC1-IP reactions are shown.

Article Snippet: HIC1 immunoprecipitation (IP) was performed using anti-HIC1 antibody (Santa Cruz Biotechnology, Cat# sc-271499) that recognizes 611-733 C-terminus region of human HIC1, together with the respective control IgG antibody, in three biological replicates.

Techniques: Isolation, Cell Culture, Immunoprecipitation, Western Blot, Control

Journal: bioRxiv

Article Title: HIC1 interacts with FOXP3 multi protein complex: a novel mechanism to regulate human regulatory T cell differentiation and function

doi: 10.1101/2023.05.15.540505

Figure Lengend Snippet: The HIC1 Interactome. Following affinity purification and mass spectrometry (AP-MS), statistical analysis of interactions using the MIST algorithm and filtering for common contaminants, a panel of interacting proteins was identified. The proteins are shown as a heat-map ( A) , clustered on the basis of their Z-score normalized LFQ intensities from the bait and control measurements. Representations of their cellular localization ( B ) and functional classes ( C ) are shown as pie charts. The interactors are further represented as an interaction network ( D ), with the enriched biological processes indicated by the use of colored circles and strength of the enrichment indicated by the size of the peripheral circles. Further representation of this data is provided in Table S4.

Article Snippet: HIC1 immunoprecipitation (IP) was performed using anti-HIC1 antibody (Santa Cruz Biotechnology, Cat# sc-271499) that recognizes 611-733 C-terminus region of human HIC1, together with the respective control IgG antibody, in three biological replicates.

Techniques: Affinity Purification, Mass Spectrometry, Protein-Protein interactions, Control, Functional Assay

Journal: bioRxiv

Article Title: HIC1 interacts with FOXP3 multi protein complex: a novel mechanism to regulate human regulatory T cell differentiation and function

doi: 10.1101/2023.05.15.540505

Figure Lengend Snippet: HIC1 interactome validation by SRM-MS, IP-WB, reverse IP-WB and PLA assay. ( A ) SRM-MS validation of the candidate HIC1 interacting partners. HIC1 IP was performed using HIC1 antibody in 72 h polarized iTreg cells and six proteins were validated by targeted proteomics. Averaged results from three replicates are presented in the form of a box plot. Statistical analysis was made by a two-tailed paired student’s T-test, **p-value represents < 0.005; and ***p-value represents < 0.0005. ( B ) Validation of HIC1 protein interaction by IP-western blot. HIC1 IP followed by WB detection with RUNX1, IKZF3, CBFB and FOXP3 antibodies was performed to validate the HIC1 interaction with RUNX1, IKZF3, CBFB and FOXP3. Total cell lysate (Input), control IP (IgG) and HIC1 IP are shown in the blots. A conformational specific secondary antibody was used to probe proteins without interference from the denatured IgG heavy (50 kDa) and light chains (25 kDa). A representative WB of three biological replicates is shown. ( C ) RUNX1-IP, IKZF3-IP, CBFB-IP and FOXP3-IP were performed using iTreg cell lysate followed by WB detection with HIC1 antibody to validate the HIC1 interaction with RUNX1, IKZF3, CBFB and FOXP3. A representative western blot of three biological replicates is shown. ( D ) Proximity ligation assay (PLA) with indicated antibody pairs in 72 h polarized iTreg cells. The GFP antibody was used as a negative control. DAPI was used to stain the nuclei. The scale bar is 7 μm. The graph shows the average number of PLA signals (spots) in each experiment (n=10 images from a representative experiment of n=3 experiments). Statistics by paired two-tailed T-test, where * ** represents p<0.01; and **** represents p<0.001.

Article Snippet: HIC1 immunoprecipitation (IP) was performed using anti-HIC1 antibody (Santa Cruz Biotechnology, Cat# sc-271499) that recognizes 611-733 C-terminus region of human HIC1, together with the respective control IgG antibody, in three biological replicates.

Techniques: Biomarker Discovery, Targeted Proteomics, Two Tailed Test, Western Blot, Control, Proximity Ligation Assay, Negative Control, Staining

Journal: bioRxiv

Article Title: HIC1 interacts with FOXP3 multi protein complex: a novel mechanism to regulate human regulatory T cell differentiation and function

doi: 10.1101/2023.05.15.540505

Figure Lengend Snippet: ChIPseq, Luciferase and ChIP-qPCR assays demonstrate the recruitment of HIC1 to the RUNX1 promoter in iTreg cells. ( A ) ChIP-Seq analysis identifies binding sites of HIC1 at RUNX1 promoter region in iTreg cells. UCSC genome browser snap shots for the RUNX1 gene that demonstrates the HIC1 binding peaks as indicated in enclosed box. ( B ) ChIP-qPCR assay was performed in iTreg cells using anti-HIC1 IgG (HIC1) and mouse IgG (IgG) as control. Input of sheared chromatin was prepared prior to immunoprecipitation. ( C ) Relative luciferase activities were measured after co-transfection of RUNX1 promoter (Runx1) or mutated Runx1 promoter (ΔRunx1) constructs along with the pRL-TK expression plasmid into iTreg cells. Statistical significance calculated using Student’s t-test (two-tailed paired) where ***denotes p value <0.0005. *denotes p value <0.05.

Article Snippet: HIC1 immunoprecipitation (IP) was performed using anti-HIC1 antibody (Santa Cruz Biotechnology, Cat# sc-271499) that recognizes 611-733 C-terminus region of human HIC1, together with the respective control IgG antibody, in three biological replicates.

Techniques: Luciferase, ChIP-qPCR, ChIP-sequencing, Binding Assay, Control, Immunoprecipitation, Cotransfection, Construct, Expressing, Plasmid Preparation, Two Tailed Test

Journal: bioRxiv

Article Title: HIC1 interacts with FOXP3 multi protein complex: a novel mechanism to regulate human regulatory T cell differentiation and function

doi: 10.1101/2023.05.15.540505

Figure Lengend Snippet: HIC1-knockout (KO) leads to reduced RUNX1 expression. ( A ) HIC1 was targeted with CRISPR-Cas9 RNPs in three individual donor cell pools, and HIC1, RUNX1 and Actin expression levels were measured by WB. ( B ) The dot plot shows the quantification of the HIC1 levels in HIC1 ablated and Control (NT) iTreg cells. ( C ) The dot plot shows the quantification of the RUNX1 levels in HIC1 silenced and Control (NT) iTreg cells. Quantification was performed using Image Studio Lite software Ver 5.2. HIC1 and RUNX1 levels were normalised to Actin levels. Statistical significance calculated using Student’s t-test (two-tailed paired) where ***denotes p value <0.0005. **denotes p value <0.005.

Article Snippet: HIC1 immunoprecipitation (IP) was performed using anti-HIC1 antibody (Santa Cruz Biotechnology, Cat# sc-271499) that recognizes 611-733 C-terminus region of human HIC1, together with the respective control IgG antibody, in three biological replicates.

Techniques: Knock-Out, Expressing, CRISPR, Control, Software, Two Tailed Test

Journal: bioRxiv

Article Title: HIC1 interacts with FOXP3 multi protein complex: a novel mechanism to regulate human regulatory T cell differentiation and function

doi: 10.1101/2023.05.15.540505

Figure Lengend Snippet: HIC1 interacts with FOXP3-RUNX1-CBFB protein complex. ( A ) FOXP3-RUNX1-CBFB-HIC1 protein complex regulates expression of TBET, IFNG and GATA3 and control Treg suppression. ( B ) HIC1 deficiency causes down-regulation of RUNX1, destabilizes the FOXP3 protein complex, increases the expression of effector T cell associated genes including TBET, GATA3 and IFNG which leads to loss of Treg suppression.

Article Snippet: HIC1 immunoprecipitation (IP) was performed using anti-HIC1 antibody (Santa Cruz Biotechnology, Cat# sc-271499) that recognizes 611-733 C-terminus region of human HIC1, together with the respective control IgG antibody, in three biological replicates.

Techniques: Expressing, Control

Journal: eLife

Article Title: Maf-family bZIP transcription factor NRL interacts with RNA-binding proteins and R-loops in retinal photoreceptors

doi: 10.7554/eLife.103259

Figure Lengend Snippet: ( A ) DHX9 and DDX5 localization to the euchromatin region (nuclear periphery in murine rods) is shown in red. Zoom-in insets show proteins distributed in puncta (arrows). Nuclei are stained with DAPI. Scale bar is 10 μM. ( B ) Proximity ligation assay (PLA) signal (magenta) using anti-DHX9 and goat IgG antibodies in the adult human retina. Scale bar = 20 μM. ONL = outer nuclear layer; INL = inner nuclear layer.

Article Snippet: The following antibodies were used: DDX5 Monoclonal antibody (Cat. No. 67025-1-Ig), DHX9 Polyclonal antibody (Cat. No. 17721-1-AP), and HNRNPA2B1 (Cat. No. 14813-1-AP), all obtained from Proteintech (Rosemont, IL, USA); HNRNPA1 (Cat.No.

Techniques: Staining, Proximity Ligation Assay

Journal: eLife

Article Title: Maf-family bZIP transcription factor NRL interacts with RNA-binding proteins and R-loops in retinal photoreceptors

doi: 10.7554/eLife.103259

Figure Lengend Snippet: ( A ) DHX9 (blue) and NRL (red) expression in adult mouse wild-type (WT) and Nrl Knockout (KO) retina. ( B ) Proximity ligation assay (PLA) signal (magenta) using anti-DHX9 and NRL antibodies in adult mouse WT and Nrl KO retina. ( C ) DDX5 (blue) and NRL (red) expression in adult mouse WT and Nrl KO retina. ( D ) PLA signal (magenta) using anti-DDX5 and NRL antibodies in adult mouse WT and Nrl KO retina. ( E ) DHX9 (blue) and NRL (red) expression in adult human retina. ( F ) PLA signal (magenta) using anti-DHX9 and NRL antibodies in the adult human retina. ( G ) DDX5 (blue) and NRL (red) expression in adult human retina. ( H ) PLA signal (magenta) using anti-DDX5 and NRL antibodies in the adult human retina. ( I ) PLA signal (magenta) using no primary antibody in the adult human retina. ( J ) PLA signal in HEK293 cells overexpressing human Xpress–NRL. DHX9 interaction with its known protein partner HNRNPU is shown in the nuclear periphery (arrow). Xpress–NRL interaction with DDX5 and DHX9 in euchromatin is shown in red (arrows). Nuclei were counterstained with DAPI (gray in human and mouse retina; blue in HEK293 cells). Scale bar = 20 μM. ONL = outer nuclear layer; INL = inner nuclear layer.

Article Snippet: The following antibodies were used: DDX5 Monoclonal antibody (Cat. No. 67025-1-Ig), DHX9 Polyclonal antibody (Cat. No. 17721-1-AP), and HNRNPA2B1 (Cat. No. 14813-1-AP), all obtained from Proteintech (Rosemont, IL, USA); HNRNPA1 (Cat.No.

Techniques: Expressing, Knock-Out, Proximity Ligation Assay

Journal: eLife

Article Title: Maf-family bZIP transcription factor NRL interacts with RNA-binding proteins and R-loops in retinal photoreceptors

doi: 10.7554/eLife.103259

Figure Lengend Snippet: ( A ) Genomic view of the human DDX5, HNRNPU , DDX3X , and THRAP loci showing Hi-C loops, RNA-Seq, ATAC-Seq, H3K27ac ChIP-Seq, CRX-ChIP-Seq, NRL-ChIP-Seq, and super-enhancer tracks (obtained from ). ( B ) Genome browser view showing Cut&Run peaks for NRL at mouse Dhx9 promoter.

Article Snippet: The following antibodies were used: DDX5 Monoclonal antibody (Cat. No. 67025-1-Ig), DHX9 Polyclonal antibody (Cat. No. 17721-1-AP), and HNRNPA2B1 (Cat. No. 14813-1-AP), all obtained from Proteintech (Rosemont, IL, USA); HNRNPA1 (Cat.No.

Techniques: Hi-C, RNA Sequencing, ChIP-sequencing

Journal: eLife

Article Title: Maf-family bZIP transcription factor NRL interacts with RNA-binding proteins and R-loops in retinal photoreceptors

doi: 10.7554/eLife.103259

Figure Lengend Snippet: ( A, B ) Co-immunoprecipitation (co-IP) of DDX5 and DHX9 from HEK293 cells overexpressing NRL. Lysates were treated for 30 min with different nucleases (as shown) before incubations with respective antibodies. Immunoprecipitation (IP) of NRL ( A ) or DHX9 ( B ) and immunoblot (IB) staining for NRL, DHX9, and DDX5 is shown. ( C, D ) Quantification of signal intensities normalized to affinity-purified NRL and DHX9 (shown in A and B, respectively) ( n = 3). Data are presented as the mean ± SEM. An unpaired two-tailed t -test was performed to compare the means of samples against controls. ( E, F ) Co-IP of DDX5 and DHX9 from nuclear fractions of bovine retinas. Lysates were treated for 30 min with different nucleases (as shown) before incubations with respective antibodies. IP of NRL ( E ) or DHX9 ( F ) and immunoblot (IB) staining for NRL, DHX9, and DDX5 is shown. ( G, H ) Quantification of signal intensities normalized to affinity-purified NRL and DHX9 (shown in E and F, respectively) ( n = 4). Data are presented as the mean ± SEM. Unpaired two-tailed t -test was performed to compare means of samples against controls. Figure 4—source data 1. PDF file containing original western blots for , indicating the relevant bands and treatments. Figure 4—source data 2. Original files for western blots displayed in .

Article Snippet: The following antibodies were used: DDX5 Monoclonal antibody (Cat. No. 67025-1-Ig), DHX9 Polyclonal antibody (Cat. No. 17721-1-AP), and HNRNPA2B1 (Cat. No. 14813-1-AP), all obtained from Proteintech (Rosemont, IL, USA); HNRNPA1 (Cat.No.

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Western Blot, Staining, Affinity Purification, Two Tailed Test

Journal: eLife

Article Title: Maf-family bZIP transcription factor NRL interacts with RNA-binding proteins and R-loops in retinal photoreceptors

doi: 10.7554/eLife.103259

Figure Lengend Snippet: Co-immunoprecipitation (co-IP) of DDX5 with DHX9 antibody in HEK293 cells overexpressing neural retina leucine (NRL) ( A ) and in bovine retina ( B ). Lysates were treated for 30 min with different nucleases (as shown) before incubations with DHX9 antibody ( n = 3 for HEK cells; n = 4 for bovine retina). Quantification of signal intensities were normalized to precipitated DHX9. Data are presented as the mean ± SEM. Unpaired two-tailed t -test was performed to compare means of samples against controls.

Article Snippet: The following antibodies were used: DDX5 Monoclonal antibody (Cat. No. 67025-1-Ig), DHX9 Polyclonal antibody (Cat. No. 17721-1-AP), and HNRNPA2B1 (Cat. No. 14813-1-AP), all obtained from Proteintech (Rosemont, IL, USA); HNRNPA1 (Cat.No.

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Two Tailed Test

Journal: eLife

Article Title: Maf-family bZIP transcription factor NRL interacts with RNA-binding proteins and R-loops in retinal photoreceptors

doi: 10.7554/eLife.103259

Figure Lengend Snippet: ( A ) Western blot of DNA:RNA hybrid immunoprecipitation (DRIP) from adult mouse retina showing immunoblot (IB) staining for DHX9, NRL, and DDX5. Retinal genomic DNA (gDNA) was digested with MseI, DdeI, Alul, MboI, incubated with RNase III with/without RNase H and immunoprecipitated with S9.6 antibody/protein G beads. Retinal nuclear lysates were incubated with antibody/bead complexes. Quantification of signal intensities of immunoprecipitated DHX9 and NRL compared to input ( n = 3). Data are presented as the mean ± SEM. Unpaired one-tailed t -test was performed to compare means of samples against controls. ( B ) Confocal image of HEK293 cells transfected with NRL and wild-type (WT) human RNase H1 or D201N catalytic dead mutant EGFP fusions (GFP-HR and GFP-dHR, respectively). Proximity ligation assay (PLA) signals using antibodies for NRL and DHX9 are shown in red. Some cells displayed nucleolar-like accumulation of PLA signal (arrows). ( C ) Confocal image of HEK293 cells transfected with NRL and GFP-dRH or GFP-RH and stained with antibodies against NRL (red). Nuclei are stained with DAPI (gray). Scale bar is 20 μM. ( D ) Quantification of cells with positive PLA signals from B. Each dot represents a ratio of number of GFP+ cells with nuclear PLA signals per image. Data are presented as the mean ± SEM. Unpaired two-tailed t -test was performed to compare means of samples against controls. ( E ). Bar graph showing percentage of EGFP+ cells harboring NRL–DHX9 PLA signals in subnuclear compartments from B. Cells were counted in four independent assays ( n = 83 and 85 cells for GFP-dHR and GFP-RH, respectively). Figure 5—source data 1. PDF file containing original western blots for , indicating the relevant bands. Figure 5—source data 2. Original files for western blots displayed in .

Article Snippet: The following antibodies were used: DDX5 Monoclonal antibody (Cat. No. 67025-1-Ig), DHX9 Polyclonal antibody (Cat. No. 17721-1-AP), and HNRNPA2B1 (Cat. No. 14813-1-AP), all obtained from Proteintech (Rosemont, IL, USA); HNRNPA1 (Cat.No.

Techniques: Western Blot, Immunoprecipitation, Staining, Incubation, One-tailed Test, Transfection, Mutagenesis, Proximity Ligation Assay, Two Tailed Test

Journal: iScience

Article Title: JAK inhibitors dampen activation of interferon-activated transcriptomes and the SARS-CoV-2 receptor ACE2 in human renal proximal tubules

doi: 10.1016/j.isci.2021.102928

Figure Lengend Snippet:

Article Snippet: Human Primary Proximal Tubule (HPPT) Cells , ATCC , Cat# PCS-400-010.

Techniques: Recombinant, RNA Sequencing, ChIP-sequencing, Hi-C, Software