rabbit anti pnuts  (Novus Biologicals)

Journal: The Journal of Biological Chemistry

Article Title: TOX High-Mobility Group Box Family Member 4 promotes DNA double-strand break repair via nonhomologous end joining

doi: 10.1016/j.jbc.2025.110174

Figure Lengend Snippet: TOX4 and PNUTS coordinate in NHEJ. A , HeLa cells were treated with siRNA targeting TOX4 or PNUTS, as indicated, for 24 h. The cell lysates were analyzed by immunoblotting for γ-H2AX, TOX4, PNUTS, and tubulin. B , NHEJ repair efficiency was measured in HeLa cells using the linearized GFP vector, as in N . Cells were treated with or without siRNA targeting TOX4, PNUTS, or KU80, as indicated. Immunoblots of GFP, TOX4, PNUTS, and β-actin are shown. C , Ku80 IP was performed in HeLa cells treated with control or TOX4 siRNA. The lysate input at 20%, control (ctr) IP with blank beads, and KU80 IP products were analyzed by immunoblotting for PNUTS, TOX4, KU80, and β-actin. NHEJ, nonhomologous end joining; PNUTS, phosphatase 1 nuclear targeting subunit; TOX4, Thymocyte Selection–Associated High-Mobility Group Box Family Member 4.

Article Snippet: Immunoprecipitation was performed using 300 μg of protein lysate along with 4 μg of TOX4 antibody, KU70 antibody (ab3108; Abcam), PNUTS antibody, or control rabbit IgG (Santa Cruz Biotechnology).

Techniques: Western Blot, Plasmid Preparation, Control, Selection

Journal: The Journal of Biological Chemistry

Article Title: TOX High-Mobility Group Box Family Member 4 promotes DNA double-strand break repair via nonhomologous end joining

doi: 10.1016/j.jbc.2025.110174

Figure Lengend Snippet: TOX4 promotes treatment resistance in cancer. A , the Cancer Genome Atlas database analysis of TOX4 gene amplification in various types of cancer and representative of amplification ( red ) and deep deletion ( blue ) are shown. B , Kaplan–Meier survival analysis of head and neck cancer was performed, in groups with high or low expression of TOX4. C , HeLa cells were treated with control or TOX4 siRNA at day 0, incubated with doxorubicin (DOX) at day 1 and maintained in culture for 3 days. Cell viability was determined in each day and normalized to that of day 1. The mean value and SD were calculated from three independent experiments. D , the clonogenic assay was performed as described in the section. The numbers of colonies were normalized to untreated control. The mean value and SD were calculated from three independent experiments. Statistical significance was analyzed using an unpaired two-tailed Student t test. E , UM-SCC-38 cells with or without TOX4 siRNA and DOX were cultured in nonadhesive dishes for anchorage-independent growth. Spheroid growth was imaged and shown. F , spheroid size, as in E , was measured, and shown. Statistical significance was determined using an unpaired two-tailed Student t test (∗ p < 0.05, ∗∗ p < 0.01). G , schematic diagram summarizing the role of TOX4 in promoting DNA-PKcs activation and NHEJ. The subsequent accumulation of TOX4, at upregulated levels several hours after DNA damage. TOX4 bound both PNUTS and DNA-PK, and was required for the association of PNUTS with DNA-PK, which modulates DNA-PKcs activation after DNA damage. DNA-PKcs, DNA-dependent protein kinase catalytic subunit; NHEJ, nonhomologous end joining; PNUTS, phosphatase 1 nuclear targeting subunit; TOX4, Thymocyte Selection–Associated High-Mobility Group Box Family Member 4.

Article Snippet: Immunoprecipitation was performed using 300 μg of protein lysate along with 4 μg of TOX4 antibody, KU70 antibody (ab3108; Abcam), PNUTS antibody, or control rabbit IgG (Santa Cruz Biotechnology).

Techniques: Amplification, Expressing, Control, Incubation, Clonogenic Assay, Two Tailed Test, Cell Culture, Activation Assay, Selection

Journal: The Journal of Biological Chemistry

Article Title: TOX High-Mobility Group Box Family Member 4 promotes DNA double-strand break repair via nonhomologous end joining

doi: 10.1016/j.jbc.2025.110174

Figure Lengend Snippet: TOX4 and PNUTS coordinate in NHEJ. A , HeLa cells were treated with siRNA targeting TOX4 or PNUTS, as indicated, for 24 h. The cell lysates were analyzed by immunoblotting for γ-H2AX, TOX4, PNUTS, and tubulin. B , NHEJ repair efficiency was measured in HeLa cells using the linearized GFP vector, as in N . Cells were treated with or without siRNA targeting TOX4, PNUTS, or KU80, as indicated. Immunoblots of GFP, TOX4, PNUTS, and β-actin are shown. C , Ku80 IP was performed in HeLa cells treated with control or TOX4 siRNA. The lysate input at 20%, control (ctr) IP with blank beads, and KU80 IP products were analyzed by immunoblotting for PNUTS, TOX4, KU80, and β-actin. NHEJ, nonhomologous end joining; PNUTS, phosphatase 1 nuclear targeting subunit; TOX4, Thymocyte Selection–Associated High-Mobility Group Box Family Member 4.

Article Snippet: SDS-PAGE and IB were carried out as previously described , using the following antibodies: KU80 (A302-627A), γ-H2AX (A300-081A), PNUTS (A300-439A), and Smc1 phosphoS957 (A304-147A) antibodies from Bethyl Laboratories; GFP (sc-9996) and KU70 (sc-56129) antibodies from Santa Cruz Biotechnology; TOX4 (ab272576), Chk2 phospho-T387 (ab195783), PAR (ab14459), DNA-PKcs (ab70250), DNA-PKcs phospho-S2056 (ab18192) antibodies from Abcam; β-actin (#4970), tubulin (#2144), caspase-3 (#9662), and Chk2 phospho-T68 (#2661) antibodies from Cell Signaling Technology.

Techniques: Western Blot, Plasmid Preparation, Control, Selection

Journal: Cell reports

Article Title: PNUTS:PP1 recruitment to Tox4 regulates chromosomal dispersal in Drosophila germline development

doi: 10.1016/j.celrep.2025.115693

Figure Lengend Snippet: (A) Domain organization of mammalian (human/rat) and fly ( Drosophila ) PNUTS (aqua) and Tox4 (pink), with key domains labeled. Sequence identity between mammalian and fly for the PNUTS N-terminal TFIIS domain and the Tox4 C-terminal domain are indicated. Domains that interact between PNUTS and Tox4 are shown by a double arrowed line. (B) Drosophila PNUTS interacts with Tox4 in the yeast two-hybrid system. PNUTS full-length bait (PNUTS 1–1135 -LexA DNA binding domain) and Tox4 prey (Tox4 179–250 -Gal4 activation domain) plasmids were tested in duplicate (clones 1 and 2) at several dilutions, as indicated. Auxotrophic growth on media without tryptophan and leucine (DO-2) or without tryptophan, leucine, and histidine with 50 mM 3-aminotriazol (DO-3+ 3-AT) is shown. C+, positive control with interacting bait and prey plasmids for Smad3 and Smurf1, respectively; C–, negative controls, as indicated. (C) Binding of Myc-tagged PNUTS to GFP-tagged Tox4 requires the Tox4 C terminus (residues 216–250) in pull-downs from Drosophila S2R+ cells. Immunoblots show total protein extract (IN) and immunoprecipitated protein extract (IP) from S2R+ cells co-transfected with GFP-tagged wild-type (Tox4 wt ) or truncated (Tox4 P216Term ) Tox4, with or without myc-tagged PNUTS. (D) 2D [ 1 H, 15 N]HSQC spectrum of 15 N-labeled PNUTS 5–160 C48S alone (red) and in complex with Tox4 571–621 (blue). (E) Binding isotherm of PNUTS 5–160 C48S with Tox4 571–621 C601S.

Article Snippet: 6.1 × 10 7 Drosophila 3 rd instar larval cDNA clones were screened in yeast using full-length PNUTS (amino acids 1–1135) protein as ‘bait’ fused to the LexA DNA binding domain in pB27 (a derivative of pBTM116) by Hybrigenics Inc.

Techniques: Labeling, Sequencing, Binding Assay, Activation Assay, Clone Assay, Positive Control, Western Blot, Immunoprecipitation, Transfection

Journal: Cell reports

Article Title: PNUTS:PP1 recruitment to Tox4 regulates chromosomal dispersal in Drosophila germline development

doi: 10.1016/j.celrep.2025.115693

Figure Lengend Snippet: (A) Structure of the Tox4 (magenta) and PNUTS NTD (Tox4 binding domain [Tox4BD], helices ɑ1-ɑ4, dark teal; Tox4 TFIIS domain, helices ɑ5-ɑ9, light teal) complex. The Tox4 PNUTS binding helix and TND-interacting motif (TIM) surface on PNUTS are labeled. Dotted box is Tox4 zinc binding pocket, shown also in the inset with zinc binding residues labeled. (B) A DALI structure similarity search identified RTR1 (lavender; PDB: 4FC8) and BS69 (gray; PDB: 5C2Y) as having weak similarity with the Tox4 C-terminal domain. Tox4, BS69, and Rtr1 structures, respectively, are shown at the top. BS69 is a dimer with the zinc binding domain at the end of a long coiled coil. Lower panel, structure-based sequence alignment of Tox4 (both the human and Drosophila sequence shown with identical residues indicated by an “*”), BS69, and Rtr1, with DALI Z scores reported, together with overlay of the zinc binding pockets with zinc binding residues labeled. (C) Interaction surfaces of Tox4 and PNUTS showing the electrostatic potential surface, with the hydrophobic (middle) and polar/charged (upper/lower) regions labeled. (D) Hydrophobic interactions between Tox4 (magenta/pink) and PNUTS (dark teal/light teal). Residues that become nearly completely buried upon complex formation are colored in dark shades. (E) N-terminal polar/charged interaction surface, with hydrogen bonds/salt bridges between Tox4 and PNUTS shown as dashed lines. (F) C-terminal polar/charged interaction surface, with inter- and intramolecular hydrogen bonds/salt bridges shown as dashed lines. (G) Consurf analysis of Tox4 (left) and PNUTS (right), with the regions at the PNUTS:Tox4 interface indicated by gray dotted lines.

Article Snippet: 6.1 × 10 7 Drosophila 3 rd instar larval cDNA clones were screened in yeast using full-length PNUTS (amino acids 1–1135) protein as ‘bait’ fused to the LexA DNA binding domain in pB27 (a derivative of pBTM116) by Hybrigenics Inc.

Techniques: Binding Assay, Labeling, Sequencing

Journal: Cell reports

Article Title: PNUTS:PP1 recruitment to Tox4 regulates chromosomal dispersal in Drosophila germline development

doi: 10.1016/j.celrep.2025.115693

Figure Lengend Snippet: (A) Maximal projection of stack of confocal images showing subcellular localization of ectopically expressed GFP-tagged Tox4 and RFP-tagged PNUTS throughout live S2R+ cells. In the absence of RFP-PNUTS (−), GFP-Tox4 wt , and GFP-Tox4 P216Term are found in both the nucleus and cytoplasm. In the presence of RFP-PNUTS (+), GFP-Tox4 wt accumulates in the nucleus, whereas the distribution of GFP-Tox4 P216Term is largely unaffected. Scale bars, 10 μm. (B) Intensity histograms showing distribution of pairs of GFP and RFP voxel intensities of cells in (A) co-transfected with GFP-Tox4 (green) and RFP-PNUTS (magenta), revealing strong colocalization of RFP-PNUTS and GFP-Tox4 wt but not GFP-Tox4 P216Term . (C) Plot showing mean ± SEM of Manders’ coefficient of the overlap between GFP and RFP in cotransfected cells. Means are derived from n = 3 experiments, shown on Beeswarm plots of individual measurements of repeated counts ( n = 10 cells/biological replicate). (D) Distribution of GFP-tagged Tox4 under the control of the endogenous tox4 promoter in Drosophila stage 10 egg chambers. Fluorescent signal from GFP-Tox4 wt (green) is clearly evident in both (smaller) somatic and (larger, polyploid) nurse cell nuclei, costained for DNA with DAPI (blue). In contrast, the signal from GFP-Tox4 P216Term can be seen less strongly in the nucleus and is diffusely localized throughout the cytoplasm. (E) Quantification of nuclear/cytoplasmic ratio. Datapoints for individual nuclei, color coded by egg chamber, are shown together with mean ± SEM ( n = 21 egg chambers, ~100 nuclei/genotype. Scale bars, 50 μm. Statistical significance was tested using pairwise t tests with Bonferroni p value correction. *** p < 0.001.

Article Snippet: 6.1 × 10 7 Drosophila 3 rd instar larval cDNA clones were screened in yeast using full-length PNUTS (amino acids 1–1135) protein as ‘bait’ fused to the LexA DNA binding domain in pB27 (a derivative of pBTM116) by Hybrigenics Inc.

Techniques: Transfection, Derivative Assay, Control

Journal: Cell reports

Article Title: PNUTS:PP1 recruitment to Tox4 regulates chromosomal dispersal in Drosophila germline development

doi: 10.1016/j.celrep.2025.115693

Figure Lengend Snippet: (A) PNUTS:Tox4 interaction interface highlighted the residue mutations to generate PNUTS TBD-dead —L12E, K43E, and V45D—shown in yellow. (B) Representative binding isotherm of PNUTS TBD-dead with Tox4 571–621 C601S. (C) Results of complementation tests with genomic PNUTS transgenes to determine their ability to rescue larval lethality of PNUTS 13B homozygous animals. A prediction of how often this genotype should be represented if there were no mutation (corresponding to a Mendelian ratio of 2:1 heterozygous/homozygous animals) is shown with a dotted line. Wild-type (PNUTS wt ) and reduced-Tox4 binding mutants (PNUTS ED and PNUTS E/ED ) all rescued PNUTS 13B homozygotes to adulthood (mean ± SEM is shown, n = 5). (D) Quantitation of ratio of eggs laid by the indicated genotype of parental flies relative to control flies ( PNUTS 13B/13B ; PNUTS wt ). Egg laying in PNUTS 13B/13B flies was strongly reduced by inducible PNUTS wt-flp-W726A and weakly reduced by PNUTS E/ED . (E) Quantitation of ratio of eggs from the indicated genotype of parental flies that hatched relative to control. Hatching was reduced to a similar extent by PNUTS wt-flp-W726A and PNUTS E/ED . (D and E) Plots show overall mean ± SEM derived from means of n = 3 experiments, superimposed on Beeswarm plots of individual measurements of repeated counts for each of the indicated genotypes. ns, not significant; * p < 0.05, ** p < 0.01, *** p < 0.001 by one-way ANOVA.

Article Snippet: 6.1 × 10 7 Drosophila 3 rd instar larval cDNA clones were screened in yeast using full-length PNUTS (amino acids 1–1135) protein as ‘bait’ fused to the LexA DNA binding domain in pB27 (a derivative of pBTM116) by Hybrigenics Inc.

Techniques: Residue, Binding Assay, Mutagenesis, Quantitation Assay, Control, Derivative Assay

Journal: Cell reports

Article Title: PNUTS:PP1 recruitment to Tox4 regulates chromosomal dispersal in Drosophila germline development

doi: 10.1016/j.celrep.2025.115693

Figure Lengend Snippet: (A and B) Strings of egg chambers of different stages (numbered, up to stage 10) stained with DAPI, which labels both somatic follicular cell nuclei surrounding each chamber and large nurse cell germline nuclei. Lower panels show magnified images of representative nurse cell nuclei from different stages. (A) In w 1118 control egg chambers, nurse cell chromosomes disperse throughout the nucleoplasm by stage 6. (B) In contrast, tox4 null / tox4 null nurse cell chromosomes fail to disperse, frequently retaining a “five-blob” structure until later stages of development (compare magnified images in A and B). Incomplete dispersal phenotypes were also observed at stage 10, with chromosomes decorating the nuclear periphery without dispersing completely throughout the nucleoplasm (arrow). Scale bars, 40 μm (ovarioles), 2 μm (magnified nuclei). (C) Quantification of non-dispersed nurse cell chromosomes in egg chambers at stage 6–7, 8–9, and 10. Violin plots show mean percentage non-dispersed nurse cell chromosomes/egg chamber for each indicated condition ( n > 15 egg chambers/stage/genotype). At stage 6–7, all conditions showed significant levels of non-dispersal except tox4 null / tox4 null rescued by GFP-tox4 w t . GFP-tox4 P216Term failed to substantially rescue dispersal phenotypes at this stage. In all conditions, the extent of non-dispersal decreased with developmental stage. However, significant non-dispersal was still observed for tox4 null / tox4 null (with or without GFP-tox4 P216Term ), and PNUTS 13B / 13B PNUTS EED -GFP at stage 10. ns, not significant; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 by Kruskal-Wallis test.

Article Snippet: 6.1 × 10 7 Drosophila 3 rd instar larval cDNA clones were screened in yeast using full-length PNUTS (amino acids 1–1135) protein as ‘bait’ fused to the LexA DNA binding domain in pB27 (a derivative of pBTM116) by Hybrigenics Inc.

Techniques: Staining, Control

Journal: Cell reports

Article Title: PNUTS:PP1 recruitment to Tox4 regulates chromosomal dispersal in Drosophila germline development

doi: 10.1016/j.celrep.2025.115693

Figure Lengend Snippet: (A) Principal-component analysis showing common response (PC1) to perturbation of PP1- or Tox4 binding to PNUTS, and divergent response (PC2) to tox4 loss of function, which together explain approximately 67% of the variance in gene expression. Data points for three independent biological repeats are shown together with the centroid in Euclidian space for each condition: w 1118 (gray), tox4 null / tox4 null (red), tox4 null / tox4 null GFP-tox4 w t (blue), PNUTS 13B / 13B ovoFLP>PNUTS wt-flp-W726A (purple), PNUTS 13B / 13B PNUTS ED (green), and PNUTS 13B / 13B PNUTS E/ED (yellow). (B) Plot showing number of differentially expressed (DE) genes compared with w 1118 control (>1.5-fold over or under-expressed, p adj <0.1) for the following conditions: (1) tox4 null / null , (2) PNUTS 13B / 13B ovoFLP>PNUTS wt-flp-W726A , (3) PNUTS 13B / 13B with either PNUTS ED or PNUTS E/ED . Overexpressed genes, red bar; underexpressed genes, blue bar. (C) Plot showing percentage of overexpressed and underexpressed genes for each condition as (B) that were found in each quartile of normal expression level derived from read counts in w 1118 control. Greater than 90% of underexpressed genes in each condition are in the top two quartiles for normal expression level, whereas >80% of overexpressed genes are more modestly expressed (2nd and 3rd quartiles). (D and E) Gene Ontology (GO) enrichment for DE genes as (B) grouped by Boolean terms, as indicated. (D) Enrichment of transcription factors, for underexpressed (>1.5-fold) and modestly overexpressed (1.5- to 2.0-fold) genes among different conditions compared with w 1118 control. The top two statistically significant GO categories ( p adj < 0.05) for transcription factors (black text) are shown in each case. (E) Enriched GO terms associated with biological functions among under-expressed genes. (F and G) The PNUTS:PP1:WDR82:TOX4 complex. (F) The PNUTS:Tox4 complex (teal and magenta, respectively) bound to the MYC TIM (PDB: 7LQT; orange). (G) Cartoon illustrating the complex between Tox4 (magenta), PNUTS (teal), PP1 (yellow), WDR82 (blue), and the MYC TIM (orange). Domain interactions for which structures have been determined are shown as cartoon and/or surfaces. Folded interaction partners (WDR82) are shown as a shape, while residues predicted to be IDRs are shown as lines.

Article Snippet: 6.1 × 10 7 Drosophila 3 rd instar larval cDNA clones were screened in yeast using full-length PNUTS (amino acids 1–1135) protein as ‘bait’ fused to the LexA DNA binding domain in pB27 (a derivative of pBTM116) by Hybrigenics Inc.

Techniques: Binding Assay, Gene Expression, Control, Expressing, Derivative Assay

Journal: Nucleic acids research

Article Title: Overlapping and distinct functions of SPT6, PNUTS, and PCF11 in regulating transcription termination.

doi: 10.1093/nar/gkaf179

Figure Lengend Snippet: Figure 1. SPT6 And IWS1 chromatin binding and transcriptional roles. ( A ) A schematic representation of SPT6 N-terminal (1–283 amino acids), core (284–1287 amino acids), and C-terminal (1288–1726 amino acids) regions. The illustration denotes N to C terminal protein domains. ( B ) Overexpressed Flag-tagged SPT6 from HEK293T total protein extracts was immunoprecipitated using an anti-Flag antibody and analyzed by western blotting using indicated antibodies. ( C ) SPT6 and IWS1 mean ChIP-seq signal intensities plotted at RefSeq-annotated genes. The solid line represents the mean. The dark area represents the standard error and light area represents the 95% confidence interval. ( D ) Heatmap representing the Pearson correlation coefficients calculated for IWS1, SPT6, and RNAPII merged ChIP-seq signals, on RefSeq-annotated genes ( ±1 kb). ( E ) SPT6 / RNAPII and IWS1 / RNAPII mean ChIP-seq signal ratios calculated on RefSeq-annotated genes in HeLa cells. The solid line represents the mean. The dark area represents the standard error and light area represents the 95% confidence interval. ( F ) Total protein extracts from HeLa cells were separated using a 5%–60% glycerol gradient. A total of 12 fractions were recovered after ultracentrifugation and analyzed by western blotting. ( G ) Western blot showing SPT6 and IWS1 depletions upon siRNA transfection in RNA-seq e xperiments. L o w er panel sho ws protein quantification relative to GAPDH and to the siCT condition ( n = 3). ( H ) SPT6 and IWS1 target genes in HeLa cells. Positive targets correspond to genes having a |FC| > | ±1.5| and a padj < 0.05. ( I ) Density plot showing the gene count of SPT6 and IWS1 readthrough targets, highlighting the distribution of their log 2 fold-change (FC) values. ( J ) SEC22B gene locus featuring RNAPII, SPT6, and IWS1 ChIP-seq profiles, as well as RNA-seq profiles upon depletion of SPT6 and IWS1 (negative strand). The arrow highlights readthrough transcription ( K ) Western blot showing SPT6 and IWS1 depletion upon siRNA transfection (see the “Materials and methods” section for details). ( L ) SEC22B readthrough and mRNA levels were assayed by RT-qPCR in three independent experiments. Values were normalized to the siCT condition arbitrarily set to 1. ( M ) SEC22B readthrough le v els w ere assa y ed b y nuclear run-on e xperiments. T he input represents total nuclear RNAs. The siSPT6 (-BrdU) condition is used to control the specificity of the anti-BrdU immunoprecipitation. Values were normalized to the siCT condition arbitrarily set to 1, and to the KDSR and 18S housekeeping genes.

Article Snippet: Construction of shRNA plasmids and production of virus-like particles Control short hairpin RNA (shRNA) sequence (shCT) or shRNA sequence targeting PNUTS (see references in Supplementary Table S1 ) were cloned into the pLK O .1- TRC cloning vector (gift from David Root, Addgene plasmid #10878; http:// n2t.net/ addgene:10878 ; RRID: Addgene_10 878) according to the manufacturer’s instructions.

Techniques: Binding Assay, Immunoprecipitation, Western Blot, ChIP-sequencing, Transfection, RNA Sequencing, Quantitative RT-PCR, Control

Journal: Nucleic acids research

Article Title: Overlapping and distinct functions of SPT6, PNUTS, and PCF11 in regulating transcription termination.

doi: 10.1093/nar/gkaf179

Figure Lengend Snippet: Figure 3. SPT6 interacts with IWS1, PNUTS, and PCF11. ( A ) Upper panel: Diagram depicting PNUTS domains: the TND (1–147 amino acids), the PP1-binding domain (348–418 amino acids) and the RNA-binding domain (674–750 amino acids). L o w er panel: Diagram depicting PCF11 domains: the CTD-interacting domain (CID) (1 4–1 42 amino acids), and the RNA-binding zinc fingers (between 1343 and 1478 amino acids). The illustration denotes N-to-C terminal protein domains. ( B ) Nuclear protein extracts from WT or IWS1-knockout (IWS1- / -) J-Lat A1 cells were used to immunoprecipitate endogenous SPT6. Co-immunoprecipitation of IWS1, PCF11 and PNUTS was assessed ( n = 3). ( C ) Overexpressed Flag-tagged WT PNUTS (WT) or Flag-tagged PNUTS–W401A (W401A), a mutant for PP1 interaction, were immunoprecipitated using an anti-Flag antibody from HEK293T total protein e xtracts. SPT6 co-immunoprecipitation w as e xamined f or both WT and mutant PNUTS ( n = 3). ( D ) HeLa cells infected with lentiviruses containing either a control shRNA (shCT) or an shRNA targeting PNUTS (shPNUTS). Cells nuclear fractions were used to immunoprecipitate endogenous SPT6 ( n = 2). “C yto ” stands for Cytoplasmic fraction, and “Nuc” stands for Nuclear fraction used as input.

Article Snippet: Construction of shRNA plasmids and production of virus-like particles Control short hairpin RNA (shRNA) sequence (shCT) or shRNA sequence targeting PNUTS (see references in Supplementary Table S1 ) were cloned into the pLK O .1- TRC cloning vector (gift from David Root, Addgene plasmid #10878; http:// n2t.net/ addgene:10878 ; RRID: Addgene_10 878) according to the manufacturer’s instructions.

Techniques: Binding Assay, RNA Binding Assay, Zinc-Fingers, Knock-Out, Immunoprecipitation, Mutagenesis, Infection, Control, shRNA

Journal: Nucleic acids research

Article Title: Overlapping and distinct functions of SPT6, PNUTS, and PCF11 in regulating transcription termination.

doi: 10.1093/nar/gkaf179

Figure Lengend Snippet: Figure 5. Differential readthrough transcripts upon depletion of SPT6, PNUTS, and PCF11. ( A ) Mean RNA-seq signal intensity shown at SPT6 readthrough targets (TES + 5 kb) in the different conditions. ( B ) Heatmap displaying differentially expressed readthrough transcripts in HeLa cells f ollo wing SPT6 and / or PNUTS depletion. Readthrough transcripts with a log 2 FC > 1 in the siSPT6 condition compared to the control condition were considered positive. The readthrough regions were identified as described in Supplementary Fig. S1 F. The heatmap plots the average log2FC scores (siRNA / siCT) of these regions. Three distinct clusters were defined to characterize the varying responses to SPT6 and / or PNUTS depletion. The analysis was conducted using three independent biological replicates. ( C ) Box-plots showing the median log 2 FC of readthrough transcripts relative to the control condition for the three clusters shown in panel (B). ( D –F ) Same as in panels (A)–(C) but for SPT6 and / or PCF11 depletion. ( G ) CTNND1 gene locus featuring RNA-seq data (positive strand) following the use of the indicated siRNAs. The lower panel (PAS) displays the locations of human PASs as defined by Zhang et al. [ 82 ]. The arrow highlights readthrough transcription. The scale is shown at the top right corner of the figure.

Article Snippet: Construction of shRNA plasmids and production of virus-like particles Control short hairpin RNA (shRNA) sequence (shCT) or shRNA sequence targeting PNUTS (see references in Supplementary Table S1 ) were cloned into the pLK O .1- TRC cloning vector (gift from David Root, Addgene plasmid #10878; http:// n2t.net/ addgene:10878 ; RRID: Addgene_10 878) according to the manufacturer’s instructions.

Techniques: RNA Sequencing, Control

Journal: Nucleic acids research

Article Title: Overlapping and distinct functions of SPT6, PNUTS, and PCF11 in regulating transcription termination.

doi: 10.1093/nar/gkaf179

Figure Lengend Snippet: Figure 6. Differential PROMPTs regulation upon depletion of SPT6, PNUTS, and PCF11. ( A ) Mean RNA-seq signal intensity shown at SPT6 PROMPT targets (TSS, -5 kb) in the different conditions. ( B ) Heatmap displaying differentially expressed PROMPTs in HeLa cells following SPT6 and / or PNUTS depletion. PROMPTs with a log 2 FC > 1 in the siSPT6 condition compared to the control condition were considered positive. PROMPT regions were identified as described in Supplementary Fig. S6 A. The heatmap plots the average log 2 FC scores (siRNA / siCT) of these regions. Three distinct clusters were defined to characterize the varying responses to SPT6 and / or PNUTS depletion. The analysis was conducted using three independent biological replicates. ( C ) B o x-plots sho wing the median log 2 FC of PR OMPTs relativ e to the control condition f or the three clusters sho wn in panel (B). ( D ) PXDN PR OMPT le v els w ere assa y ed b y R T-qPCR in three independent e xperiments. Values w ere normaliz ed to the siCT condition arbitrarily set to 1. ( E –G ) Same as in panels (A)–(C) but for SPT6 and / or PCF11 depletion. ( H ) Same as in panel (D) but for the GGCT gene. ( I ) PVT1 gene locus featuring RNA-seq data (positive strand is in positive values and negative strand in negative values) following the use of the indicated siRNAs. The lower panel (PAS) displays the locations of human PASs as defined by Zhang et al. [ 82 ]. The arrow highlights PROMPTs. The scale is shown at the top right corner of the figure.

Article Snippet: Construction of shRNA plasmids and production of virus-like particles Control short hairpin RNA (shRNA) sequence (shCT) or shRNA sequence targeting PNUTS (see references in Supplementary Table S1 ) were cloned into the pLK O .1- TRC cloning vector (gift from David Root, Addgene plasmid #10878; http:// n2t.net/ addgene:10878 ; RRID: Addgene_10 878) according to the manufacturer’s instructions.

Techniques: RNA Sequencing, Control

Journal: Nucleic acids research

Article Title: Overlapping and distinct functions of SPT6, PNUTS, and PCF11 in regulating transcription termination.

doi: 10.1093/nar/gkaf179

Figure Lengend Snippet: Figure 7. Model for the regulation of transcription termination by SPT6, PNUTS, and PCF11 at promoters and gene ends. At transcription termination sites, SPT6, PNUTS, and PCF11 pre v ent readthrough transcription. At gene promoters, SPT6 and PNUTS establish an initial block to limit PROMPTs. When this primary block is remo v ed, PCF11 promotes PROMPTs, either directly or by inhibiting the recruitment of another PROMPT repressor. Created in BioRender. Bejjani, F. (2025) https:// BioRender.com/ f07e489.

Article Snippet: Construction of shRNA plasmids and production of virus-like particles Control short hairpin RNA (shRNA) sequence (shCT) or shRNA sequence targeting PNUTS (see references in Supplementary Table S1 ) were cloned into the pLK O .1- TRC cloning vector (gift from David Root, Addgene plasmid #10878; http:// n2t.net/ addgene:10878 ; RRID: Addgene_10 878) according to the manufacturer’s instructions.

Techniques: Blocking Assay