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Novus Biologicals anti 128 ints6
Anti 128 Ints6, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 92 stars, based on 2 article reviews

anti 128 ints6 - by Bioz Stars, 2026-05

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( A ) The distributions of nonsense, frameshift, and splicing variants in INTS 6 identified in NDDs are shown in a protein model and gene model, respectively. ( B ) The distribution of missense variants in INTS6 identified in NDDs is shown in a protein model. Protein tolerance landscape for missense variants in INTS6 was visualized via MetaDome20. All variants in INTS6 are predicted to be “intolerant” for aa substitutions. The density plot of ultrarare missense variants in gnomAD is shown. ( C ) Comparison of the distribution of combined annotation-dependent depletion (CADD) and MPC scores between de novo missense variants in NDDs and ultrarare missense variants in gnomAD database. Data are reported as mean ± SEM. P values were determined from a 2-tailed, unpaired Mann-Whitney test. ( D ) Comparison of SIFT, PolyPhen-2, and AlphaMissense prediction between de novo missense variants in NDDs and ultrarare missense variants in the gnomAD database. SIFT: D (deleterious), T (tolerated); PolyPhen-2: D (probably damaging), P (possibly damaging), B (benign); AlphaMissense: P (likely pathogenic), B (likely benign), A (ambiguous). ( E ) Left: Ribbon diagram of the INTS-PP2A complex bound to paused Pol II (PDB:7PKS). The disease-associated protein INTS6 and its interacting proteins are labeled. Right: Close-up view of NDD-related variants on INTS6 (red spheres), highlighting the importance of these residues in mediating protein-protein interactions or maintaining the structural integrity of INTS6.

Journal: The Journal of Clinical Investigation

Article Title: Disrupting integrator complex subunit INTS6 causes neurodevelopmental disorders and impairs neurogenesis and synapse development

doi: 10.1172/JCI191729

Figure Lengend Snippet: ( A ) The distributions of nonsense, frameshift, and splicing variants in INTS 6 identified in NDDs are shown in a protein model and gene model, respectively. ( B ) The distribution of missense variants in INTS6 identified in NDDs is shown in a protein model. Protein tolerance landscape for missense variants in INTS6 was visualized via MetaDome20. All variants in INTS6 are predicted to be “intolerant” for aa substitutions. The density plot of ultrarare missense variants in gnomAD is shown. ( C ) Comparison of the distribution of combined annotation-dependent depletion (CADD) and MPC scores between de novo missense variants in NDDs and ultrarare missense variants in gnomAD database. Data are reported as mean ± SEM. P values were determined from a 2-tailed, unpaired Mann-Whitney test. ( D ) Comparison of SIFT, PolyPhen-2, and AlphaMissense prediction between de novo missense variants in NDDs and ultrarare missense variants in the gnomAD database. SIFT: D (deleterious), T (tolerated); PolyPhen-2: D (probably damaging), P (possibly damaging), B (benign); AlphaMissense: P (likely pathogenic), B (likely benign), A (ambiguous). ( E ) Left: Ribbon diagram of the INTS-PP2A complex bound to paused Pol II (PDB:7PKS). The disease-associated protein INTS6 and its interacting proteins are labeled. Right: Close-up view of NDD-related variants on INTS6 (red spheres), highlighting the importance of these residues in mediating protein-protein interactions or maintaining the structural integrity of INTS6.

Article Snippet: Ints6 flox/flox mice were generated by Cyagen Biotechnology using the CRISPR-Cas9 method, following the strategy outlined in .

Techniques: Comparison, MANN-WHITNEY, Labeling, Protein-Protein interactions

Journal: The Journal of Clinical Investigation

Article Title: Disrupting integrator complex subunit INTS6 causes neurodevelopmental disorders and impairs neurogenesis and synapse development

doi: 10.1172/JCI191729

Figure Lengend Snippet: ( A ) Heatmap and spatial distribution of RNAPII binding at the TSS of genes analyzed by CUT&Tag in WT and cKO E15.5 mice. ( B ) Average distribution profile of RNAPII across gene regions, including TSSs and TESs. ( C ) Spatial distribution and heatmap representation of the distance of RNAPII binding around the TSS of RIP-Seq genes. ( D ) Average distribution profile of RNAPII across coding sequences (CDS) regions of RIP-Seq. The gradient of blue to white color ( A – D ) indicates high to low counts in the corresponding region. ( E ) A Venn diagram illustrating the overlap between DGEs ( P < 0.05) identified in RNA-Seq and CUT&Tag data sets. ( F ) Heatmap analysis of the relative expression levels of 2,374 genes in the overlap of RNA-Seq and CUT&Tag. Upregulated genes are depicted in blue; downregulated genes are shown in red. ( G ) Bar graph depicting enriched KEGG pathways identified from the overlap data. ( H ) Bubble plot depicting enriched GO terms identified from the overlap data. ( I ) Browser tracks of CUT&Tag profiles for the genes related to the cell cycle at E15.5 days of embryonic development, comparing expression levels in WT and INTS6 cKO mice. ( J ) Western blot analysis of total RNAPII and Ser2P in HEK293T cells transfected with either WT, missense variants ( n = 5), or LGD variants ( n = 8). P values were determined from Friedman with Dunnett’s multiple comparisons test. ( K ) Statistical analysis of the effects of CDK9i on the growth of WT ( n = 27 DMSO-treated; n = 14 CDK9i-treated); cHET ( n = 23 DMSO-treated, n = 18 CDK9i-treated) and cKO ( n = 12 DMSO-treated, n = 19 CDK9i-treated) neurosphere. P values were determined from a 2-tailed unpaired t test and Mann-Whitney test. * P < 0.05, ** P < 0.01. CON, control. Data are reported as mean ± SEM.

Article Snippet: Ints6 flox/flox mice were generated by Cyagen Biotechnology using the CRISPR-Cas9 method, following the strategy outlined in .

Techniques: Binding Assay, RNA Sequencing, Expressing, Western Blot, Transfection, MANN-WHITNEY, Control

( A ) Heatmaps depicting the movement of WT ( n = 14) or cHET ( n = 14) mice in a 3-chamber social interaction test. Preference scores were calculated as (S – E)/(S + E) for social versus empty interactions and (S2 – S1)/(S2 + S1) for stranger versus original mouse interactions. Data are reported as mean ± SEM. P values were determined from a 2-tailed unpaired t test. ( B ) Morris water maze test of spatial learning and memory in Ints6 cHET mice. Latent time (s) during training trials, time in platform quadrant, and distance traveled are measured ( n = 14). Data are reported as mean ± SEM. P values were determined from 2-way ANOVA with Bonferroni’s multiple comparisons test and a 2-tailed unpaired Mann-Whitney test. ( C ) Elevated cross-maze experiments were performed with WT ( n = 13) and cHET ( n = 14) mice to evaluate anxiety-related behaviors. The experiments statistically analyzed the movement distance and dwell time in both the open and closed arms of the maze. Data are reported as mean ± SEM. P values were determined from a 2-tailed unpaired Mann-Whitney test. ( D ) Path-tracking images from an open field test, showing movement patterns of WT ( n = 14) and cHET ( n = 15) mice. Bar graph representing the distance traveled and the time spent in the central area of the open field over 10 minutes. Data are reported as mean ± SEM. P values were determined from a 2-tailed unpaired t test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. snRNP, small nuclear ribonucleoprotein.

Journal: The Journal of Clinical Investigation

Article Title: Disrupting integrator complex subunit INTS6 causes neurodevelopmental disorders and impairs neurogenesis and synapse development

doi: 10.1172/JCI191729

Figure Lengend Snippet: ( A ) Heatmaps depicting the movement of WT ( n = 14) or cHET ( n = 14) mice in a 3-chamber social interaction test. Preference scores were calculated as (S – E)/(S + E) for social versus empty interactions and (S2 – S1)/(S2 + S1) for stranger versus original mouse interactions. Data are reported as mean ± SEM. P values were determined from a 2-tailed unpaired t test. ( B ) Morris water maze test of spatial learning and memory in Ints6 cHET mice. Latent time (s) during training trials, time in platform quadrant, and distance traveled are measured ( n = 14). Data are reported as mean ± SEM. P values were determined from 2-way ANOVA with Bonferroni’s multiple comparisons test and a 2-tailed unpaired Mann-Whitney test. ( C ) Elevated cross-maze experiments were performed with WT ( n = 13) and cHET ( n = 14) mice to evaluate anxiety-related behaviors. The experiments statistically analyzed the movement distance and dwell time in both the open and closed arms of the maze. Data are reported as mean ± SEM. P values were determined from a 2-tailed unpaired Mann-Whitney test. ( D ) Path-tracking images from an open field test, showing movement patterns of WT ( n = 14) and cHET ( n = 15) mice. Bar graph representing the distance traveled and the time spent in the central area of the open field over 10 minutes. Data are reported as mean ± SEM. P values were determined from a 2-tailed unpaired t test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. snRNP, small nuclear ribonucleoprotein.

Article Snippet: Ints6 flox/flox mice were generated by Cyagen Biotechnology using the CRISPR-Cas9 method, following the strategy outlined in .

Techniques: MANN-WHITNEY

Figure 1. INTS6 localizes to DSBs in a DNA:RNA hybrid-dependent manner. ( A ) Left: Scans of representative EMSA experiments of INTS6 with 61- or 21 -mer ssDNA, 61 -mer dsDNA, R-loops and DNA:RNA hybrids. Right: Graph representing quantification of EMSA experiments (n = 3). ( B ) Left: Scans of representative EMSA experiments of the tetrameric SOSS1 complex with 61- or 21-mer ssDNA, 61-mer dsDNA, R-loops and DNA:RNA hybrids. Right: Graph representing quantification of EMSA experiments (n = 3). ( C ) Laser striping of cells transiently transfected with INTS6-GFP plasmid with or without RNase H1-RFP plasmid. White lines indicate laser stripes. R epresentativ e conf ocal microscop y images and quantification (n ≥20) sho w GFP and RFP signals at the indicated time points. Error bars: mean ± SEM. ( D ) PLA of INTS6 and S9.6 in cells without IR, with IR or with IR and RNaseH1 treatment. IR = 10 Gy, samples were collected 10 min post-IR. Here, we used modified PLA protocol ( 55 ), which includes RNaseT1 (digest ssRNA) and RNase III (digest dsRNA) treatment during slide preparation process. Top: R epresentativ e conf ocal microscop y images; bottom: quantification of top, error bar = mean ± SD, significance was determined using non-parametric Mann–Whitney test. **** P ≤0.0 0 01. Scale bar = 10 μm. Single antibodies were used as a negative control. ( E ) PLA of INTS6 and γH2AX in cells transiently transfected with RNAseH1 wt -GFP or RNAseH1 WKKD -GFP (binding and cat alytic) or RNAseH1 D210N −GFP (cat alytic) mut ants with or without IR. IR = 1 0 Gy , samples were collected 1 0 min post-IR. L eft: representativ e conf ocal microscopy images; right: quantification of left, error bar = mean ± SD, significance was determined using non-parametric Mann–Whitney test. **** P ≤0.0 0 01, * P ≤0.05. Scale bar = 10 μm. Single antibodies were used as a negative control.

Journal: Nucleic acids research

Article Title: Tetrameric INTS6-SOSS1 complex facilitates DNA:RNA hybrid autoregulation at double-strand breaks.

doi: 10.1093/nar/gkae937

Figure Lengend Snippet: Figure 1. INTS6 localizes to DSBs in a DNA:RNA hybrid-dependent manner. ( A ) Left: Scans of representative EMSA experiments of INTS6 with 61- or 21 -mer ssDNA, 61 -mer dsDNA, R-loops and DNA:RNA hybrids. Right: Graph representing quantification of EMSA experiments (n = 3). ( B ) Left: Scans of representative EMSA experiments of the tetrameric SOSS1 complex with 61- or 21-mer ssDNA, 61-mer dsDNA, R-loops and DNA:RNA hybrids. Right: Graph representing quantification of EMSA experiments (n = 3). ( C ) Laser striping of cells transiently transfected with INTS6-GFP plasmid with or without RNase H1-RFP plasmid. White lines indicate laser stripes. R epresentativ e conf ocal microscop y images and quantification (n ≥20) sho w GFP and RFP signals at the indicated time points. Error bars: mean ± SEM. ( D ) PLA of INTS6 and S9.6 in cells without IR, with IR or with IR and RNaseH1 treatment. IR = 10 Gy, samples were collected 10 min post-IR. Here, we used modified PLA protocol ( 55 ), which includes RNaseT1 (digest ssRNA) and RNase III (digest dsRNA) treatment during slide preparation process. Top: R epresentativ e conf ocal microscop y images; bottom: quantification of top, error bar = mean ± SD, significance was determined using non-parametric Mann–Whitney test. **** P ≤0.0 0 01. Scale bar = 10 μm. Single antibodies were used as a negative control. ( E ) PLA of INTS6 and γH2AX in cells transiently transfected with RNAseH1 wt -GFP or RNAseH1 WKKD -GFP (binding and cat alytic) or RNAseH1 D210N −GFP (cat alytic) mut ants with or without IR. IR = 1 0 Gy , samples were collected 1 0 min post-IR. L eft: representativ e conf ocal microscopy images; right: quantification of left, error bar = mean ± SD, significance was determined using non-parametric Mann–Whitney test. **** P ≤0.0 0 01, * P ≤0.05. Scale bar = 10 μm. Single antibodies were used as a negative control.

Article Snippet: The Open Reading Frame (ORF) of INTS6 was cloned into plasmid 438C (pFastBac His6 MBP Asn10 TEV cloning vector with BioBrick Polypromoter LIC subcloning, Addgene plasmid #55220).

Techniques: Transfection, Plasmid Preparation, Modification, MANN-WHITNEY, Negative Control, Binding Assay, Microscopy

Figure 2. INTS6 facilitates PP2A recruitment to DSBs to dephosphorylate RNAPII. ( A ) PLA of PP2A and γH2AX in WT or INTS6 knockdown cells with or without IR. IR = 10 Gy, samples were collected 10 min post-IR. Left: Representative confocal microscopy images; right: quantification of left, error bar = mean ± SD, significance was determined using non-parametric Mann–Whitney test., **** P ≤0.0 0 01, *** P ≤0.001, * P ≤0.05. Scale bar = 10 μm. Single antibodies were used as a negative control. ( B ) PLA of PP2A and γH2AX in cells with or without transient RNaseH1-GFP expression, with or without IR. IR = 1 0Gy , samples were collected 10 min post-IR. Left: Representative confocal microscopy images; right: quantification of left, error bar = mean ± SD, significance was determined using non-parametric Mann–Whitney test. **** P ≤0.0 0 01, * P ≤0.05. Scale bar = 10 μm. Single antibodies were used as a negative control. ( C ) PLA of S5P and γH2AX in wildtype or INTS6 knockdown cells with or without IR. IR = 10 Gy, samples were collected 10 min post-IR. Left: Representative confocal microscopy images; right: quantification of left, error bar = mean ± SD, significance was determined using non-parametric Mann–Whitney test. **** P ≤0.0 0 01, * P ≤0.05. Scale bar = 10 μm. Single antibodies were used as a negative control. ( D ) PLA of S5P and γH2AX with or without IR in the presence or absence of PP2A inhibitor (LB-100, 2.5 μM, 2 h). IR = 10 Gy, samples were collected 10 min post-IR. Left: Representative confocal microscopy images; right: quantification of left, error bar = mean ± SD, significance was determined using non-parametric Mann–Whitney test. **** P ≤0.0 0 01, ** P ≤0.01. Scale bar = 10 μm. Single antibodies were used as a negative control. ( E ) Left: Drawing of ChIP probes positions around DS1. Right: drawing showing position of ChIP probes in GAPDH gene. ( F ) Bar charts showing RNAPII, S2P and S5P ChIP signals at DS1 in the absence or presence of PP2A inhibitor (LB-100, 2.5 μM, 4 h). n ≥3. Error bar = mean ± SD, significance was determined using unpaired Student’s t -test, **** P ≤0.0 0 01, *** P ≤0.001,** P ≤0.01* P ≤0.05.

Journal: Nucleic acids research

Article Title: Tetrameric INTS6-SOSS1 complex facilitates DNA:RNA hybrid autoregulation at double-strand breaks.

doi: 10.1093/nar/gkae937

Figure Lengend Snippet: Figure 2. INTS6 facilitates PP2A recruitment to DSBs to dephosphorylate RNAPII. ( A ) PLA of PP2A and γH2AX in WT or INTS6 knockdown cells with or without IR. IR = 10 Gy, samples were collected 10 min post-IR. Left: Representative confocal microscopy images; right: quantification of left, error bar = mean ± SD, significance was determined using non-parametric Mann–Whitney test., **** P ≤0.0 0 01, *** P ≤0.001, * P ≤0.05. Scale bar = 10 μm. Single antibodies were used as a negative control. ( B ) PLA of PP2A and γH2AX in cells with or without transient RNaseH1-GFP expression, with or without IR. IR = 1 0Gy , samples were collected 10 min post-IR. Left: Representative confocal microscopy images; right: quantification of left, error bar = mean ± SD, significance was determined using non-parametric Mann–Whitney test. **** P ≤0.0 0 01, * P ≤0.05. Scale bar = 10 μm. Single antibodies were used as a negative control. ( C ) PLA of S5P and γH2AX in wildtype or INTS6 knockdown cells with or without IR. IR = 10 Gy, samples were collected 10 min post-IR. Left: Representative confocal microscopy images; right: quantification of left, error bar = mean ± SD, significance was determined using non-parametric Mann–Whitney test. **** P ≤0.0 0 01, * P ≤0.05. Scale bar = 10 μm. Single antibodies were used as a negative control. ( D ) PLA of S5P and γH2AX with or without IR in the presence or absence of PP2A inhibitor (LB-100, 2.5 μM, 2 h). IR = 10 Gy, samples were collected 10 min post-IR. Left: Representative confocal microscopy images; right: quantification of left, error bar = mean ± SD, significance was determined using non-parametric Mann–Whitney test. **** P ≤0.0 0 01, ** P ≤0.01. Scale bar = 10 μm. Single antibodies were used as a negative control. ( E ) Left: Drawing of ChIP probes positions around DS1. Right: drawing showing position of ChIP probes in GAPDH gene. ( F ) Bar charts showing RNAPII, S2P and S5P ChIP signals at DS1 in the absence or presence of PP2A inhibitor (LB-100, 2.5 μM, 4 h). n ≥3. Error bar = mean ± SD, significance was determined using unpaired Student’s t -test, **** P ≤0.0 0 01, *** P ≤0.001,** P ≤0.01* P ≤0.05.

Article Snippet: The Open Reading Frame (ORF) of INTS6 was cloned into plasmid 438C (pFastBac His6 MBP Asn10 TEV cloning vector with BioBrick Polypromoter LIC subcloning, Addgene plasmid #55220).

Techniques: Knockdown, Confocal Microscopy, MANN-WHITNEY, Negative Control, Expressing

Figure 3. INTS6 depletion leads to the accumulation of DARTs. ( A ) Metagene plot shows chrRNA-seq sense and antisense coverage in no damage (-4OHT), damage (siCtrl + 4OHT) and INTS6 knockdown (siINTS6 + 4OHT) cells around 2.5 kb flank region of cut Asi SI sites (details in Supplementary Table S4 ). The reference genome is human hg19. ( B ) Metagene plot shows chrRNA-seq sense and antisense coverage in no damage (-4OHT), damage (siCtrl + 4OHT) and INTS6 knockdown (siINTS6 + 4OHT) cells around 2.5 kb flank region of uncut Asi SI sites (details in Supplementary Table S4 ). The reference genome is human hg19. ( C ) Box plots show log2 fold change of chrRNA-seq co v erage of sense reads and antisense reads upon INTS6 knockdown with damage induction compared to control with damage induction for cut Asi SI ( ± 500 bp). Two-sample Wilco x on test is used for statistical testing of medians between sense and antisense log2 fold change distribution. ( D ) Heatmaps show antisense nascent RNA (chrRNA-seq) read co v erage across annotated Asi SI sites (details in Supplementary Table S4 ) sorted based on their clea v age efficiency. ( E ) B o x plot shows chrRNA-seq antisense coverage in ± 500 bp flank of Asi SI cut sites (details in Supplementary Table S4 ) in -4OHT, siCtrl + 4OHT and siINTS6 + 4OHT samples. Two-sample Wilcoxon test is used with medians test. Error bar = mean ± SD. ( F, G ) Representative snapshots of individual genes showing sense and antisense chrRNA-seq coverage in INTS6 knockdown and control with damage induction around 2.5 kb flank region of Asi SI cut. The specific loci information is listed on top of the snapshots. The reference genome is human hg19.

Journal: Nucleic acids research

Article Title: Tetrameric INTS6-SOSS1 complex facilitates DNA:RNA hybrid autoregulation at double-strand breaks.

doi: 10.1093/nar/gkae937

Figure Lengend Snippet: Figure 3. INTS6 depletion leads to the accumulation of DARTs. ( A ) Metagene plot shows chrRNA-seq sense and antisense coverage in no damage (-4OHT), damage (siCtrl + 4OHT) and INTS6 knockdown (siINTS6 + 4OHT) cells around 2.5 kb flank region of cut Asi SI sites (details in Supplementary Table S4 ). The reference genome is human hg19. ( B ) Metagene plot shows chrRNA-seq sense and antisense coverage in no damage (-4OHT), damage (siCtrl + 4OHT) and INTS6 knockdown (siINTS6 + 4OHT) cells around 2.5 kb flank region of uncut Asi SI sites (details in Supplementary Table S4 ). The reference genome is human hg19. ( C ) Box plots show log2 fold change of chrRNA-seq co v erage of sense reads and antisense reads upon INTS6 knockdown with damage induction compared to control with damage induction for cut Asi SI ( ± 500 bp). Two-sample Wilco x on test is used for statistical testing of medians between sense and antisense log2 fold change distribution. ( D ) Heatmaps show antisense nascent RNA (chrRNA-seq) read co v erage across annotated Asi SI sites (details in Supplementary Table S4 ) sorted based on their clea v age efficiency. ( E ) B o x plot shows chrRNA-seq antisense coverage in ± 500 bp flank of Asi SI cut sites (details in Supplementary Table S4 ) in -4OHT, siCtrl + 4OHT and siINTS6 + 4OHT samples. Two-sample Wilcoxon test is used with medians test. Error bar = mean ± SD. ( F, G ) Representative snapshots of individual genes showing sense and antisense chrRNA-seq coverage in INTS6 knockdown and control with damage induction around 2.5 kb flank region of Asi SI cut. The specific loci information is listed on top of the snapshots. The reference genome is human hg19.

Article Snippet: The Open Reading Frame (ORF) of INTS6 was cloned into plasmid 438C (pFastBac His6 MBP Asn10 TEV cloning vector with BioBrick Polypromoter LIC subcloning, Addgene plasmid #55220).

Techniques: Knockdown, Control

Figure 4. INTS6 associates with SETX. ( A ) Silver stain of affinity-purified Integrator complex. The integrator complex was purified from nuclear lysate of HEK293Tcells, stably o v ere xpressing FLAG-INTS6. Mock indicates the same FLAG-IP purification steps from parental HEK293T cells. The indicated Integrator subunits were assigned as identified by Baillat et al. ( 31 ). ( B ) Affinity-purified Integrator complex mass spectrometry analyses were performed on nuclear lysate of HEK293T cells stably overexpressing FLAG-INTS6 or mock FLAG-IP purification steps from parental HEK293T cells. The values represent intensity-based absolute quantification (iBAQ) intensities and the unique peptides. ( C ) Affinity-purified Integrator complex followed by western blot showing indicated proteins. ( D ) PLA of SETX and INTS6 with or without IR treatment. IR = 10 Gy, samples were collected 10 min post-IR. Left: R epresentativ e conf ocal microscop y images; right: quantification of lef t, error bar = mean ± SD , significance was determined using non-parametric Mann–Whitney test. **** P ≤0.0 0 01. Scale bar = 10 μm. Single antibodies were used as a negative control. ( E ) Immunoprecipitation of SETX from cells with or without IR treatment (IR = 1 0 Gy , samples were collected 10 min post-IR), f ollo w ed b y Western blot sho wing signals for SETX, INTS6, INTS3 and hSSB1. KDa indicates size of the proteins. Bar charts show quantifications of three independent blots, error bar = mean ± SD, significance was determined using unpaired Student’s t -test, * P ≤0.05.

Journal: Nucleic acids research

Article Title: Tetrameric INTS6-SOSS1 complex facilitates DNA:RNA hybrid autoregulation at double-strand breaks.

doi: 10.1093/nar/gkae937

Figure Lengend Snippet: Figure 4. INTS6 associates with SETX. ( A ) Silver stain of affinity-purified Integrator complex. The integrator complex was purified from nuclear lysate of HEK293Tcells, stably o v ere xpressing FLAG-INTS6. Mock indicates the same FLAG-IP purification steps from parental HEK293T cells. The indicated Integrator subunits were assigned as identified by Baillat et al. ( 31 ). ( B ) Affinity-purified Integrator complex mass spectrometry analyses were performed on nuclear lysate of HEK293T cells stably overexpressing FLAG-INTS6 or mock FLAG-IP purification steps from parental HEK293T cells. The values represent intensity-based absolute quantification (iBAQ) intensities and the unique peptides. ( C ) Affinity-purified Integrator complex followed by western blot showing indicated proteins. ( D ) PLA of SETX and INTS6 with or without IR treatment. IR = 10 Gy, samples were collected 10 min post-IR. Left: R epresentativ e conf ocal microscop y images; right: quantification of lef t, error bar = mean ± SD , significance was determined using non-parametric Mann–Whitney test. **** P ≤0.0 0 01. Scale bar = 10 μm. Single antibodies were used as a negative control. ( E ) Immunoprecipitation of SETX from cells with or without IR treatment (IR = 1 0 Gy , samples were collected 10 min post-IR), f ollo w ed b y Western blot sho wing signals for SETX, INTS6, INTS3 and hSSB1. KDa indicates size of the proteins. Bar charts show quantifications of three independent blots, error bar = mean ± SD, significance was determined using unpaired Student’s t -test, * P ≤0.05.

Article Snippet: The Open Reading Frame (ORF) of INTS6 was cloned into plasmid 438C (pFastBac His6 MBP Asn10 TEV cloning vector with BioBrick Polypromoter LIC subcloning, Addgene plasmid #55220).

Techniques: Silver Staining, Affinity Purification, Purification, Stable Transfection, Mass Spectrometry, Quantitative Proteomics, Western Blot, MANN-WHITNEY, Negative Control, Immunoprecipitation

Figure 5. INTS6 is required for SETX recruitment to DSBs and clearance of DNA:RNA hybrids. ( A ) PLA of SETX and γH2AX with or without IR in mock or siINTS6 cells. IR = 10 Gy, samples were collected 10 min post-IR. Left: Representative confocal microscopy images; right: quantification of left, error bar = mean ± SD, significance was determined using non-parametric Mann–Whitney test. **** P ≤0.0 0 01. Scale bar = 10 μm. Single antibodies were used as a negative control. ( B, C ) Bar charts showing SETX ChIP signals over DS1 ( B ) and DS2 ( C ) loci in the presence or absence of INTS6. n = 3. Error bar = mean ± SD, significance was determined using Student’s t -test, unpaired, ** P ≤0.01. ( D ) PLA of S9.6 and γH2AX with or without IR in mock or siINTS6 cells. IR = 10 Gy, samples were collected 10 min post-IR. Left: Representative confocal microscopy images; right: quantification of top, error bar = mean ± SD, significance was determined using non-parametric Mann–Whitney test. **** P ≤0.0 0 01. Scale bar = 10 μm. Single antibodies were used as a negative control. ( E, F ) Bar charts showing DRIP signals over DS1 ( E ) and DS2 ( F ) loci in the presence or absence of INTS6 and RNaseH1 treatment. n = 3. Error bar = mean ± SD, significance was determined using unpaired Student’s t -test, *** P ≤0.001, ** P ≤0.01, * P ≤0.05.

Journal: Nucleic acids research

Article Title: Tetrameric INTS6-SOSS1 complex facilitates DNA:RNA hybrid autoregulation at double-strand breaks.

doi: 10.1093/nar/gkae937

Figure Lengend Snippet: Figure 5. INTS6 is required for SETX recruitment to DSBs and clearance of DNA:RNA hybrids. ( A ) PLA of SETX and γH2AX with or without IR in mock or siINTS6 cells. IR = 10 Gy, samples were collected 10 min post-IR. Left: Representative confocal microscopy images; right: quantification of left, error bar = mean ± SD, significance was determined using non-parametric Mann–Whitney test. **** P ≤0.0 0 01. Scale bar = 10 μm. Single antibodies were used as a negative control. ( B, C ) Bar charts showing SETX ChIP signals over DS1 ( B ) and DS2 ( C ) loci in the presence or absence of INTS6. n = 3. Error bar = mean ± SD, significance was determined using Student’s t -test, unpaired, ** P ≤0.01. ( D ) PLA of S9.6 and γH2AX with or without IR in mock or siINTS6 cells. IR = 10 Gy, samples were collected 10 min post-IR. Left: Representative confocal microscopy images; right: quantification of top, error bar = mean ± SD, significance was determined using non-parametric Mann–Whitney test. **** P ≤0.0 0 01. Scale bar = 10 μm. Single antibodies were used as a negative control. ( E, F ) Bar charts showing DRIP signals over DS1 ( E ) and DS2 ( F ) loci in the presence or absence of INTS6 and RNaseH1 treatment. n = 3. Error bar = mean ± SD, significance was determined using unpaired Student’s t -test, *** P ≤0.001, ** P ≤0.01, * P ≤0.05.

Article Snippet: The Open Reading Frame (ORF) of INTS6 was cloned into plasmid 438C (pFastBac His6 MBP Asn10 TEV cloning vector with BioBrick Polypromoter LIC subcloning, Addgene plasmid #55220).

Techniques: Confocal Microscopy, MANN-WHITNEY, Negative Control

Figure 6. INTS6-dependent accumulation of DARTs correlates with DNA:RNA hybrids at DSBs. ( A ) Heatmaps show the chrRNA-seq coverage in siINTS6 + 4OHT, siCtrl + 4OHT, -4OHT, SETX + 4OHT ChIP-seq co v erage and S9.6 + 4OHT DRIP-seq co v erage across cut Asi SI sites (details in Supplementary Table S4 ) sorted by their cleavage efficiency. The reference genome is human hg19. ( B ) Box plots show log2 fold change of total chrRNA-seq co v erage of INTS6 knockdown with damage induction compared to control with damage induction in 500 bp bins centered at DSB, 1, 2 and 3 kb a w a y from DSB (cut Asi SI sites, details in Supplementary Table S4 ). The reference genome is human hg19. ( C ) Metagene plots show S9.6 DRIP-seq co v erage and SETX co v erage upon damage induction along with chrRNA-seq sense and antisense co v erage around 2.5 kb flank region of cut Asi SI sites (details in Supplementary Table S4 ). The reference genome is human hg19. ( D ) Metagene plots show S9.6 DRIP-seq coverage and SETX co v erage upon damage induction along with chrRNA-seq sense and antisense co v erage around 2.5 kb flank region of uncut Asi SI sites (n = 20). The reference genome is human hg19. ( E, F ) R epresentativ e snapshots of individual genes showing DRIP-seq co v erage and SETX co v erage upon damage induction along with sense and antisense chrRNA-seq co v erage in siINTS6 and control with damage induction around 2.5 kb flank region of Asi SI cut. The specific loci information is listed on top of the snapshot respectively. The reference genome is human hg19.

Journal: Nucleic acids research

Article Title: Tetrameric INTS6-SOSS1 complex facilitates DNA:RNA hybrid autoregulation at double-strand breaks.

doi: 10.1093/nar/gkae937

Figure Lengend Snippet: Figure 6. INTS6-dependent accumulation of DARTs correlates with DNA:RNA hybrids at DSBs. ( A ) Heatmaps show the chrRNA-seq coverage in siINTS6 + 4OHT, siCtrl + 4OHT, -4OHT, SETX + 4OHT ChIP-seq co v erage and S9.6 + 4OHT DRIP-seq co v erage across cut Asi SI sites (details in Supplementary Table S4 ) sorted by their cleavage efficiency. The reference genome is human hg19. ( B ) Box plots show log2 fold change of total chrRNA-seq co v erage of INTS6 knockdown with damage induction compared to control with damage induction in 500 bp bins centered at DSB, 1, 2 and 3 kb a w a y from DSB (cut Asi SI sites, details in Supplementary Table S4 ). The reference genome is human hg19. ( C ) Metagene plots show S9.6 DRIP-seq co v erage and SETX co v erage upon damage induction along with chrRNA-seq sense and antisense co v erage around 2.5 kb flank region of cut Asi SI sites (details in Supplementary Table S4 ). The reference genome is human hg19. ( D ) Metagene plots show S9.6 DRIP-seq coverage and SETX co v erage upon damage induction along with chrRNA-seq sense and antisense co v erage around 2.5 kb flank region of uncut Asi SI sites (n = 20). The reference genome is human hg19. ( E, F ) R epresentativ e snapshots of individual genes showing DRIP-seq co v erage and SETX co v erage upon damage induction along with sense and antisense chrRNA-seq co v erage in siINTS6 and control with damage induction around 2.5 kb flank region of Asi SI cut. The specific loci information is listed on top of the snapshot respectively. The reference genome is human hg19.

Article Snippet: The Open Reading Frame (ORF) of INTS6 was cloned into plasmid 438C (pFastBac His6 MBP Asn10 TEV cloning vector with BioBrick Polypromoter LIC subcloning, Addgene plasmid #55220).

Techniques: ChIP-sequencing, Knockdown, Control

Figure 7. INTS6 is required for efficient DNA damage repair. ( A ) Left: Representative images of the clonogenic assay in control and INTS6 knockdown cells. The cells were stained and counted after 10 days of growing. Right: Quantification of left. * P ≤0.05, ** P ≤0.01. ( B ) MTT assay to show cell viability (%) at indicated time points in siCtrl and siINTS6 cells with or without 2 Gy IR treatment. Error bar = mean ± SD, significance was determined using unpaired Student’s t -test, * P ≤0.05. ( C ) Left: Drawing of DR-GFP HR reporter strategy. Right: Bar chart shows the efficiency of HR repair in DR-GFP HeLa reporter cells, as measured by FACS. BRCA1 knockdown was used as the positive control. ****P ≤0.0 0 01, ***P ≤0.001. ( D ) Top: R epresentativ e images of comet assay in siCtrl, siINTS6, siRAD51 and si53BP1 cells. siRAD51 and si53BP1 cells were used as positive controls. IR = 5 Gy. Error bar = 100 μm. Bottom: Quantification of top. **** P ≤0.0001, ** P ≤0.01. ( E ) Model: INTS6 as part of tetrameric SOSS1 complex binds to DNA:RNA hybrids at DSBs and recruits PP2A to dephosphorylate RNAPII. Depletion of INTS6 results in increased levels of DARTs and DNA:RNA hybrids. INTS6 interacts with SETX and is required for its recruitment to damaged sites. SETX, in turn, resolves DNA:RNA hybrids at DSBs facilitating their INTS6-dependent autoregulation. Image was created with Biorender.

Journal: Nucleic acids research

Article Title: Tetrameric INTS6-SOSS1 complex facilitates DNA:RNA hybrid autoregulation at double-strand breaks.

doi: 10.1093/nar/gkae937

Figure Lengend Snippet: Figure 7. INTS6 is required for efficient DNA damage repair. ( A ) Left: Representative images of the clonogenic assay in control and INTS6 knockdown cells. The cells were stained and counted after 10 days of growing. Right: Quantification of left. * P ≤0.05, ** P ≤0.01. ( B ) MTT assay to show cell viability (%) at indicated time points in siCtrl and siINTS6 cells with or without 2 Gy IR treatment. Error bar = mean ± SD, significance was determined using unpaired Student’s t -test, * P ≤0.05. ( C ) Left: Drawing of DR-GFP HR reporter strategy. Right: Bar chart shows the efficiency of HR repair in DR-GFP HeLa reporter cells, as measured by FACS. BRCA1 knockdown was used as the positive control. ****P ≤0.0 0 01, ***P ≤0.001. ( D ) Top: R epresentativ e images of comet assay in siCtrl, siINTS6, siRAD51 and si53BP1 cells. siRAD51 and si53BP1 cells were used as positive controls. IR = 5 Gy. Error bar = 100 μm. Bottom: Quantification of top. **** P ≤0.0001, ** P ≤0.01. ( E ) Model: INTS6 as part of tetrameric SOSS1 complex binds to DNA:RNA hybrids at DSBs and recruits PP2A to dephosphorylate RNAPII. Depletion of INTS6 results in increased levels of DARTs and DNA:RNA hybrids. INTS6 interacts with SETX and is required for its recruitment to damaged sites. SETX, in turn, resolves DNA:RNA hybrids at DSBs facilitating their INTS6-dependent autoregulation. Image was created with Biorender.

Article Snippet: The Open Reading Frame (ORF) of INTS6 was cloned into plasmid 438C (pFastBac His6 MBP Asn10 TEV cloning vector with BioBrick Polypromoter LIC subcloning, Addgene plasmid #55220).

Techniques: Clonogenic Assay, Control, Knockdown, Staining, MTT Assay, Positive Control, Single Cell Gel Electrophoresis

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