atrx h300 antibody (Santa Cruz Biotechnology)
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Santa Cruz Biotechnology
atrx h300 antibody
Atrx H300 Antibody, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/h300+antibody/anti+gr/pmc12264150-447-7-9
Average 90 stars, based on 1 article reviews
Atrx H300 Antibody, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/h300+antibody/anti+gr/pmc12264150-447-7-9
Average 90 stars, based on 1 article reviews
atrx h300 antibody - by Bioz Stars,
2026-09
90/100 stars
Images
1) Product Images from "A chromatin-remodeling-independent role for ATRX in protecting centromeric cohesion"
Article Title: A chromatin-remodeling-independent role for ATRX in protecting centromeric cohesion
Journal: The EMBO Journal
doi: 10.1038/s44318-025-00465-6
Figure Legend Snippet: ( A ) Schematic diagram illustrating sister chromatid cohesion mediated by the ring-shaped cohesin complex. ( B ) Nocodazole-arrested mitotic HeLa cells stably expressing Myc-Pds5A or Myc-Pds5B were immunoprecipitated using anti-Myc beads or control IgG, followed by immunoblotting for ATRX and the Myc tag. ( C ) HEK-293T cells co-expressing ATRX-GFP with Myc-Pds5A or Myc-Pds5B were immunoprecipitated using anti-GFP beads or control IgG, followed by immunoblotting for GFP and the Myc tag. ( D ) HEK-293T cells expressing SFB-ATRX were immunoprecipitated using anti-Flag beads or control IgG, followed by immunoblotting for the Flag tag, Pds5B, SMC1A, and RAD21.
Techniques Used: Stable Transfection, Expressing, Immunoprecipitation, Control, Western Blot, FLAG-tag
Figure Legend Snippet: ( A , B ) HEK-293T cells expressing Myc-Pds5B were subjected to immunoprecipitation using anti-Myc beads or control IgG, followed by mass spectrometry ( A ) or immunoblotting with antibodies against the Myc tag, ATRX, and RAD21 ( B ). ( C , D ) HEK-293T cells expressing Myc-Pds5B and/or ATRX-GFP were immunoprecipitated using anti-Myc ( C ) or anti-GFP ( D ) beads, followed by immunoblotting for the Myc tag, GFP, GAPDH, or α-Tubulin as indicated. ( E ) HeLa cells stably expressing ATRX-GFP were transfected with control or Pds5B-targeting siRNA. Forty-eight hours post-transfection, cells were immunoprecipitated using anti-GFP beads and analyzed by immunoblotting for GFP, Pds5B, SMC1A, SMC3, and RAD21. ( F – I ) HEK-293T cells co-expressing ATRX-GFP and Myc-tagged SMC1A ( F ), SMC3 ( G ), RAD21 ( H ), or SA2 ( I ) were immunoprecipitated using anti-GFP beads and immunoblotted for GFP and the Myc tag. Myc-Pds5B co-transfection was used as a positive control. .
Techniques Used: Expressing, Immunoprecipitation, Control, Mass Spectrometry, Western Blot, Stable Transfection, Transfection, Cotransfection, Positive Control
Figure Legend Snippet: ( A ) HEK-293T cells co-expressing ATRX-GFP and the indicated Pds5B-Flag truncations were immunoprecipitated using anti-GFP beads and analyzed by immunoblotted for GFP, the Flag tag, and α-Tubulin. ( B ) Schematic representation of GST-Pds5B (1–300) binding to ATRX mutants. ( C – G ) Pull-down assays of the indicated ATRX-GFP truncations using GST or GST-Pds5B (1–300), followed by immunoblotting for GFP and Coomassie Brilliant Blue (CBB) staining. ( H ) HEK-293T cells expressing ATRX-GFP (WT or Y1419A) and/or Myc-Pds5B were immunoprecipitated using anti-GFP beads and analyzed by immunoblotting for GFP, the Myc tag, and α-Tubulin. ( I ) Alignment of ATRX sequences from multiple species surrounding the conserved RSYK motif. ( J ) AlphaFold3 model of the ATRX (RSYKQ) peptide bound to Pds5B (1–300). ATRX (RSYKQ) is shown as sticks, and Pds5B as an electrostatic surface. The interaction between Y1419 of ATRX and Pds5B residues F88, A92, V138, and I143 is highlighted. .
Techniques Used: Expressing, Immunoprecipitation, FLAG-tag, Binding Assay, Western Blot, Staining
Figure Legend Snippet: ( A ) HEK-293T cells expressing ATRX-GFP were subjected to pull-down using GST or GST-Pds5B (1–300), followed by immunoblotting for GFP and CBB staining. ( B – E ) Recombinant GFP-ATRX (1394–1443)-2xStrep (wild-type or mutants) proteins were subjected to pull-down using GST or GST-Pds5B (1–300), followed by immunoblotting for GFP and CBB staining. ( F ) HEK-293T cells expressing Myc-Pds5B were subjected to pull-down using GFP-2xStrep or GFP-ATRX (1394–1443)-2xStrep (WT or Y1419A), followed by immunoblotting for the Myc tag, and CBB staining. ( G ) Cartoon presentation of the structure for ATRX (RSYKQ) binding to Pds5B (1–300). ATRX and Pds5B residues are shown in yellow and gray, respectively. The binding details of ATRX residues R1417, S1418, Y1419, K1420, and Q1421 with Pds5B residues Q47, F88, A92, V138, and I143 are depicted. ( H , I ) HEK-293T cells expressing ATRX-GFP were subjected to pull-down using GST or GST-Pds5B (1–300) (WT or mutants), followed by immunoblotting for GFP and CBB staining. ( J ) HEK-293T cells expressing ATRX-GFP (WT, R1417A or R1417E) were subjected to pull-down using GST or GST-Pds5B (1–300), followed by immunoblotting for GFP and CBB staining.
Techniques Used: Expressing, Western Blot, Staining, Recombinant, Binding Assay
Figure Legend Snippet: ( A – C ) HeLa cells were transfected with control or ATRX siRNA duplexes. Forty-eight hours post-transfection, cells were either analyzed by immunoblotting for ATRX and GAPDH ( A ) or treated with MG132 and then fixed at the indicated time points for DAPI staining. The percentage of mitotic cells in prometaphase, metaphase, and pseudo-metaphase with misaligned chromosomes was quantified from more than 560 cells per condition across three independent experiments. p values from left to right: ns p = 1.04E−01, ns p = 0.2203, * p = 4.93E−02, * p = 4.10E−02, ** p = 2.40E−03, * p = 0.018, ** p = 5.50E−03, ** p = 4.60E−03, ** p = 3.60E−03, **** p = 3.50E−05, **** p = 5.00E−06, **** p = 3.50E−05, **** p = 3.00E−06, **** p = 6.70E−05, **** p = 2.00E−06 ( B ). Representative images are shown, with arrows indicating misaligned chromosomes ( C ). ( D ) HeLa cells stably expressing H2B-GFP were transfected with control or ATRX siRNA and synchronized in early S phase with thymidine. Seven hours after thymidine release, cells were treated with STLC for 5 h. Mitotic cells were then collected and released into MG132-containing medium for live imaging of mitosis progression. The time from STLC washout to metaphase alignment and from metaphase to chromosome scattering or the imaging endpoint was recorded. See Movies and . ( E , F ) HeLa cells were transfected with control or ATRX siRNAs. Forty-eight hours post-transfection, cells were treated with MG132 for 8 h. Mitotic cells were collected for chromosome spread preparation, stained with CENP-C antibodies and DAPI. The percentage of mitotic cells with predominantly separated or unseparated sister chromatids was determined from more than 930 cells per condition across three independent experiments. p values from left to right: ** p = 3.04E−03, *** p = 4.16E−04, ** p = 4.53E−03 ( E ). Representative images are shown ( F ). ( G , H ) HeLa cells were transfected as above, then fixed and stained with DAPI. The percentage of cells displaying lagging chromosomes was quantified from more than 490 cells per condition across three independent experiments. p values from left to right: *** p = 8.13E−04, ** p = 5.62E−03, * p = 2.54E−02 ( G ). Representative images are shown, with arrows indicating lagging chromosomes ( H ). ( I ) HeLa cells were transfected as described above and treated with STLC for 5 h. Two hours after STLC release, cells were fixed and stained with DAPI. The percentage of anaphase cells with lagging chromosomes was quantified from more than 700 cells per condition across three independent experiments. p values from left to right: ** p = 2.86E−03, ** p = 4.19E−03, *** p = 4.63E−04. Data information: Statistics were performed using unpaired Student’s t-test ( B , E , G , I ). Means and standard deviations (SDs) are shown ( B , E , G , I ). ns, no significance. Scale bars, 10 µm ( C , F , H ). .
Techniques Used: Transfection, Control, Western Blot, Staining, Stable Transfection, Expressing, Imaging
Figure Legend Snippet: ( A ) HeLa cells stably expressing H2B-GFP were transfected with control or ATRX siRNA, followed by live imaging of mitosis progression. Selected movie frames are shown. Time is indicated in hours: minutes. Arrows point to scattering chromosomes. Related to Fig. . ( B , C ) HeLa cells were transfected with control or ATRX siRNA. Forty-eight hours post-transfection, cells were treated with MG132 for 2 h, and mitotic chromosome spreads were stained for CENP-C and DAPI. Representative super-resolution images are shown ( B ). Cell lysates were analyzed by immunoblotting for ATRX and GAPDH ( C ). ( D – G ) HeLa cells transfected as described above were treated with nocodazole for 3 h, and mitotic chromosome spreads were stained for CENP-C and DAPI. The inter-KT distance was measured on more than 2000 chromosomes from 95 cells per condition across two independent experiments, and the means and ranges are plotted ( D ). Inter-KT distances from two individual experiments are shown. p values from left to right: **** p < 1.00E−15, **** p < 1.00E−15, **** p < 1.00E−15 ( E ). **** p < 1.00E−15, **** p < 1.00E−15, **** p < 1.00E−15 ( F ). Representative images are shown ( G ). ( H – L ) HeLa cells were transfected with the indicated siRNAs, co-transfected with NLS-dCas9-EGFP-expressing vector and the sgRNA-expressing vector, and synchronized using double thymidine treatment. Eight hours after release from thymidine, cells were stained for GFP and DAPI. Representative images are shown ( H ). The sister loci distance was measured in more than 450 cells per condition across two independent experiments, and the means and ranges are plotted ( I ). Sister loci distances from two individual experiments are shown. p values from left to right: ns p = 6.12E−01, **** p < 1.00E−15 ( J ). ns p = 3.95E−01, **** p < 1.00E−15 ( K ). Cell lysates were analyzed by immunoblotting for ATRX, Sororin, and GAPDH ( L ). Data information: Statistics were performed using unpaired Student’s t-test ( E , F , J , K ). Means and SDs are shown ( E , F , J , K ). ns, no significance. Scale bars, 10 µm ( A , B , G , H ).
Techniques Used: Stable Transfection, Expressing, Transfection, Control, Imaging, Staining, Western Blot, Plasmid Preparation
Figure Legend Snippet: ( A ) HeLa cells were transfected with control or ATRX siRNAs. Forty-eight hours post-transfection, cells were treated with MG132 for 2 h. Mitotic cells were collected for chromosome spread preparation and subjected to immunofluorescence staining with ATRX and CENP-C antibodies, and DAPI. ( B ) HeLa and HP1α/γ DKO cells were treated with MG132 for 2 h, and mitotic chromosome spreads were stained for ATRX, ACA, and DAPI. ( C , D ) HeLa cells were transfected with control or ATRX siRNA. Forty-eight hours post-transfection, cells were treated with MG132 for 3 h, and mitotic chromosome spreads were stained for ATRX, ACA, and DAPI. Representative super-resolution images are shown ( C ). The number of centromeric ATRX foci was quantified from more than 299 chromosomes per condition across two independent experiments ( D ). ( E ) HeLa cells stably expressing siRNA-resistant ATRX-GFP (WT or Y1419A) were analyzed by immunoblotting for ATRX, GFP, and GAPDH. ( F ) HeLa cells stably expressing ATRX-GFP (WT or Y1419A) were arrested in mitosis with nocodazole, immunoprecipitated using anti-GFP beads, and analyzed by immunoblotting for Pds5B, HP1α, and GFP. S. exp., short exposure; L. exp., long exposure. ( G , H ) HeLa cells stably expressing ATRX-GFP (WT or Y1419A) were treated with MG132 for 3 h, and mitotic chromosome spreads were stained for GFP, ACA, and DAPI. Representative super-resolution images are shown ( G ). The number of centromeric ATRX foci was quantified from more than 46 chromosomes ( H ). Data information: Scale bars, 10 µm ( A , B , C , G ). .
Techniques Used: Transfection, Control, Immunofluorescence, Staining, Stable Transfection, Expressing, Western Blot, Immunoprecipitation
Figure Legend Snippet: ( A ) HeLa cells stably expressing Myc-Pds5B were synchronized using double thymidine treatment. Eight hours after release from thymidine, cells were stained for ATRX, the Myc tag, ACA, and DAPI. Representative images are shown. ( B ) HeLa cells were synchronized in G2 phase as described above and stained for ATRX, HP1α, ACA, and DAPI. Representative images are shown. ( C ) HeLa and HP1α/γ DKO cells were stained for ATRX, HP1α, CENP-C, and DAPI. Representative images are shown. ( D , E ) HeLa cells were treated with nocodazole for 3 h or with MG132 for 1, 3, and 6 h, and mitotic chromosome spreads were stained for ATRX, ACA, and DAPI. The fluorescence intensity ratio of ATRX to ACA was quantified on more than 230 chromosomes from 15–20 cells. p values from left to right: ns p = 6.59E−02, ns p = 1.91E−01, ns p = 9.35E−01, ns p = 2.07E−01 ( D ). Representative images are shown ( E ). ( F – H ) HeLa cells stably expressing Sororin-GFP were treated with nocodazole for 3 h or with MG132 for 1, 3, and 6 h, and mitotic chromosome spreads were stained for GFP, ACA, and DAPI. The fluorescence intensity ratio of Sororin-GFP to ACA was quantified on more than 195 chromosomes from 15 to 20 cells. p values from left to right: **** p < 1E−15, **** p < 1E−15, **** p < 1E−15, **** p < 1E−15 ( F ). Representative images are shown ( G ). Cell lysates were analyzed by immunoblotting for GFP and α-Tubulin ( H ). Data information: Statistics were performed using unpaired Student’s t-test ( D , F ). Means and SDs are shown ( D , F ). N.S., no significance. Scale bars, 10 µm ( A , B , C , E , G ).
Techniques Used: Stable Transfection, Expressing, Staining, Fluorescence, Western Blot
Figure Legend Snippet: ( A ) HeLa cells were transfected with control or ATRX siRNAs. Forty-eight hours post-transfection, cells were analyzed by immunoblotting for ATRX, RAD21, SMC1A, SMC3, SA2, Pds5B, Sororin, Wapl, and GAPDH. ( B , C ) HeLa and HeLa cells stably expressing ATRX-GFP (WT or Y1419A) were transfected as described above, treated with nocodazole for 3 h, and mitotic chromosome spreads were stained for GFP, ACA, and DAPI. The inter-KT distance was measured on more than 1750 chromosomes from 80 cells per condition across two independent experiments. p values from left to right: **** p < 1.00E−15. ns p = 1.01E−01, **** p < 1.00E−15 ( B ). **** p < 1.00E−15 ( J ). ns p = 5.10E−01, **** p < 1.00E−15 ( C ). Related to Fig. . ( D , E ) HeLa and HeLa cells stably expressing ATRX-GFP (WT or Y1419A) were transfected with control or ATRX siRNA. Forty-eight hours post-transfection, cells were subjected to mitotic index analysis. The mitotic index was determined from more than 940 cells per condition across two independent experiments ( D ). Representative images of cells are shown, with round cells classified as mitotic ( E ). ( F ) HeLa and HeLa cells stably expressing ATRX-GFP (WT or Y1419A) transfected as described above were treated with STLC for 5 h. Two hours after STLC release, cells were fixed and stained with DAPI. The percentage of anaphase cells with lagging chromosomes was quantified from more than 560 cells per condition across three independent experiments. p values from left to right: **** p = 5.30E−05. **** p = 5.00E−05, ns p = 9.92E−01, ns p = 2.09E−01. **** p = 2.44E−04. **** p = 2.29E−04. Data information: Statistics were performed using unpaired Student’s t-test ( B , C , F ). Means and SDs are shown ( B , C , F ). ns, no significance. Scale bars, 10 µm ( E ).
Techniques Used: Transfection, Control, Western Blot, Stable Transfection, Expressing, Staining
Figure Legend Snippet: ( A – C ) HeLa and HeLa cells stably expressing ATRX-GFP (WT or Y1419A) were transfected with control or ATRX siRNAs. Forty-eight hours post-transfection, cells were treated with MG132 for 8 h, and mitotic chromosome spreads were stained for GFP, ACA, and DAPI. The percentage of mitotic cells exhibiting predominantly separated or unseparated sister chromatids was quantified from more than 500 cells per condition across three independent experiments. p values from left to right: *** p = 8.95E−04, *** p = 7.60E−04, ns p = 3.31E−01, ns p = 7.75E−02, *** p = 3.77E−04, **** p = 9.60E−05 ( A ). Representative images are shown ( B ). Cell lysates were immunoblotted for ATRX, GFP, and GAPDH ( C ). ( D , E ) Cells transfected with siRNAs as described above were treated with nocodazole for 3 h, and mitotic chromosome spreads were stained for GFP, ACA, and DAPI. The inter-KT distance was measured on more than 1750 chromosomes from 80 cells per condition across two independent experiments (see Fig. ). Means and ranges are plotted ( D ). Representative images are shown ( E ). ( F ) Cells transfected with siRNAs as described above were treated with MG132 and fixed at the indicated time points for DAPI staining. The percentages of mitotic cells in prometaphase, metaphase, and pseudo-metaphase exhibiting misaligned chromosomes were determined from more than 620 cells per condition across three independent experiments. p values from left to right: ns p = 1.40E−01. ns p = 6.74E−02, ** p = 1.71E−03, ** p = 1.48E−03. ns p = 6.43E−02. ** p = 6.81E−03, *** p = 4.86E−04, **** p = 4.70E−05 (HeLa). ns p = 5.39E−02. * p = 1.32E−02, *** p = 6.11E−04, *** p = 5.09E−04. * p = 2.47E−02. * p = 2.26E−02, ** p = 1.60E−03, ** p = 1.30E−03 (ATRX-GFP). ns p = 1.47E−01. * p = 1.99E−02, ** p = 1.64E−03, ** p = 1.25E−03. * p = 4.43E−02. ** p = 2.78E−03, *** p = 1.38E−04, **** p = 4.25E−07 (ATRX-Y1419A-GFP). ( G , H ) Cells transfected with siRNAs as described above were fixed and stained with DAPI. The percentage of anaphase cells exhibiting lagging chromosomes was determined from more than 530 cells per condition across three independent experiments. p values from left to right: ** p = 1.69E−03, *** p = 1.86E−04, ns p = 5.64E−01, ns p = 2.70E−01, ** p = 1.06E−03, ** p = 1.16E−03 ( G ). Representative images are shown, with arrows indicating lagging chromosomes ( H ). Data information: Statistics were performed using unpaired Student’s t-test ( A , F , G ). Means and SDs are shown ( A , F , G ). ns, no significance. Scale bars, 10 µm ( B , E , H ). .
Techniques Used: Stable Transfection, Expressing, Transfection, Control, Staining
Figure Legend Snippet: ( A ) HeLa cells stably expressing CB-GFP or CB-ATRX (1394–1443)-GFP (WT or Y1419A) were analyzed by immunoblotting for GFP, ATRX, and α-Tubulin. ( B – D ) HeLa cells stably expressing the indicated proteins were transfected with control or ATRX siRNA. Forty-eight hours post-transfection, cells were treated with MG132 for 8 h, and mitotic chromosome spreads were stained with DAPI. The percentage of mitotic cells exhibiting predominantly separated or unseparated sister chromatids was quantified from more than 770 cells per condition across three independent experiments. p values from left to right: *** p = 6.69E−04, ns p = 5.30E−01, ** p = 1.04E−03 ( B ). Representative images are shown ( C ). Cell lysates were analyzed by immunoblotting for GFP, ATRX, and α-Tubulin ( D ). ( E – H ) Cells transfected as described above were treated with nocodazole for 3 h, and mitotic chromosome spreads were stained for CENP-A and DAPI. The inter-KT distance was measured on more than 2000 chromosomes from 90 cells per condition across two independent experiments, and the means and ranges are plotted ( E ). Inter-KT distances from two individual experiments are shown. p values from left to right: **** p < 1.00E−15, * p = 3.62E−02, **** p = 7.27E−06 ( F ). **** p < 1.00E−15, ns p = 1.11E−01, **** p < 1.00E−15 ( G ). Representative images are shown ( H ). ( I ) Cells transfected as described above were treated with MG132 and fixed at the indicated time points for DAPI staining. The percentages of mitotic cells in prometaphase, metaphase, and pseudo-metaphase with misaligned chromosomes were determined from more than 670 cells per condition across three independent experiments. p values from left to right: ns p = 6.74E−02. ns p = 6.02E−02, ** p = 3.47E−03, ** p = 2.39E−03. * p = 2.21E−02. ** p = 6.22E−03, *** p = 3.34E−04, **** p = 6.14E−07 (CB-GFP). ** p = 1.20E−03. * p = 1.1E−02, ** p = 1.21E−03, ** p = 1.55E−03. * p = 4.76E−02. * p = 1.50E−02, *** p = 1.25E−04, *** p = 1.10E−04 (CB-ATRX (1394–1443)-GFP). ns p = 1.23E−01. * p = 3.33E−02, ** p = 1.15E−03, *** p = 6.70E−04. * p = 1.36E−02. ** p = 2.20E−03, **** p = 3.70E−05, **** p = 6.32E−06 (CB-ATRX (1394–1443)-GFP-Y1419A). ( J ) Cells transfected as described above were fixed and stained with DAPI. The percentage of anaphase cells exhibiting lagging chromosomes was quantified from more than 650 cells per condition across three independent experiments. p values from left to right: **** p = 4.40E−05, * p = 1.10E−02, **** p = 3.90E−05. Data information: Statistics were performed using unpaired Student’s t-test ( B , F , G , I , J ). Means and SDs are shown ( B , F , G , I , J ). ns, no significance. Scale bars, 10 µm ( C , H ). .
Techniques Used: Stable Transfection, Expressing, Western Blot, Transfection, Control, Staining
Figure Legend Snippet: ( A – C ) HeLa cells were transfected with control siRNA, ATRX siRNA, and/or Wapl siRNA. Forty-eight hours post-transfection, cells were treated with MG132 for 8 h, and mitotic chromosome spreads were stained for CENP-C and DAPI. The percentage of mitotic cells exhibiting predominantly separated or unseparated sister chromatids was quantified from 1000 cells per condition across three independent experiments. p values from left to right: *** p = 1.05E−04, * p = 4.10E−02 ( A ). Representative images are shown ( B ). Cell lysates were analyzed by immunoblotting for ATRX, Wapl, and GAPDH ( C ). ( D – G ) HeLa cells transfected as described above were treated with nocodazole for 3 h, and mitotic chromosome spreads were stained for CENP-C and DAPI. The inter-KT distance was measured on more than 2000 chromosomes from 95 cells per condition across two independent experiments, and the means and ranges are plotted ( D ). Inter-KT distances from two individual experiments are shown. p values from left to right: **** p < 1.00E−15, **** p < 1.00E−15, ns p = 8.92E−02 ( E ). **** p < 1.00E−15, **** p < 1.00E−15, ns p = 4.73E−02 ( F ). Representative images are shown ( G ). ( H, I ) HeLa cells transfected as described above were treated with MG132 for 2 h, and mitotic chromosome spreads were stained for ATRX, ACA, and DAPI. Representative images are shown ( H ). Cell lysates were analyzed by immunoblotting for Wapl and α-Tubulin ( I ). ( J ) HeLa cells transfected as described above were treated with MG132 and fixed at the indicated time points for DAPI staining. The percentages of mitotic cells in prometaphase, metaphase, and pseudo-metaphase exhibiting misaligned chromosomes were determined from more than 700 cells per condition across three independent experiments. p values from left to right: * p = 2.73E−02, ns p = 3.80E−01, ns p = 9.86E−01, ** p = 2.36E−03, ns p = 7.80E−01, ns p = 4.34E−01, *** p = 1.85E−04, ns p = 7.17E−02, ns p = 3.57E−01, *** p = 1.21E−04, ns p = 6.58E−01, ns p = 9.20E−01, **** p = 2.90E−05, ns p = 6.78E−01, ns p = 9.71E−01. Data information: Statistics were performed using unpaired Student’s t-test ( A , E , F , J ). Means and SDs are shown ( A , E , F , J ). ns, no significance. Scale bars, 10 µm ( B , G , H ). .
Techniques Used: Transfection, Control, Staining, Western Blot
Figure Legend Snippet: ( A ) HEK-293T cells expressing Myc-Pds5B were subjected to pull-down with GST or Wapl (1–28)-GST, followed by immunoblotting for the Myc tag and CBB staining. ( B ) Multiple sequence alignment of ATRX, Wapl, Sororin, Haspin, and PD-L1. ( C ) HEK-293T cells expressing Myc-Pds5B were subjected to pull-down with GST or GST-Pds5B (1–300) (WT or mutants), followed by immunoblotting for GFP and CBB staining. ( D ) HEK-293T cells expressing Myc-Pds5B were subjected to pull-down with GST or Wapl (1–28)-GST in the presence of GFP-2xStrep or increasing amounts of GFP-ATRX (1394–1443)-2xStrep (WT or Y1419A), followed by immunoblotting for the Myc tag, GFP, and CBB staining. ( E ) MBP-Pds5B (1–300) was subjected to pull-down with GST or Wapl (1–28)-GST in the presence of GFP-2xStrep or increasing amounts of GFP-ATRX (1394–1443)-2xStrep (WT or Y1419A), followed by immunoblotting for MBP, GFP, and CBB staining. ( F ) HeLa cells stably expressing Myc-Pds5B were immunoprecipitated using anti-Myc beads or control IgG in the presence of GFP-2xStrep or increasing amounts of GFP-ATRX (1394–1443)-2xStrep (WT or Y1419A), followed by immunoblotting for Wapl, GFP, GAPDH, and the Myc tag. S. exp., short exposure; L. exp., long exposure. ( G ) Nocodazole-arrested mitotic HeLa cells were immunoprecipitated using the ATRX antibody or control IgG, followed by immunoblotting for ATRX, Pds5B, and Wapl. ( H ) Nocodazole-arrested mitotic HeLa cells stably expressing Wapl-GFP were immunoprecipitated using anti-GFP beads or control IgG, followed by immunoblotting for GFP, Pds5B, and ATRX. ( I ) HeLa cells stably expressing Myc-Pds5B, synchronized in G2-phase by double thymidine block/release or arrested in mitosis by nocodazole, were immunoprecipitated using anti-Myc beads or control IgG, followed by immunoblotting for the Myc tag, Wapl, Sororin, ATRX, and GAPDH. .
Techniques Used: Expressing, Western Blot, Staining, Sequencing, Stable Transfection, Immunoprecipitation, Control, Blocking Assay
Figure Legend Snippet: ( A – C ) HeLa and Haspin KO cells were transfected with control or ATRX siRNA. Forty-eight hours post-transfection, cells were treated with MG132 for 2, 4, and 6 h, and mitotic chromosome spreads were stained with DAPI. The percentage of mitotic cells with predominantly separated or unseparated sister chromatids was quantified from more than 720 cells per condition across three independent experiments. p values from left to right: *** p = 8.95E−04, ** p = 2.53E−03, ** p = 4.86E−03, * p = 1.38E−02, *** p = 2.19E−04, **** p = 4.70E−05, *** p = 6.23E−04, ** p = 2.88E−03, *** p = 1.93E−04, *** p = 1.19E−04, *** p = 2.55E−04, *** p = 4.02E−04 ( A ). Representative images are shown ( B ). Cell lysates were analyzed by immunoblotting for ATRX and GAPDH ( C ). ( D ) Cells transfected as described above were treated with MG132 and fixed at the indicated time points for DAPI staining. The percentage of mitotic cells in prometaphase, metaphase, and pseudo-metaphase with misaligned chromosomes was quantified from more than 740 cells per condition across three independent experiments. p values from left to right: ** p = 2.41E−03, ** p = 2.20E−03, ** p = 4.81E−03, ** p = 2.70E−03, ** p = 3.79E−03, ** p = 2.21E−03, ** p = 4.51E−03, ** p = 3.71E−03, *** p = 3.87E−04, *** p = 1.42E−04, **** p = 2.50E−05, *** p = 1.08E−04. ( E ) Cells transfected as described above were fixed and stained with DAPI. The percentage of anaphase cells with lagging chromosomes was determined from more than 650 cells per condition across three independent experiments. p values from left to right: **** p = 3.70E−05, **** p = 5.10E−05, *** p = 8.31E−04, ** p = 2.42E−03. ( F ) Model for the role of ATRX in antagonizing Wapl and maintaining centromeric cohesion during mitosis. ATRX is enriched at the bipartite subdomains of the inner centromere, where it binds to Pds5B to antagonize Wapl binding, thereby preventing Wapl-mediated release of centromeric cohesin. Note that another pool of cohesin, in which Pds5B binds to Haspin, is also present at the inner centromere. Data information: Statistics were performed using unpaired Student’s t-test ( A , D , E ). Means and SDs are shown ( A , D , E ). Scale bars, 10 µm ( B ). .
Techniques Used: Transfection, Control, Staining, Western Blot, Binding Assay
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![Fig. 2. MutSβ deficiency induces R-loop-dependent replication stress. (A to F) <t>MSH3</t> depletion induces replication fork stalling and micronucleation in U2OS cells. (A) Top: Workflow of DNA fiber assays. Bottom: Representative images of replication tracts on DNA fibers of cells transfected with indicated siRNAs. (B) Quantification of the lengths of replication tracts (CldU + IdU) in the images represented in (A) (n ≥ 311). (C) Representative images of symmetric and asymmetric replication tracts of sister forks observed on DNA fiber spreads in (A). (D) Plot of the values of IdU tract length ratio of sister forks (sister fork ratio) obtained for indicated conditions (n ≥ 120). (E) Top: Workflow of cytokinesis-block micronucleus assay. Bottom: Representative images of binucleated cells with or without micronucleus (red arrow). (F) Quantification of the percentage of micronucleus-positive binucleated cells for indicated conditions. Data are means ± SEM (n = 3). (G to K) RNase H1 overexpression rescues replication fork stalling and micronucleation in MSH3-depleted cells. (G) Western blot analysis of extracts of U2OS T-REx [RNH1-GFP] cells transfected with indicated siRNAs and treated with (+) or without (−) doxycycline (Dox; 1 ng/ml) to induce expression of GFP-tagged RNase H1 (RNH1-GFP). (H) Workflow of DNA fiber assays. (I) Quantification of the lengths of DNA replication tracts for indicated conditions (n ≥ 402). (J) Plot of the values of sister fork ratio obtained for indicated conditions (n ≥ 211). (K) Top: Workflow of cytokinesis-block micronucleus assay. Bottom: Quantification of the percentage of micronucleus-positive binucleated cells for indicated conditions. Data are means ± SEM (n = 3). Kruskal-Wallis test followed by Dunn’s multiple comparisons test was used in (B), (D), (I), and (J). ****P < 0.0001. One-way ANOVA with Tukey’s mul- tiple comparisons correction was used in (F) and (K). ***P < 0.001 and *P < 0.05. All DNA fiber experiments were performed three times. Red lines represent median val- ues. Scale bars, 10 μm. ns, not significant.](https://pub-med-unpaywalled-images-cdn.bioz.com/pub_med_ids_ending_with_4687/pm38324687/pm38324687__page3_image1.jpg)
![Fig. 3. MutSβ is required for replication restart at R-loop–stalled replication forks. (A) Top: Workflow of DNA fiber assays with U2OS cells. The PARP inhibitor olaparib [10 μM; PARP inhibition (PARPi)/Pi] was added 2 hours before replication tract labeling and was also present during the labeling. PDS (10 μM) or CPT (100 nM) were added together with IdU. Bottom: Representative images of DNA replication tracts of U2OS cells transfected with control siRNA (siLuc) and treated as indicated. Scale bar, 10 μm. (B) Effect of depletion of MSH3 or MSH6 on replication fork progression in U2OS cells treated as indicated. Scatter plot of the IdU/CldU tract length ratio is shown. At least 328 replication tracts were scored in three independent experiments for each condition. Red lines represent median values. ****P < 0.0001 (Kruskal-Wallis test followed by Dunn’s multiple comparisons test). ns, not significant.](https://pub-med-unpaywalled-images-cdn.bioz.com/pub_med_ids_ending_with_4687/pm38324687/pm38324687__page4_image1.jpg)
