Journal: Cell Cycle

Article Title: S100A11 plays a role in homologous recombination and genome maintenance by influencing the persistence of RAD51 in DNA repair foci

doi: 10.1080/15384101.2016.1220457

Figure Lengend Snippet: S100A11 interacts with RAD51 at sites of DSB repair. (A) HaCaT cells synchronized in S phase were treated with bleomycin (12.5 µg/ml) for 30 min and released in fresh medium for 90 min or were untreated (control) followed by immunostaining with antibodies against S100A11 (green) and γH2AX (red). Nuclear DNA was detected using DAPI (blue). Bar, 10 µm. (B) S phase HaCaT cells that were analyzed by laser scanning microscopy for S100A11 (green) and RAD51 (red) at sites of DNA damage 2 hour after bleomycin treatment. Bar, 10 µm. An area marked by a rectangle (light blue) in the overlay image (merge) is shown enlarged below the respective image. The intensities of the immunofluorescences in one cell derived from the RAD51 signal (red) and the S100A11 signal (green) are shown in a linescan (left side of the enlarged overlay). (C) Quantification of the colocalization of S100A11 with γH2AX (white Whisker box), RAD51 (gray Whisker box) and γH2AX/RAD51 (black Whisker box) in HaCaT cells after DSB induction. Colocalization events were determined in nuclei of cells treated as described in A. Thirty nuclei from 2 independent experiments were analyzed. Data are displayed as mean values (±SD). (D) Co-immunoprecipitation (CoIP) experiments between S100A11 and RAD51. A specific anti-S100A11 antibody precipitated RAD51 from whole cell extracts of S phase HaCaT cells treated with bleomycin (12.5 µg/ml) (lane 4) or untreated S phase cells (lane 6). In a control, an unspecific antibody used in CoIP experiments was unable to precipitate RAD51 from the same extracts (lanes 3 and 5). (E) The authenticity of the RAD51/S100A11 interaction was confirmed by pull-down with overexpressed proteins. U2OS cells were transfected with a plasmid encoding FLAG-S100A11 alone or together with a His-RAD51 encoding plasmid followed by synchronization in S phase and induction of DSBs by bleomycin treatment. Pull-down (pd) was carried out from cell extracts of the transfected cells for His-tagged proteins binding to Talon resins, and immunoblotting was performed using the antibodies as indicated. As loading control (load), 5% of the cell extract was used.

Article Snippet: Anti-S100A11 rabbit polyclonal antibody ( {"type":"entrez-nucleotide","attrs":{"text":"BC001410","term_id":"33876180"}} BC001410 ; Protein Tech Group) at 1:1,000, anti-S100A11 sheep polyclonal antibody (PAS9364; Randox Laboratories Inc.) at 1:2,000, anti-RAD51 rabbit polyclonal antibody (sc-8349; Santa Cruz) at 1:200, anti-RAD51 goat polyclonal antibody (sc-7410; Santa Cruz) at 1:200, anti-GAPDH monoclonal antibody (5174; Cell Signaling) at 1.1,000, normal mouse IgG (500-M00; Pepro Tech Inc.) at 1:1,000, and anti-FLAG mouse monoclonal antibody (F1804; Sigma) at 1:500 were used in coimmunoprecipitation experiments and immunoblotting.

Techniques: Control, Immunostaining, Laser-Scanning Microscopy, Derivative Assay, Whisker Assay, Immunoprecipitation, Transfection, Plasmid Preparation, Binding Assay, Western Blot

Journal: Cell Cycle

Article Title: S100A11 plays a role in homologous recombination and genome maintenance by influencing the persistence of RAD51 in DNA repair foci

doi: 10.1080/15384101.2016.1220457

Figure Lengend Snippet: S100A11 is functionally required for complete DSB repair. (A) Down-regulation of S100A11 in HaCaT cells treated with specific siRNA against S100A11 (lane 2: S100A11 siRNA#6, lane 3: S100A11 siRNA#7) was confirmed by immunoblotting using a specific antibody against S100A11. As control, HaCaT cells transfected with nonspecific control (nsc) siRNA (lane1) were used. GAPDH served as loading control. (B) Experimental setup. HaCaT cells were transfected with the indicated siRNA and subsequently synchronized in S phase by double-thymidine block. DNA damage was induced by treatment with 12.5 µg/ml bleomycin for 30 min. Cells were harvested after different time points (2 and 8 h repair) for immunostaining against S100A11 and γH2AX. (C) γH2AX foci persist in damaged S100A11 knock-down cells. Control represents cells without bleomycin treatment. The number of γH2AX foci per cell is shown as Whisker graphs. Mean values represent 3 independent experiments with n > 30 cells analyzed for each condition. (D) Analysis of the percentage of individual cells possessing γH2AX foci. HaCaT cells were treated as described in B. Mean values represent analysis of n > 90 cells for each condition in 3 independent experiments. (E) RAD51 foci (white) persist upon DSB damage after S100A11 knock-down. HaCaT cells were treated as described in B and analyzed by immunostaining against RAD51 and S100A11. Nuclear DNA was detected using DAPI (blue). Bar, 10 µm. (F) Quantification of percentage of individual cell showing RAD51 foci. Mean values represent analysis of n > 85 cells for each condition in 3 independent experiments. * P < 0.05, *** P < 0.001.

Article Snippet: Anti-S100A11 rabbit polyclonal antibody ( {"type":"entrez-nucleotide","attrs":{"text":"BC001410","term_id":"33876180"}} BC001410 ; Protein Tech Group) at 1:1,000, anti-S100A11 sheep polyclonal antibody (PAS9364; Randox Laboratories Inc.) at 1:2,000, anti-RAD51 rabbit polyclonal antibody (sc-8349; Santa Cruz) at 1:200, anti-RAD51 goat polyclonal antibody (sc-7410; Santa Cruz) at 1:200, anti-GAPDH monoclonal antibody (5174; Cell Signaling) at 1.1,000, normal mouse IgG (500-M00; Pepro Tech Inc.) at 1:1,000, and anti-FLAG mouse monoclonal antibody (F1804; Sigma) at 1:500 were used in coimmunoprecipitation experiments and immunoblotting.

Techniques: Western Blot, Control, Transfection, Blocking Assay, Immunostaining, Knockdown, Whisker Assay

Journal: Cell Cycle

Article Title: S100A11 plays a role in homologous recombination and genome maintenance by influencing the persistence of RAD51 in DNA repair foci

doi: 10.1080/15384101.2016.1220457

Figure Lengend Snippet: S100A11 stimulates the strand exchange activity of RAD51. (A) left panel Scheme of the strand exchange reaction between circular ssDNA and linearized dsDNA. right panel Strand exchange by human RAD51 requires Ca2+. RAD51, derived from 2 distinct purification procedures, (lanes 2–3 and 5–6: 3 µM) was incubated with 24 µM circular ΦX174 ssDNA in strand exchange buffer containing 2 mM of either magnesium or calcium acetate for 15 min at 37°C followed by incubation with 2.4 µM RPA for 5 min and addition of 24 µM linearized ΦX174 dsDNA to initiate strand exchange reaction for 2 h at 37°C. Lane M: constructed joint molecule DNA product derived from ssDNA/dsDNA annealing used as marker (B) left panel S100A11 enhances RAD51-mediated strand exchange. RAD51 (lanes 4–6: 3 µM) alone or with S100A11 (lane 5: 2 µM, lane 6: 4 µM) was incubated as described in (A) in strand exchange buffer containing calcium acetate (2 mM). As negative control, S100A11 (lane 7: 4 µM) was incubated alone. The joint molecule product (jm) was visualized by GelStar staining. right panel Quantification of S100A11-stimulated joint molecule formation by RAD51. Average values of 3 independent experiments are shown with standard derivation. (C) Dialysis of S100A11 abrogated the stimulating effect of S100A11 on RAD51 activity. RAD51 (lanes 4–8 and 10) together with undialyzed S100A11 (lane 5), S100A11 dialyzed in EGTA containing buffer (lanes 7–9), or S100A11 dialyzed in buffer without EGTA (lane 10), was incubated with 24 µM circular ΦX174 ssDNA in strand exchange buffer containing 2 mM of either magnesium or calcium acetate for 15 min at 37°C followed by incubation with 2.4 µM RPA for 5 min and addition of 24 µM linearized ΦX174 dsDNA to initiate strand exchange reaction for 2 h at 37°C. *P < 0.05.

Article Snippet: Anti-S100A11 rabbit polyclonal antibody ( {"type":"entrez-nucleotide","attrs":{"text":"BC001410","term_id":"33876180"}} BC001410 ; Protein Tech Group) at 1:1,000, anti-S100A11 sheep polyclonal antibody (PAS9364; Randox Laboratories Inc.) at 1:2,000, anti-RAD51 rabbit polyclonal antibody (sc-8349; Santa Cruz) at 1:200, anti-RAD51 goat polyclonal antibody (sc-7410; Santa Cruz) at 1:200, anti-GAPDH monoclonal antibody (5174; Cell Signaling) at 1.1,000, normal mouse IgG (500-M00; Pepro Tech Inc.) at 1:1,000, and anti-FLAG mouse monoclonal antibody (F1804; Sigma) at 1:500 were used in coimmunoprecipitation experiments and immunoblotting.

Techniques: Activity Assay, Derivative Assay, Purification, Incubation, Construct, Marker, Negative Control, Staining

Journal: Cell Cycle

Article Title: S100A11 plays a role in homologous recombination and genome maintenance by influencing the persistence of RAD51 in DNA repair foci

doi: 10.1080/15384101.2016.1220457

Figure Lengend Snippet: A S100A11 mutant without Ca2+-binding impairs DSB repair. (A) left panel Immunostaining of U2OS cells for γH2AX in cells expressing recombinant S100A11. Cells expressing S100A11ΔCa display significantly increased γH2AX levels 8 h after DSB induction. Cells transfected with a plasmid encoding S100A11wt or S100A11ΔCa, respectively, were synchronized in S phase, followed by treatment with 12.5 µg/ml bleomycin for 30 min. Cells were harvested 8 h after induction of DNA damage and analyzed by immunostaining against γH2AX (white). Nuclear DNA was detected using DAPI (blue). Bar, 10 µm. right panel Quantification of the percentage of cells showing γH2AX foci after S100A11wt (n = 88) or S100A11ΔCa (n = 99) transfection. The results of 3 independent experiments are presented. Data are shown as the mean ±SD. (B) Recombinant S100A11ΔCa expression increases RAD51 foci persistence. left panel U2OS cells were treated as above and analyzed by immunostaining against RAD51 (white). Nuclear DNA was detected using DAPI (blue). Bar, 10 µM. right panel Quantification of percentage of individual cells showing RAD51 foci. Mean values represent analysis of cells expressing S100A11wt (n = 94) or S100A11ΔCa (n = 99) of 3 independent experiments. (C) Expression of S100A11wt (lane 2) or S100A11ΔCa (lane 3) in pcDNA4-transfected U2OS cells was confirmed by immunoblotting against S100A11. An empty plasmid was used for mock-transfection of U2OS cells (lane 1). GAPDH served as loading control. (D) S100A11ΔCa mutant failed to interact with RAD51. U2OS cells were transfected with a plasmid encoding FLAG-S100A11 (wild-type or ΔCa mutant, respectively) alone or together with a His-RAD51 encoding plasmid followed by synchronization in S phase and induction of DSBs by bleomycin treatment. Pull-down (pd) was carried out from cell extracts of the transfected cells using Talon resins to precipitate His-tagged proteins together with interacting partners. For analysis of the pulled-down interacting proteins, immunoblotting was performed using the antibodies as indicated. As loading control (load), 5% of the cell extract was used. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Anti-S100A11 rabbit polyclonal antibody ( {"type":"entrez-nucleotide","attrs":{"text":"BC001410","term_id":"33876180"}} BC001410 ; Protein Tech Group) at 1:1,000, anti-S100A11 sheep polyclonal antibody (PAS9364; Randox Laboratories Inc.) at 1:2,000, anti-RAD51 rabbit polyclonal antibody (sc-8349; Santa Cruz) at 1:200, anti-RAD51 goat polyclonal antibody (sc-7410; Santa Cruz) at 1:200, anti-GAPDH monoclonal antibody (5174; Cell Signaling) at 1.1,000, normal mouse IgG (500-M00; Pepro Tech Inc.) at 1:1,000, and anti-FLAG mouse monoclonal antibody (F1804; Sigma) at 1:500 were used in coimmunoprecipitation experiments and immunoblotting.

Techniques: Mutagenesis, Binding Assay, Immunostaining, Expressing, Recombinant, Transfection, Plasmid Preparation, Western Blot, Control

Journal: Cell Cycle

Article Title: S100A11 plays a role in homologous recombination and genome maintenance by influencing the persistence of RAD51 in DNA repair foci

doi: 10.1080/15384101.2016.1220457

Figure Lengend Snippet: Downregulation of S100A11 results in restricted recombination capacity, chromosomal aberrations, and reduced cell viability. (A) S100A11 knock-down leads to a significant decrease in SCE. (top) Representative examples show multiple SCEs (left nsc siRNA, right S100A11 siRNA; white arrows) and (bottom) quantification of SCE per metaphase of 2 independent experiments. HaCaT cells with or without S100A11 siRNA were treated with bleomycin (12.5 µg/ml) for 30 min, allowed to recover for 2 h and transferred to medium containing 25 µM BrdU. After 4 h, 0.2 µg/ml Colcemid was added in fresh medium for 18 h to collect cells in metaphase. At least 40 metaphases per data point were analyzed. Error bars represent standard errors of the mean. (B-D) S100A11 knock-down leads to a significant increase of chromosomal abberations. HaCaT cells transfected with S100A11 siRNA or nsc siRNA were treated with 12.5 µg/ml bleomycin for 30 min. After further culture for 20 h, 0.1 µg/ml Colcemid was added for another 6 h to enrich cells in metaphase. The number of chromosome breaks (B) and complex chromosome aberrations (CCA) (C) are shown. Chromosome breaks and CCA were scored in n = 160 metaphase cells for each condition for 2 independent experiments. Error bars represent the standard errors of means. (D) Representative metaphase nuclei containing chromosomal aberrations as detected in S100A11-depleted HaCaT cells. Arrows (red) point to chromatid breaks; arrowheads (blue) point to radial figures. These aberrations are typical for DSBs occurred after DNA replication. (E) S100A11 knock-down leads to a significant loss of cell viability after bleomycin treatment. HaCaT cells transfected with specific S100A11 siRNA or control nsc siRNA for 72 h were treated with bleomycin (12.5 µg/ml) for 30 min. After this, medium was exchanged and the cells cultured for another 10 days. Then, the number of colonies formed was determined using Clono-Counter software in 3 independent experiments. HaCaT cells transfected with the control nsc siRNA without bleomycin treatment were used as control. * P < 0.05, ** P < 0.01, ***P < 0.001.

Article Snippet: Anti-S100A11 rabbit polyclonal antibody ( {"type":"entrez-nucleotide","attrs":{"text":"BC001410","term_id":"33876180"}} BC001410 ; Protein Tech Group) at 1:1,000, anti-S100A11 sheep polyclonal antibody (PAS9364; Randox Laboratories Inc.) at 1:2,000, anti-RAD51 rabbit polyclonal antibody (sc-8349; Santa Cruz) at 1:200, anti-RAD51 goat polyclonal antibody (sc-7410; Santa Cruz) at 1:200, anti-GAPDH monoclonal antibody (5174; Cell Signaling) at 1.1,000, normal mouse IgG (500-M00; Pepro Tech Inc.) at 1:1,000, and anti-FLAG mouse monoclonal antibody (F1804; Sigma) at 1:500 were used in coimmunoprecipitation experiments and immunoblotting.

Techniques: Knockdown, Transfection, Control, Cell Culture, Software

Journal: bioRxiv

Article Title: Xist Repeat A coordinates an assembly of SR proteins to recruit SPEN and induce gene silencing

doi: 10.1101/2025.05.21.655143

Figure Lengend Snippet: (A) Strategy to identify Repeat A-interacting proteins in vitro . (B) Proteins enriched by WT over ΔGC-core Repeat A ( p < 0.05, two-sided t-test, n=3). (C) GO terms of GC-core-specific proteins (top 20). (D) Relative recovery of SR proteins across Repeat A RNAs. Columns, independent experiments. (E) RMCE- Xist WT RIP-seq with antibodies against controls, SPEN, and SR and/or RNA-processing proteins. (F) RIP-seq signal within Repeat A relative to total Xist . Dots, independent experiments. See .

Article Snippet: SBP-tagged proteins were first diluted into room-temp RBNQ Binding Buffer with Random RNA (3 mM MgCl 2 , 25 mM Tris-HCl pH 8.05, 150 mM KCl, 1 mg/mL BSA [Thermo Fisher AM2616], 2 U/mL SUPERase-In [Thermo Fisher Scientific AM2694], 50 nM random 16-nt RNA [IDT, bases randomized by machine-mixing, purified by standard desalting]) to a range of different protein concentrations (∼1-1400 nM).

Techniques: In Vitro

Journal: bioRxiv

Article Title: Xist Repeat A coordinates an assembly of SR proteins to recruit SPEN and induce gene silencing

doi: 10.1101/2025.05.21.655143

Figure Lengend Snippet: (A) Promega Diamond-stained urea-PAGE gels of in-vitro-transcribed, internally biotinylated Repeat A RNAs used for biotinylated RNA pulldown experiments. (B) Representative silver-stained SDS-PAGE gel of proteins recovered by biotinylated Repeat A mutant pulldowns or no-RNA control pulldown. The remaining 85% of each sample was analyzed by quantitative proteomics. (C) Validation of quantitative proteomics results by biotinylated RNA pulldown and western blotting for the proteins RBM15, U2AF2, and SRSF1. (D) Quantification of the proportion of RIP-seq signal in within Repeat A relative to total reads per million. Dots, values from independent experiments.

Article Snippet: SBP-tagged proteins were first diluted into room-temp RBNQ Binding Buffer with Random RNA (3 mM MgCl 2 , 25 mM Tris-HCl pH 8.05, 150 mM KCl, 1 mg/mL BSA [Thermo Fisher AM2616], 2 U/mL SUPERase-In [Thermo Fisher Scientific AM2694], 50 nM random 16-nt RNA [IDT, bases randomized by machine-mixing, purified by standard desalting]) to a range of different protein concentrations (∼1-1400 nM).

Techniques: Staining, In Vitro, SDS Page, Mutagenesis, Control, Quantitative Proteomics, Biomarker Discovery, Western Blot

Journal: bioRxiv

Article Title: Xist Repeat A coordinates an assembly of SR proteins to recruit SPEN and induce gene silencing

doi: 10.1101/2025.05.21.655143

Figure Lengend Snippet: (A) Genotyping PCR for independent clonal lines of RMCE- Xist WT Δ Spen cells or non-targeting gRNA-treated control cells generated by CRISPR-Cas9. PCR1 was performed using primers flanking either side of the deletion; PCR2 and PCR3 were performed using primers flanking the upstream and downstream Spen gRNA target sites. (B) Confirmation of expression of WT and mutant Halo-SPEN-V5 proteins by IP-WB or WB. (C) Xist silencing activity determined by RNA-seq, as in , for non-targeting control cells and Δ Spen cells with and without WT Halo-SPEN-V5 expression. Asterisks, significant differences by two-sided t-test. (D) Quantification of exon 1 aberrant splicing for each RMCE- Xist cell line (dots), as done in . Asterisks, values significantly different from WT by two-sided t-test. (E and F) Quantification of RS/SR and RE/ER dipeptide content in each IDR of SPEN by percentage of residues (E) or overall residue count (F). (G) Gene ontology analysis of the 75 proteins recovered with SPEN RRM1-4 two-fold more than with RRM2-4 in two independent experiments, showing the 10 most significantly enriched terms. (H) Gene ontology analysis of the 44 non-chromosome 1 (chr1) genes with expression significantly correlated with SPEN across tissue types in the TCGA Tumor dataset (FDR < 0.1). An additional 59 chr1 genes were significantly correlated with SPEN in this dataset but were omitted from this analysis because their correlations with SPEN , a gene on chr1, cannot be decoupled from cancer-associated copy-number variations.

Article Snippet: SBP-tagged proteins were first diluted into room-temp RBNQ Binding Buffer with Random RNA (3 mM MgCl 2 , 25 mM Tris-HCl pH 8.05, 150 mM KCl, 1 mg/mL BSA [Thermo Fisher AM2616], 2 U/mL SUPERase-In [Thermo Fisher Scientific AM2694], 50 nM random 16-nt RNA [IDT, bases randomized by machine-mixing, purified by standard desalting]) to a range of different protein concentrations (∼1-1400 nM).

Techniques: Control, Generated, CRISPR, Expressing, Mutagenesis, Activity Assay, RNA Sequencing, Residue

Journal: bioRxiv

Article Title: Xist Repeat A coordinates an assembly of SR proteins to recruit SPEN and induce gene silencing

doi: 10.1101/2025.05.21.655143

Figure Lengend Snippet: (A) Xist abundance across genotypes (RNA-seq). Dots, independent clonal lines. (B) Halo-SPEN-V5-interacting proteins ranked by proportion of peptides recovered. (C) GO terms of SPEN-interacting proteins (top 10). (D) V5 IP, Halo and SRSF1 western blots (no-rescue vs. Halo-SPEN-V5 ESCs). (E) SPEN domains and deletion mutants. IDRs, MobiDB. (F) SPEN-Repeat A association for WT vs. mutants by input-normalized Halo RIP-qPCR. Dots, qPCR triplicates (≥2 independent experiments). Asterisks, significantly different from WT. Crosses, significantly different from RRM1-4. (G) Enrichment of proteins, by class, in RRM1-4 vs. RRM2-4 IP-MS/MS. “(0),” no recovery with RRM2-4. (H) Peptide counts of SR and/or speckle proteins in RRM1-4 or RRM2-4 IP-MS/MS. Dots, independent experiments. (I) Motifs from top 1500 non- Xist , non- Spen Halo RIP-seq peaks across genotypes. (J) Significant SPEN -correlated genes from GTEx (guilt-by-association; p < 0.01 and FDR < 0.1, see Methods). (K) Overlap of significant SPEN -correlated genes (FDR < 0.1) from the GTEx, TCGA Tumor (non-chr1), and TCGA Normal datasets. (L) GO terms of SPEN -correlated genes from GTEx (top 10 and 14 th ). Excepting (J), p -values, two-sided t-tests: (*/†), p < 0.05; (**), p < 0.01. See .

Article Snippet: SBP-tagged proteins were first diluted into room-temp RBNQ Binding Buffer with Random RNA (3 mM MgCl 2 , 25 mM Tris-HCl pH 8.05, 150 mM KCl, 1 mg/mL BSA [Thermo Fisher AM2616], 2 U/mL SUPERase-In [Thermo Fisher Scientific AM2694], 50 nM random 16-nt RNA [IDT, bases randomized by machine-mixing, purified by standard desalting]) to a range of different protein concentrations (∼1-1400 nM).

Techniques: RNA Sequencing, Western Blot, Protein-Protein interactions

Journal: bioRxiv

Article Title: Xist Repeat A coordinates an assembly of SR proteins to recruit SPEN and induce gene silencing

doi: 10.1101/2025.05.21.655143

Figure Lengend Snippet: (A) O-MAP schematic; HRP, horseradish peroxidase. (B) Proteins differentially recovered by O-MAP from WT and Δ Spen cells ( p < 0.05, two-sided t-test, n=4). (C) Repeat A association (input-normalized RIP-qPCR) in non-targeting-control and Δ Spen ESCs derived from RMCE- Xist WT, and RMCE- Xist AWCG+ΔGG cells. Values relative to non-targeting controls. Dots, qPCR triplicates from ≥2 independent experiments. Values above non-targeting-control bars, experiment-averaged IP/input relative to IgG control. (D) RIP-qPCR as in (C), shown relative to IgG. (E) Xist abundance (RNA-seq) in RMCE- Xist WT ESCs treated with non-targeting control siRNA (siNT) and 1000, 25, or 10 ng/mL dox, or protein-targeting siRNAs and 1000 ng/mL dox. Values relative to siNT-1000. Dots, independent experiments. (F) Xist silencing activity as in , for siRNA-treated cells. (G) Median silencing activity vs. Xist abundance. Δ Spen data, , S5C. Dashed line, silencing expected for corresponding levels of Xist in siNT ESCs. P -values, two-sided t-tests relative to WT or non-targeting controls: (*) indicates p < 0.05; (**) indicates p < 0.01. See .

Article Snippet: SBP-tagged proteins were first diluted into room-temp RBNQ Binding Buffer with Random RNA (3 mM MgCl 2 , 25 mM Tris-HCl pH 8.05, 150 mM KCl, 1 mg/mL BSA [Thermo Fisher AM2616], 2 U/mL SUPERase-In [Thermo Fisher Scientific AM2694], 50 nM random 16-nt RNA [IDT, bases randomized by machine-mixing, purified by standard desalting]) to a range of different protein concentrations (∼1-1400 nM).

Techniques: Control, Derivative Assay, RNA Sequencing, Activity Assay

Journal: bioRxiv

Article Title: Xist Repeat A coordinates an assembly of SR proteins to recruit SPEN and induce gene silencing

doi: 10.1101/2025.05.21.655143

Figure Lengend Snippet: (A) SRSF1-Repeat A association in SM33 or RMCE- Xist ESCs by input-normalized RIP-qPCR. Srsf2 mRNA, control. Dots, qPCR triplicates from two independent experiments. (B) SPEN- vs. SRSF1-Repeat A association (RIP-qPCR). r , p : data fit to linear regression. (C) RBNQ with SRSF1 RRM1-2 and Repeat A. Dots, values from two independent experiments. Error bars, standard deviation of qPCR triplicates. (D) RBNQ across Repeat A RNAs using fixed SRSF1 RRM1-2 concentration. Dots, qPCR triplicates values, ≥2 independent experiments. (E) FKBP1A F36V -SRSF1 degradation by dTAG V -1 in cells derived from RMCE- Xist WT. (F) SPEN-Repeat A association in WT or FKBP1A F36V - Srsf1 cells by input-normalized RIP-qPCR. Dots, qPCR triplicates from independent clonal lines. (G) Xist abundance (RNA-seq) in FKBP1A F36V - Srsf1 cells, with/without dox and with/without dTAG V -1. Dots, independent lines, relative to dox(+), dTAG V -1(-) condition. (H) Xist silencing activity as in , for FKBP1A F36V - Srsf1 cells treated as in (G). (I) MCP-MS2 tethering to assess SPEN recruitment to Xist ΔRepA-XB-6x(U-spacer-3xMS2). (J) SPEN-6x(U-spacer-3xMS2) association across MCP genotypes by input-normalized RIP-qPCR. Dots, qPCR triplicates from ≥2 independent experiments. (K) Xist abundance (RNA-seq) across genotypes. Dots, independent experiments. (L) Xist silencing activity as in across MCP genotypes. “n.s.,” not significantly different. P -values, two-sided t-tests relative to WT or EGFP unless specified by black bars: (*), p < 0.05; (**), p < 0.01. See .

Article Snippet: SBP-tagged proteins were first diluted into room-temp RBNQ Binding Buffer with Random RNA (3 mM MgCl 2 , 25 mM Tris-HCl pH 8.05, 150 mM KCl, 1 mg/mL BSA [Thermo Fisher AM2616], 2 U/mL SUPERase-In [Thermo Fisher Scientific AM2694], 50 nM random 16-nt RNA [IDT, bases randomized by machine-mixing, purified by standard desalting]) to a range of different protein concentrations (∼1-1400 nM).

Techniques: Control, Standard Deviation, Concentration Assay, Derivative Assay, RNA Sequencing, Activity Assay

Journal: bioRxiv

Article Title: Xist Repeat A coordinates an assembly of SR proteins to recruit SPEN and induce gene silencing

doi: 10.1101/2025.05.21.655143

Figure Lengend Snippet: (A) Levels of Repeat A recovery by input-normalized FLAG RIP-qPCR from RMCE- Xist WT cells expressing no FLAG-tagged construct and RMCE- Xist WT and ΔCCUGC cells expressing SRSF2-3xFLAG. Dots, qPCR triplicate values from one experiment. (B) Coomassie blue-stained SDS-PAGE gel of two independent preps of SBP-tagged SRSF1 RRM1-2 used for replicates of equilibrium RBNQ experiments. (C) Genotyping PCR for parental RMCE- Xist WT control cells or independent clonal lines of FKBP1A F36V - Srsf1 cells generated by CRISPR-Cas9 and homology-directed repair. PCR was performed using primers flanking either side of the FKBP1A F36V degron tag insertion. (D) Quantification of exon 1 aberrant splicing for each RMCE- Xist cell line (dots), as done in . Asterisks, values significantly different from WT by two-sided t-test. (E) Western blot confirmation of expression of untagged EGFP or various MCP-tagged proteins in RMCE- Xist ΔRepA-XB-6x(U-spacer-3xMS2) cells. (F) Confirmation of MCP-tagged protein tethering by input-normalized RIP-qPCR using antibodies against SRSF1 (left) or FLAG (right). Cells expressing untagged EGFP served as a negative control. Dots, qPCR triplicate values from two independent experiments. Asterisks, significantly different from untagged EGFP control by two-sided t-test. (G) Levels of SPEN association with WT, mutant, or synthetic Repeat A in RMCE- Xist WT, ΔU- spacer, or ΔGC-core cells or RMCE- Xist ΔRepA-XB-6x(U-spacer-3xMS2) cells, determined by input-normalized RIP-qPCR. Dots, qPCR triplicate values from two independent experiments (one that included ΔGC-core). Asterisk, significantly different from WT by two-sided t-test (two-sided one-sample t-test for ΔGC-core). (H and I) Plots of Xist RNA abundance as a function of SPEN-Repeat A association level (H) or SRSF1-Repeat A association level (I). Values of r and p reflect fit to a linear regression models with the indicated data. The ss234 deletion reduces Xist abundance independent of Repeat A and was thus omitted.

Article Snippet: SBP-tagged proteins were first diluted into room-temp RBNQ Binding Buffer with Random RNA (3 mM MgCl 2 , 25 mM Tris-HCl pH 8.05, 150 mM KCl, 1 mg/mL BSA [Thermo Fisher AM2616], 2 U/mL SUPERase-In [Thermo Fisher Scientific AM2694], 50 nM random 16-nt RNA [IDT, bases randomized by machine-mixing, purified by standard desalting]) to a range of different protein concentrations (∼1-1400 nM).

Techniques: Expressing, Construct, Staining, SDS Page, Control, Generated, CRISPR, Western Blot, Negative Control, Mutagenesis

Journal: bioRxiv

Article Title: Xist Repeat A coordinates an assembly of SR proteins to recruit SPEN and induce gene silencing

doi: 10.1101/2025.05.21.655143

Figure Lengend Snippet: (A) Repeats 6 and 7 depicted according to structural model from Lu et al. , SPEN is recruited to Repeat A predominantly by proteins that bind motifs in the GC-rich core, whereas the U-rich spacers play minor roles. SPEN and GC-rich-core-bound proteins enable association with factors involved in transcriptional elongation and RNA processing. (B) Sensing of SR protein-rich transcriptional hubs by Repeat A and SPEN.

Article Snippet: SBP-tagged proteins were first diluted into room-temp RBNQ Binding Buffer with Random RNA (3 mM MgCl 2 , 25 mM Tris-HCl pH 8.05, 150 mM KCl, 1 mg/mL BSA [Thermo Fisher AM2616], 2 U/mL SUPERase-In [Thermo Fisher Scientific AM2694], 50 nM random 16-nt RNA [IDT, bases randomized by machine-mixing, purified by standard desalting]) to a range of different protein concentrations (∼1-1400 nM).

Techniques: