Journal: Nature Communications

Article Title: RECQ4-MUS81 interaction contributes to telomere maintenance with implications to Rothmund-Thomson syndrome

doi: 10.1038/s41467-025-56518-1

Figure Lengend Snippet: A Increasing concentrations of individual RECQ helicases (RECQ4, RECQ5, BLM, and RECQ1) were incubated with 3’-flap DNA substrate (6 nM) in the presence of MUS81-EME1 (0.5 nM) for 20 min. The reaction mixtures were resolved on a native PAGE gel. B Quantification of data in (A); n = 3 independent experiments; data are means ± SD. ( C ) Interaction of purified MBP-RECQ4 (1-400) and MBP-RECQ4 (1-400) Δ3 with GST-MUS81-EME1. The flow (F) and bound (B) fractions were analysed by SDS-PAGE, followed by Coomassie blue staining. D MUS81-EME1 (0.5 nM) and increasing amounts of RECQ4 (WT), RECQ4 (1-322), RECQ4 (1-400), RECQ4 (1-400) Δ3, and RECQ4 (455-1208) were incubated with 3’flap DNA substrate (6 nM) for 20 min at 37 °C before analysis by native gel electrophoresis. E Quantification of data in (D); n = 3 independent experiments; data are means ± SD. F RECQ4 (1-400) (25 nM) was either preincubated (Preincubation) with 3’-flap DNA substrate (6 nM) followed by the addition of MUS81-EME1 (0.5 nM) or mixed with DNA and MUS81-EME1 without any preincubation (No preincubation). Reactions were incubated for the indicated time at 37 °C prior to analysis by native gel electrophoresis. n = 3 independent experiments; data are means ± SD. Source data are provided as a Source data file.

Article Snippet: Primary antibodies against MUS81 (1:1000, mouse, sc-53382, Santa Cruz) and cyclin A (1:1000, rabbit, sc-596, Santa Cruz) were diluted in DMEM medium containing 10% FBS and 0.05% sodium azide (filtered through a 0.2 μm filter) and incubated at room temperature for 90 min. Respective secondary antibodies and DAPI (0.5 μg/mL) were diluted in DMEM and set at room temperature for 30 min. After staining, coverslips were washed three times with PBS and additionally twice in distilled water, dried, and mounted with a Mowiol-based mounting medium containing 12% Mowiol 4-88 (81381, Sigma-Aldrich), 30% glycerol (G5516, Sigma-Aldrich), 0.12 M Tris-HCl pH 8.5 (RES3098T-B701X, Sigma-Aldrich).

Techniques: Incubation, Clear Native PAGE, Purification, SDS Page, Staining, Nucleic Acid Electrophoresis

Journal: Nature Communications

Article Title: RECQ4-MUS81 interaction contributes to telomere maintenance with implications to Rothmund-Thomson syndrome

doi: 10.1038/s41467-025-56518-1

Figure Lengend Snippet: A Immunoprecipitation (IP) was done from whole cell extracts expressing EGFP, EGFP-RECQ4 -WT, and the mutant EGFP-REQ4-Δ3. Proteins from each cell extract (500 µg) were incubated with GFP beads for 1 h and then resolved on SDS-PAGE, followed by western blotting detecting corresponding proteins. Input (IP), and FT fractions were run on different gels respectively. B Quantification of MUS81 binding from data represented in (A); n = 3 independent experiments; data are means ± SD; one-way ANOVA followed by Tukey’s multiple comparison test; p -value were adjusted by GraphPad Prism software; WT vs Δ3 ** p = 0.0011. C Cell cycle-specific interaction between RECQ4 and MUS81. IP was done from whole cell extracts expressing EGFP-RECQ4-WT synchronised in different cell cycle phases. Protein from each extract (500 µg) was incubated with GFP beads for 1 h and then resolved on SDS-PAGE, followed by western blotting detecting corresponding proteins. D Graphical representation and representative images of binucleated cells by immunofluorescence with DAPI. Created in BioRender. Ashraf, R. (2025) https://BioRender.com/n17i536 . Quantification of the average number of micronuclei per binucleated cell for each treatment; n = 3 independent experiments; data are means ± SD; one-way ANOVA followed by Tukey’s multiple comparison test; p -value were adjusted by GraphPad Prism software; siControl vs siRECQ4 ** p = 0.0018; siControl vs siMUS81 *** p = 0.0003; siControl vs siMUS81/siRecq4 *** p = 0.0003. E Representative images and a graphical depiction of anaphase cells with bulky bridges. Created in BioRender. Ashraf, R. (2025) https://BioRender.com/n17i536 . Quantification of the average number of bulky bridges per anaphase cell (DAPI positive bridges); n = 3 independent experiments; data are means ± SD; one-way ANOVA followed by Tukey’s multiple comparison test; p -value were adjusted by GraphPad Prism software; siControl vs siRECQ4 ** p = 0.0015; siRecq4 vs WT/siRecq4 * p = 0.0155; WT/siRecq4 vs Δ3/siRECQ4 ** p = 0.0025; WT/siRecq4 vs siMus81 * p = 0.0135; WT/siRecq4 vs siRecq4/siMus81 ** p = 0.0094. Source data are provided as a Source data file.

Article Snippet: Primary antibodies against MUS81 (1:1000, mouse, sc-53382, Santa Cruz) and cyclin A (1:1000, rabbit, sc-596, Santa Cruz) were diluted in DMEM medium containing 10% FBS and 0.05% sodium azide (filtered through a 0.2 μm filter) and incubated at room temperature for 90 min. Respective secondary antibodies and DAPI (0.5 μg/mL) were diluted in DMEM and set at room temperature for 30 min. After staining, coverslips were washed three times with PBS and additionally twice in distilled water, dried, and mounted with a Mowiol-based mounting medium containing 12% Mowiol 4-88 (81381, Sigma-Aldrich), 30% glycerol (G5516, Sigma-Aldrich), 0.12 M Tris-HCl pH 8.5 (RES3098T-B701X, Sigma-Aldrich).

Techniques: Immunoprecipitation, Expressing, Mutagenesis, Incubation, SDS Page, Western Blot, Binding Assay, Comparison, Software, Immunofluorescence

Journal: Nature Communications

Article Title: RECQ4-MUS81 interaction contributes to telomere maintenance with implications to Rothmund-Thomson syndrome

doi: 10.1038/s41467-025-56518-1

Figure Lengend Snippet: A Quantification of MUS81 foci from IF analysis in a panel of ALT-positive and ALT-negative cell lines. n = minimum 900 cells; data are means ± SD; one-way ANOVA followed by Tukey’s multiple comparison test; p -value were adjusted by GraphPad Prism software; **** p < 0.0001. B – D IF analysis of MUS81 foci with indicated siRNA for 48 h. n = minimum 5000 cells; data are means ± SD; Mann Whitney test; (B) U2OS; **** p < 0.0001. (C) LM216J; **** p < 0.0001. (D) Saos-2; ns p = 0.13. E QIBC analysis of MUS81 foci in Flp-In T-REx U2OS cells expressing either EGFP-REQ4-WT or EGFP-RECQ4-Δ3, with indicated siRNA (48 h). n = minimum 5000 cells; data are means ± SD; one-way ANOVA followed by Tukey’s multiple comparison test; p -value were adjusted by GraphPad Prism software; WT set: siControl vs siRECQ4 **** p < 0.0001; siRecq4 vs WT/siRecq4 **** p < 0.0001; Δ3 set: siControl vs siRECQ4 **** p < 0.0001; siRecq4 vs Δ3/siRECQ4 * p = 0.0268. F Quantification of MUS81 foci in LM216J cells transiently transfected with indicated constructs and treated with the specified siRNA for 48 h. n = minimum 200 cells; data are means ± SD; one-way ANOVA followed by Tukey’s multiple comparison test; p -value were adjusted by GraphPad Prism software; siControl vs siRECQ4 **** p < 0.0001; siRecq4 vs WT/siRecq4 **** p < 0.0001; WT/siRecq4 vs Δ3/siRECQ4 **** p < 0.0001. G Representative IF images of MUS81 foci and their colocalization with centromere (CEN) and telomere (TEL) in U2OS and LM216J cells. Scale bar = 5 µm. H – I Quantification of data in (G). n = 50 cells in each cell line; data are means ± SD. J Quantification of (APBs) foci in Flp-In T-REx U2OS cells expressing either EGFP-REQ4-WT or EGFP-RECQ4-Δ3, with indicated siRNA (48 h). n = minimum 500 cells; data are means ± SD; one-way ANOVA followed by Tukey’s multiple comparison test; p -value were adjusted by GraphPad Prism software; siControl vs siRECQ4 **** p < 0.0001; siRecq4 vs WT/siRecq4 **** p < 0.0001; WT/siRecq4 vs Δ3/siRECQ4 **** p < 0.0001. K Representative IF images of APBs foci. Source data are provided as a Source data file.

Article Snippet: Primary antibodies against MUS81 (1:1000, mouse, sc-53382, Santa Cruz) and cyclin A (1:1000, rabbit, sc-596, Santa Cruz) were diluted in DMEM medium containing 10% FBS and 0.05% sodium azide (filtered through a 0.2 μm filter) and incubated at room temperature for 90 min. Respective secondary antibodies and DAPI (0.5 μg/mL) were diluted in DMEM and set at room temperature for 30 min. After staining, coverslips were washed three times with PBS and additionally twice in distilled water, dried, and mounted with a Mowiol-based mounting medium containing 12% Mowiol 4-88 (81381, Sigma-Aldrich), 30% glycerol (G5516, Sigma-Aldrich), 0.12 M Tris-HCl pH 8.5 (RES3098T-B701X, Sigma-Aldrich).

Techniques: Comparison, Software, MANN-WHITNEY, Expressing, Transfection, Construct

Journal: Nature Communications

Article Title: RECQ4-MUS81 interaction contributes to telomere maintenance with implications to Rothmund-Thomson syndrome

doi: 10.1038/s41467-025-56518-1

Figure Lengend Snippet: A – C The average number of UFBs per anaphase cell in clinically affected (RTS-CA) and unaffected (RTS-CU) patient fibroblasts, along with normal fibroblasts as indicated. n = 3 independent experiments; data are means ± SD; one-way ANOVA followed by Tukey’s multiple comparison test; p -value were adjusted by GraphPad Prism software. (A) Fragile sites; Normal vs RTS-CU ns p = 0.9931; normal vs RTS-CA *** p = 0.0003 (B) Centromeric; Normal vs RTS-CU ns p = 0.1527; normal vs RTS-CA **** p < 0.0001. (C) Telomeric; Normal vs RTS-CU ns p = 0.0960; normal vs RTS-CA **** p < 0.0001. D Immunoprecipitation (IP) from whole cell extracts as shown in Fig. except EGFP-RECQ4-RTS-CA mutant was also included. Proteins from each cell extract (500 µg) were incubated with GFP beads for 1 h and then resolved on SDS-PAGE, followed by western blotting detecting corresponding proteins. Input, IP, and FT fractions were run on different gels respectively. E – G The average number of UFBs per anaphase cell in Flp-In T-REx U2OS cells expressing EGFP-RECQ4-RTS-CA in combination with siControl or siRECQ4; n = 3 independent experiments; data are means ± SD.; one-way ANOVA followed by Tukey’s multiple comparison test; p -value were adjusted by GraphPad Prism software. (E) Fragile sites; siControl vs siRecq4 ** p = 0.0023; siRecq4 vs RTS-CA/siRecq4 ** p = 0.0013. (F) Centromeric; siControl vs siRecq4 *** p = 0.0002; siRecq4 vs RTS-CA/siRecq4 *** p = 0.0002. (G) Telomeric; siControl vs siRecq4 * p = 0.0259; siRecq4 vs RTS-CA/siRecq4 * p = 0.0201. H Quantification of the MUS81 foci in Cyclin A positive Flp-In T-REx U2OS (EGFP-RECQ4-RTS-CA) cells in combination with siRNA as indicated from QIBC analysis in Supplementary Fig. ; n = minimum 5000 cells; data are means ± SD; one-way ANOVA followed by Tukey’s multiple comparison test; p -value were adjusted by GraphPad Prism software; siControl vs siRecq4 **** p < 0.0001; siControl vs RTS-CA/siRecq4 **** p < 0.0001. Source data are provided as a Source data file.

Article Snippet: Primary antibodies against MUS81 (1:1000, mouse, sc-53382, Santa Cruz) and cyclin A (1:1000, rabbit, sc-596, Santa Cruz) were diluted in DMEM medium containing 10% FBS and 0.05% sodium azide (filtered through a 0.2 μm filter) and incubated at room temperature for 90 min. Respective secondary antibodies and DAPI (0.5 μg/mL) were diluted in DMEM and set at room temperature for 30 min. After staining, coverslips were washed three times with PBS and additionally twice in distilled water, dried, and mounted with a Mowiol-based mounting medium containing 12% Mowiol 4-88 (81381, Sigma-Aldrich), 30% glycerol (G5516, Sigma-Aldrich), 0.12 M Tris-HCl pH 8.5 (RES3098T-B701X, Sigma-Aldrich).

Techniques: Comparison, Software, Immunoprecipitation, Mutagenesis, Incubation, SDS Page, Western Blot, Expressing

Journal: Nature Communications

Article Title: RECQ4-MUS81 interaction contributes to telomere maintenance with implications to Rothmund-Thomson syndrome

doi: 10.1038/s41467-025-56518-1

Figure Lengend Snippet: MUS81 interacts with RECQ4 in the S phase and stimulates MUS81-EME2 to resolve replication, recombination intermediates/stalled replication forks. During the G2/M transition, RECQ4 further activates MUS81 in the form of MUS81-EME1 which helps in the resolution of Holliday junction/late replication intermediates, at this stage MUS81 foci are visible and colocalise predominantly with telomeres and partially with centromeres. The proper operation and activation of MUS81 complexes play a crucial role in ensuring a seamless transition from prometaphase to anaphase by preventing misalignment and the build-up of UFBs (with TUBs being more vulnerable in ALT-positive cells). These actions ultimately prevent the formation of micronuclei, which can lead to aneuploidy and disease development, including RTS. Loss of RECQ4 in the background of GEN1 knockout makes the cells very sick further proposing GEN1 as an essential resolution pathway which possibly is the final resolution check during the progression of the cell cycle as GEN1 is only accessible to DNA during the mitotic phase. Conversely, the depletion of both BLM and RECQ4 leads to an increase in unresolved SCEs, as evidenced by the presence of more UFBs per cell. Scale bar = 5 µm. Created in BioRender. Ashraf, R. (2025) https://BioRender.com/o78n251 .

Article Snippet: Primary antibodies against MUS81 (1:1000, mouse, sc-53382, Santa Cruz) and cyclin A (1:1000, rabbit, sc-596, Santa Cruz) were diluted in DMEM medium containing 10% FBS and 0.05% sodium azide (filtered through a 0.2 μm filter) and incubated at room temperature for 90 min. Respective secondary antibodies and DAPI (0.5 μg/mL) were diluted in DMEM and set at room temperature for 30 min. After staining, coverslips were washed three times with PBS and additionally twice in distilled water, dried, and mounted with a Mowiol-based mounting medium containing 12% Mowiol 4-88 (81381, Sigma-Aldrich), 30% glycerol (G5516, Sigma-Aldrich), 0.12 M Tris-HCl pH 8.5 (RES3098T-B701X, Sigma-Aldrich).

Techniques: Activation Assay, Knock-Out