halo mdc1  (Bio-Rad)

Journal: bioRxiv

Article Title: The PST repeat region of MDC1 is a tunable multivalent chromatin tethering domain

doi: 10.1101/2025.01.10.632395

Figure Lengend Snippet: (A) Model of MDC1 recruitment to chromatin and HILO microscopy for single-molecule imaging. (B-C) Fluorescence gels of cell lysates from (B) U2OS and (C) RPE1 expressing HaloTagged MDC1 from its endogenous locus labeled with JFX650 HaloTag-ligand. (D-E) Static fraction of Halo-MDC1 molecules in interphase (I) and mitosis (M) derived from single-particle tracking of MDC1 in (D) U2OS and (E) RPE1 cells (N = 3 biological replicates, ≥ 20 cells per replicate, Mean ± SD, two-tailed T-test). (F-G) Diffusion coefficient of (F) mobile and (G) static Halo-MDC1 molecules in interphase (I) and mitosis (M) derived from single-particle tracking of MDC1 in U2OS cells (N = 3 biological replicates, ≥ 20 cells per replicate, Mean ± S.D., two-tailed T-test). (H-I) Diffusion coefficient of (H) mobile and (I) static Halo-MDC1 molecules in interphase (I) and mitosis (M) derived from single-particle tracking of MDC1 in RPE1 cells (N = 3 biological replicates, ≥ 20 cells per replicate, Mean ± S.D., two-tailed T-test). (J) Domain organization of MDC1, sequence alignment of the thirteen 41 amino acid PST repeats, and consensus sequence surrounding the two conserved TP sites within the PST repeats. (K) Phosphorescence imaging and protein staining of and SDS PAGE gel loaded with samples of the wild type (WT) and non-phosphorylatable (T>A) GST-tagged PST repeat domain treated with CDK1-Cyclin B and ATP γ- 32 P. (L) Images of mitotic U2OS cells with MDC1 knock-out (arrested with nocodazole) transiently expressing HaloTagged wildtype MDC1 (WT) and MDC1 variants with phospo-mimicking (T>D) and non-phosphorylatable (T>A) mutations in the TP sites within the PST repeat region.

Article Snippet: In-gel fluorescent detection of Halo-MDC1 was performed using the Cy5.5 filter on a BioRad Chemidoc.

Techniques: Microscopy, Imaging, Fluorescence, Expressing, Labeling, Derivative Assay, Single-particle Tracking, Two Tailed Test, Diffusion-based Assay, Sequencing, Staining, SDS Page, Knock-Out

Journal: bioRxiv

Article Title: The PST repeat region of MDC1 is a tunable multivalent chromatin tethering domain

doi: 10.1101/2025.01.10.632395

Figure Lengend Snippet: (A-C) Displacement histograms and two state fit of single particle tracking analysis of Halo-MDC1 expressed from its endogenous locus in (A,B) U2OS cells and (C) RPE1 cells. (D) Fluorescence gel of cell lysates from U2OS expressing Halo-MDC1 from its endogenous locus and U2OS ΔMDC1 cells transiently expressing the indicated HaloTagged MDC1 variants labeled with JF646 HaloTag-ligand.

Article Snippet: In-gel fluorescent detection of Halo-MDC1 was performed using the Cy5.5 filter on a BioRad Chemidoc.

Techniques: Single-particle Tracking, Fluorescence, Expressing, Labeling

Journal: bioRxiv

Article Title: The PST repeat region of MDC1 is a tunable multivalent chromatin tethering domain

doi: 10.1101/2025.01.10.632395

Figure Lengend Snippet: (A) Fluorescence gel of cell lysates of U2OS ΔMDC1 cells transiently expressing HaloTagged (JFX650) wildtype MDC1 and truncations of the PST repeats. (B-D) Single particle tracking analysis of HaloTagged wildtype MDC1 and truncations of the PST repeats in interphase U2OS ΔMDC1 cells including the (B) fraction of static particles and the diffusion coefficients of (C) mobile and (D) static MDC1 molecules (N = 3 biological replicates, ≥ 20 cells per replicate, Mean ± S.D.). (E-G) Single particle tracking analysis of HaloTagged wildtype MDC1 (WT), MDC1 with a deletion of the PST repeat region (ΔPST) and MDC1 variants with phospo-mimicking (T>D) and non-phosphorylatable (T>A) mutations in the TP sites within the PST repeat region in interphase U2OS ΔMDC1 cells including the (E) fraction of static particles and the diffusion coefficients of (F) mobile and (G) static MDC1 molecules (N = 3 biological replicates, ≥ 20 cells per replicate, Mean ± S.D.).

Article Snippet: In-gel fluorescent detection of Halo-MDC1 was performed using the Cy5.5 filter on a BioRad Chemidoc.

Techniques: Fluorescence, Expressing, Single-particle Tracking, Diffusion-based Assay

Journal: bioRxiv

Article Title: The PST repeat region of MDC1 is a tunable multivalent chromatin tethering domain

doi: 10.1101/2025.01.10.632395

Figure Lengend Snippet: (A-B) Images of fixed interphase and mitotic U2OS cells expressing Halo-MDC1 (JFX650) immuno-stained with (A) p-PST antibody #1 or (B) p-PST antibody #2 and Hoechst to label DNA. (C) Images of fixed, mitotic U2OS cells expressing Halo-MDC1 and U2OS ΔMDC1 cells stably expressing HaloTagged MDC1 (JFX650) variants untreated or treated with zeocin. Cells were immuno-stained with p-PST antibody #1 and Hoechst to label DNA. (D-E) Images of fixed U2OS cells expressing Halo-MDC1 (JFX650) (D) untreated or (E) treated with zeocin to induce DNA breaks in various phases of mitosis and early G1-Phase. Cells were immuno-stained with p-PST antibody #1, a γH2AX antibody, and Hoechst to label DNA.

Article Snippet: In-gel fluorescent detection of Halo-MDC1 was performed using the Cy5.5 filter on a BioRad Chemidoc.

Techniques: Expressing, Staining, Stable Transfection

Journal: bioRxiv

Article Title: The PST repeat region of MDC1 is a tunable multivalent chromatin tethering domain

doi: 10.1101/2025.01.10.632395

Figure Lengend Snippet: (A) Fluorescence gel of cell lysates from U2OS expressing Halo-MDC1 from its endogenous locus and U2OS ΔMDC1 cells expressing doxycycline (Dox.) inducible HaloTagged MDC1 variants from an expression cassette integrated using Sleeping Beauty transposase. The HaloTag was labeled with JFX650 HaloTag-ligand. (B-C) Images of fixed U2OS cells expressing Halo-MDC1 (JFX650) (B) untreated or (C) treated with zeocin to induce DNA breaks in various phases of mitosis and early G1-Phase. Cells were immuno-stained with p-PST antibody #2 and a γH2AX antibody and Hoechst to label DNA. (D) Images of fixed U2OS cells expressing Halo-MDC1 (JFX650) treated with zeocin to induce DNA breaks in various phases of mitosis and early G1-Phase. Cells were immuno-stained with antibodies against 53BP1 and γH2AX and Hoechst to label DNA.

Article Snippet: In-gel fluorescent detection of Halo-MDC1 was performed using the Cy5.5 filter on a BioRad Chemidoc.

Techniques: Fluorescence, Expressing, Labeling, Staining

Journal: bioRxiv

Article Title: The PST repeat region of MDC1 is a tunable multivalent chromatin tethering domain

doi: 10.1101/2025.01.10.632395

Figure Lengend Snippet: (A) Fluorescence gels of cell lysates from U2OS expressing HaloTagged MDC1 or MDC ΔPST from its endogenous locus labeled with JFX650 HaloTag-ligand. (B) Images of fixed, mitotic U2OS cells expressing HaloTagged MDC1 or MDC ΔPST from its endogenous locus untreated or treated with zeocin to introduce DNA breaks. Cells were labeled with JFX650 and Hoechst and immuno-stained with antibodies targeting TOPBP1 and γH2AX. (C) Images of fixed, mitotic U2OS cells expressing Halo-MDC1 and U2OS ΔMDC1 cells stably expressing HaloTagged MDC1 (JFX650) variants untreated or treated with zeocin. Cells were immuno-stained an antibody targeting CIP2A and stained with Hoechst to label DNA. (D) Quantification of the accumulation of knock-in HaloTagged MDC1 and MDC ΔPST at laser micro-irraditation induced DNA damage in living mitotic U2OS cells.

Article Snippet: In-gel fluorescent detection of Halo-MDC1 was performed using the Cy5.5 filter on a BioRad Chemidoc.

Techniques: Fluorescence, Expressing, Labeling, Introduce, Staining, Stable Transfection, Knock-In

Journal: bioRxiv

Article Title: The PST repeat region of MDC1 is a tunable multivalent chromatin tethering domain

doi: 10.1101/2025.01.10.632395

Figure Lengend Snippet: (A) Genome editing strategy to knock-in a cDNA encoding MDC1 lacking its PST repeat region into the endogenous MDC1 locus. Colored arrows indicate primer positions. (B-C) PCR using genomic DNA from clonal U2OS cell lines subjected to genome editing using the indicated primer pairs. In (B) successful genome editing of all alleles is expected to lead no product amplification due to the large insertion of the cDNA and selection cassette. In (C) successful insertion of the donor sequence is validated using a reverse primer oriented downstream of the right homology arm and a forward primer in the donor. (D) Growth curve of U2OS cells expressing Halo-MDC1 and Halo-MDC1 ΔPST from the endogenous MDC1 locus, and MDC1 knock-out cells (ΔMDC1).

Article Snippet: In-gel fluorescent detection of Halo-MDC1 was performed using the Cy5.5 filter on a BioRad Chemidoc.

Techniques: Knock-In, Amplification, Selection, Sequencing, Expressing, Knock-Out

Journal: bioRxiv

Article Title: The PST repeat region of MDC1 is a tunable multivalent chromatin tethering domain

doi: 10.1101/2025.01.10.632395

Figure Lengend Snippet: (A) Still images from live cell timelapse imaging of U2OS ΔMDC1 stably expressing HaloTagged MDC1 variants labeled with JFX650. (B) Quantification of the time from nuclear envelope breakdown to anaphase onset of U2OS ΔMDC1 cells stably expressing HaloTagged MDC1 variants. (C) Quantification of the time from nuclear envelope breakdown to anaphase onset, the chromosome segregation phenotype, and Halo-MDC1 expression level of U2OS ΔMDC1 cells stably expressing HaloTagged MDC1 variants labeled with JFX650. The chromosome segregation phenotype was determined by manual inspection.

Article Snippet: In-gel fluorescent detection of Halo-MDC1 was performed using the Cy5.5 filter on a BioRad Chemidoc.

Techniques: Imaging, Stable Transfection, Expressing, Labeling

Journal: bioRxiv

Article Title: The PST repeat region of MDC1 is a tunable multivalent chromatin tethering domain

doi: 10.1101/2025.01.10.632395

Figure Lengend Snippet: (A) Quantification of clonogenic survival assays using the PARP inhibitor olaparib of U2OS cells expressing HaloTagged MDC1 or MDC ΔPST from its endogenous locus. (N = 3 biological replicates plated in triplicate, Mean ± S.D., Two-way ANOVA) (B) Images of fixed interphase U2OS cells expressing HaloTagged MDC1 or MDC ΔPST from its endogenous locus untreated or treated with zeocin to induce DNA breaks. Cells were stained with JFX650 HaloTag-ligand and Hoechst, and immuno-labeled with antibodies targeting RAD51 and γH2AX. (C) Images of fixed interphase U2OS cells expressing Halo-MDC1 from its endogenous locus, MDC1 knock-out cells (ΔMDC1), and U2OS ΔMDC1 stably expressing HaloTagged MDC1 variants untreated or treated with zeocin to induce DNA breaks. Cells were stained with JFX650 HaloTag-ligand and Hoechst, and immuno-labeled with an antibody targeting RAD51. (D) Quantification of the number of RAD51 foci of the experiment in (C). An individual replicate is plotted on the left (red line = median, >100 cells per condition) and 3 biological replicates are plotted on the right (Median ± S.D., Two-way ANOVA). (E) Images of fixed interphase U2OS cells expressing Halo-MDC1 from its endogenous locus, MDC1 knock out cells (ΔMDC1), and U2OS ΔMDC1 cells stably expressing HaloTagged MDC1 variants untreated or treated with zeocin to induce DNA breaks. Cells were stained with JFX650 HaloTag-ligand and Hoechst, and immuno-labeled with an antibody targeting 53BP1. (F) Quantification of the number of 53BP1 foci of the experiment in (E). An individual replicate is plotted on the left (red line = median, >100 cells per condition) and 3 biological replicates are plotted on the right (Median ± S.D., Two-way ANOVA). (G-H) Quantification of the accumulation of (G) Halo-MDC1 and Halo-MDC1 ΔPST and (H) ubiquitin Reader1.0 at laser micro-irradiation induced DNA lesions in U2OS ΔMDC1 cells stably expressing indicated HaloTagged MDC1 variants (N = 10-19 cells, Mean ± 95% CI). (H) Quantification of the maximal accumulation of ubiquitin Reader1.0 at laser micro-irradiation induced DNA lesions in U2OS ΔMDC1 cells stably expressing indicated HaloTagged MDC1 variants (N = 10-19 cells, Mean ± 95% CI). (G) Quantification of HR efficiency using the DR-GFP reporter stably integrated into the AAVS1 locus of U2OS ΔMDC1 cells. I-Sce and MDC1 variants were transiently transfected (N = 3-4 biological replicates, Mean ± S.D., Two-way ANOVA).

Article Snippet: In-gel fluorescent detection of Halo-MDC1 was performed using the Cy5.5 filter on a BioRad Chemidoc.

Techniques: Expressing, Staining, Labeling, Knock-Out, Stable Transfection, Irradiation, Transfection

Journal: bioRxiv

Article Title: The PST repeat region of MDC1 is a tunable multivalent chromatin tethering domain

doi: 10.1101/2025.01.10.632395

Figure Lengend Snippet: (A-B) Quantification of the accumulation of (A) Halo-MDC1 or (B) Halo-MDC1 ΔPST labeled with JFX650 HaloTag-ligand and ubiquitin Reader1.0 at laser micro-irradiation induced DNA lesions in U2OS ΔMDC1 cells stably expressing indicated HaloTagged MDC1 variants (N = 16-19 cells, Mean ± S.E.M.). (C) Accumulation of ubiquitin Reader1.0 at laser micro-irradiation induced DNA lesions in U2OS ΔMDC1 cells stably expressing indicated HaloTagged MDC1 variants. Each trace represents an individual cell.

Article Snippet: In-gel fluorescent detection of Halo-MDC1 was performed using the Cy5.5 filter on a BioRad Chemidoc.

Techniques: Labeling, Irradiation, Stable Transfection, Expressing