Journal: PLoS ONE

Article Title: Acetylation of Lysine 382 and Phosphorylation of Serine 392 in p53 Modulate the Interaction between p53 and MDC1 In Vitro

doi: 10.1371/journal.pone.0078472

Figure Lengend Snippet: (a) A scheme of the different domains of p53. (b) A scheme of the different domains of MDC1. Note that in A, and B the images are not to scale as p53 is about 5 times smaller than MDC1. (c) 293T control cells transfected with empty vector or cells overexpressing HA-tagged p53 were either untreated (-) or treated with NCS (+). Following 1 hr incubation, the proteins were extracted and subjected to co-IP using anti-HA antibodies. Control for the co-IP was done with anti-GST antibodies. Detection was done using antibodies directed against endogenous MDC1 and phopsho-Ser15 of p53 (ph-p53). (d) The tBRCT domain of MDC1 directly interacts with p53. GST pull-down assay was performed with GST-p53 and His-tBRCT or His-FHA. All recombinant proteins were expressed in bacteria. Proteins were separated on a SDS gel and stained with Coomassie blue. (e) Endogenous p53 and MDC1 interact. Protein extracts prepared from 293 cells that were induced with 5 Gray of ionizing radiation and left for recovery for 1 hr were used in an IP experiment using antibodies directed against MDC1-tBRCT (α-BRCT) or against MDC1-FHA (α-FHA). Bound proteins were detected in Western blot using anti-p53 antibodies. Inputs present 5% of the extract used in the experiment.

Article Snippet: Anti-MDC1 antibodies included rabbit and sheep anti-MDC1 directed against the FHA and tBRCT domains of MDC1 [ ] and mouse anti-MDC1, clone MDC1–50 (Sigma-Aldrich).

Techniques: Transfection, Plasmid Preparation, Incubation, Co-Immunoprecipitation Assay, Pull Down Assay, Recombinant, SDS-Gel, Staining, Western Blot

Journal: PLoS ONE

Article Title: Acetylation of Lysine 382 and Phosphorylation of Serine 392 in p53 Modulate the Interaction between p53 and MDC1 In Vitro

doi: 10.1371/journal.pone.0078472

Figure Lengend Snippet: (a) His-tBRCT retrieves p53 fragments consisting a.a. 318-393: Bacterially expressed His-tBRCT was incubated with different radio-labeled fragments of p53-HA expressed in reticulocytes (for details see schematic representations below). Following His pull-down reactions the labeled p53 fragments (in the input or those retrieved by His-tBRCT) were visualized by autoradiography. (b) p53 fragments containing a.a 318-393 bind tBRCT-MDC1: Fragments of p53 fused to GST (for details see schematic representations below) were expressed in bacteria and purified. Following incubation with radio-labeled His-tBRCT and GST pull-down reactions, His-tBRCT visualized by using autoradiography. Input is 5% of His-tBRCT added to the reaction. The same gels were used for autoradiography and Coomassie blue staining in B. (c) GST pull-down using a.a. 318-393 of p53 fused to GST (GST-p53Cter) for His-FHA or His-tBRCT, followed by Coomassie blue staining.

Article Snippet: Anti-MDC1 antibodies included rabbit and sheep anti-MDC1 directed against the FHA and tBRCT domains of MDC1 [ ] and mouse anti-MDC1, clone MDC1–50 (Sigma-Aldrich).

Techniques: Incubation, Labeling, Autoradiography, Purification, Staining

Journal: PLoS ONE

Article Title: Acetylation of Lysine 382 and Phosphorylation of Serine 392 in p53 Modulate the Interaction between p53 and MDC1 In Vitro

doi: 10.1371/journal.pone.0078472

Figure Lengend Snippet: (a) Cartoon representations of the p53 CTD peptides (Acetylated and phosphorylated peptide in blue or unmodified peptide in red), MDC1-tBRCT in cyan and phosphorous atom in gold. Left - initial conformations; right - representative snapshots of the molecular dynamics simulations. (b-e) Potential energy of the interactions between p53 CTD peptides and MDC1-tBRCT; The Lennard-Jones and the electrostatic contributions of each residue are shown in white and black, respectively. The peptides: (b) Ac-K382 and pS392. (c) Ac-K382. (d) pS392. (e) Unmodified. Error bars represent the standard deviation of the mean for the sum of the interactions.

Article Snippet: Anti-MDC1 antibodies included rabbit and sheep anti-MDC1 directed against the FHA and tBRCT domains of MDC1 [ ] and mouse anti-MDC1, clone MDC1–50 (Sigma-Aldrich).

Techniques: Standard Deviation

Journal: PLoS ONE

Article Title: Acetylation of Lysine 382 and Phosphorylation of Serine 392 in p53 Modulate the Interaction between p53 and MDC1 In Vitro

doi: 10.1371/journal.pone.0078472

Figure Lengend Snippet: (a) MD derived interactions. tBRCT is shown in surface representation in light gray and the p53 CTD peptide is shown as a blue ribbon. Zoom-in panels of the Ac-K382 and pS392 are shown below; note that in the zoom-in panels the viewer angle is slightly rotated for visualization convenience. Red arrow points K1936 in MDC1. (b) Following genotoxic stress p53 (blue) undergoes K382 acetylation (red pentagon) and S392 phosphorylation (yellow pentagon). These residues mediate the interaction with MDC1 (gray) through its tBRCT domain (light gray).

Article Snippet: Anti-MDC1 antibodies included rabbit and sheep anti-MDC1 directed against the FHA and tBRCT domains of MDC1 [ ] and mouse anti-MDC1, clone MDC1–50 (Sigma-Aldrich).

Techniques: Derivative Assay

Journal: bioRxiv

Article Title: MDC1 counteracts restrained replication fork restart and its loss causes chemoresistance in BRCA1/2-deficient mammary tumors

doi: 10.1101/2022.08.18.504391

Figure Lengend Snippet: A) TIDE analysis showing the modification rate of the Mdc1 gene in the KB2P1.21 cells expressing non-targeting (NT) or Mdc1 -targeting gRNA. B) Immunofluorescence of MDC1 (green) in the presence or absence of irradiation. Scale bars represent 10 µm. C) Quantification of MDC1 foci formation in absence or presence of irradiation in KB2P1.21 cells. Median of measured values is shown. D) Competition assay in control and Mdc1 -targeted polyclonal cell lines with two PARP inhibitors. Representative images (left) and TIDE analysis of relative change in frequency of frame-shift modifications in Mdc1 gene compared to the KB2P1.21 cells expressing NT gRNA (right). Data represent Mean ± SD of three biological replicates. E) Western blotting of PARP1 and γ-Tubulin in NT gRNA and Mdc1 KO B5 and G4 cells. Similar results were observed in two independent experiments. F) Clonogenic survival assay with representative images (left) and quantification of cell survival (right). Mean + SEM of three independent experiments is shown. G) TIDE analysis demonstrating the Mdc1 -targeting efficiency in KB2P3.4 cells. H) Competition assay in polyclonal KB2P3.4 cell lines with representative images (left) and quantification of change in the frequency of frame-shift modifications following the treatment with PARPi (right). Mean ± SD of three independent experiments is shown. I) TIDE analysis showing the modification rate of the MDC1 gene in the RPE-1 cells expressing non-targeting (NT) or MDC1 -targeting gRNA.

Article Snippet: Then, click reaction was performed by adding the click reaction buffer (100 mM Tris pH 8, 4 mM CuSO4, 100 mM sodium ascorbate, 50 µM biotin-azide) to the samples and incubating at 37 °C for 2 h. Slides were then incubated in the blocking buffer (PBS, BSA (2% w/v), glycine (0.15% w/v), Triton X-100 (0.1% v/v)) for 1 h at 37 °C, followed by incubation with primary antibodies mouse anti-MDC1 (#M2444, Sigma Aldrich, diluted 1:200) and rabbit anti-biotin (D5A7, #5597, Cell Signaling, diluted 1:1000) overnight at 4 °C.

Techniques: Modification, Expressing, Immunofluorescence, Irradiation, Competitive Binding Assay, Western Blot, Clonogenic Cell Survival Assay

Journal: bioRxiv

Article Title: MDC1 counteracts restrained replication fork restart and its loss causes chemoresistance in BRCA1/2-deficient mammary tumors

doi: 10.1101/2022.08.18.504391

Figure Lengend Snippet: A) TIDE analysis showing the frequency of WT and modified Mdc1 alleles in KB1P-G3 cells. B) Clonogenic survival assay of KB1P-G3 cells expressing NT, or Mdc1 -targeting gRNAs upon treatment with PARP inhibitors. Representative images (left) and quantification (right) are shown. Mean ± SEM of at least three independent experiments is shown. C) Clonogenic survival assay with representative images (left) and quantification of cell survival (right). Mean + SD of at least two independent experiments is shown. D) TIDE analysis showing the rate of modifications in Mdc1 gene in tumors derived from the KB2P 3D organoids. E) Kaplan-Meier curve of overall survival of mice treated with vehicle or PARPi olaparib.

Article Snippet: Then, click reaction was performed by adding the click reaction buffer (100 mM Tris pH 8, 4 mM CuSO4, 100 mM sodium ascorbate, 50 µM biotin-azide) to the samples and incubating at 37 °C for 2 h. Slides were then incubated in the blocking buffer (PBS, BSA (2% w/v), glycine (0.15% w/v), Triton X-100 (0.1% v/v)) for 1 h at 37 °C, followed by incubation with primary antibodies mouse anti-MDC1 (#M2444, Sigma Aldrich, diluted 1:200) and rabbit anti-biotin (D5A7, #5597, Cell Signaling, diluted 1:1000) overnight at 4 °C.

Techniques: Modification, Clonogenic Cell Survival Assay, Expressing, Derivative Assay

Journal: bioRxiv

Article Title: MDC1 counteracts restrained replication fork restart and its loss causes chemoresistance in BRCA1/2-deficient mammary tumors

doi: 10.1101/2022.08.18.504391

Figure Lengend Snippet: A) Quantification of RAD51 foci formation in RPE-1 cells 4h upon irradiation with 10 Gy. Mean + SD of two independent experiments is shown. B) DNA fiber assay in KB2P1.21 cells with representative images of individual fibers. Median of track lengths is shown. Similar results were obtained from 2 independent experiments. C) The total replication speed assay. Median of EdU intensity measured in 500 KB2P1.21 cells using a cytometer is shown. Similar results were obtained from 2 independent experiments. D) Representative images and analysis of proximity ligation assay showing the interaction of MDC1 with PCNA (red) in EdU-positive cells (green). Median of values from three independent experiments is shown. Scale bars represent 5 µm. E) Western blotting of DDR markers before and after 1 mM HU for 24 h in WT and MDC1 knockout RPE-1 cells. Similar results were observed in two independent experiments. F) Quantification and representative images of γH2AX foci in the KB2P1.21 cells after 24 h treatment with DMSO, 300 nM mitomycin C (MMC), 1 µM olaparib or 100 nM cisplatin. Mean ± SD of four independent experiments is shown. The scale bar represents 10 µm. G) Percentage of micronuclei positive KB2P1.21 cells after 48 h treatment with DMSO or olaparib. Mean ± SD of values from two independent experiments is shown. A representative image with examples of micronuclei; the scale bar represents 10 µm.

Article Snippet: Then, click reaction was performed by adding the click reaction buffer (100 mM Tris pH 8, 4 mM CuSO4, 100 mM sodium ascorbate, 50 µM biotin-azide) to the samples and incubating at 37 °C for 2 h. Slides were then incubated in the blocking buffer (PBS, BSA (2% w/v), glycine (0.15% w/v), Triton X-100 (0.1% v/v)) for 1 h at 37 °C, followed by incubation with primary antibodies mouse anti-MDC1 (#M2444, Sigma Aldrich, diluted 1:200) and rabbit anti-biotin (D5A7, #5597, Cell Signaling, diluted 1:1000) overnight at 4 °C.

Techniques: Irradiation, Cytometry, Proximity Ligation Assay, Western Blot, Knock-Out

Journal: bioRxiv

Article Title: MDC1 counteracts restrained replication fork restart and its loss causes chemoresistance in BRCA1/2-deficient mammary tumors

doi: 10.1101/2022.08.18.504391

Figure Lengend Snippet: A) DNA fiber assay showing RF degradation in KB2P1.21 cells upon HU-induced RF stalling. Minimum of 120 forks per sample were analyzed and the IdU/CldU ration was calculated. Median of values measured from two independent experiments is shown. B) DNA fiber assay showing reduced RF speed in KB2P1.21 Mdc1 knockout lines B5 and G4. Track lengths of at least 100 forks were measured. Median of track lengths is shown. Similar results were observed in at least three biological replicates. C) Track lengths analysis in KB1P-G3 cells expressing NT, or Mdc1 -targeting gRNA. Minimum of 100 forks per sample were analyzed and median of values measured from two independent experiments is shown. D) DNA fiber assay in MDC1 WT, MDC1 KO and GFP-tagged MDC1 WT complemented RPE-1 cells. The median of track lengths from two independent experiments is shown. E) Immunofluorescence analysis showing the loss of MDC1 IRIF formation in RPE-1 MDC1 knockout cell line. The scale bar represents 10 µm.

Article Snippet: Then, click reaction was performed by adding the click reaction buffer (100 mM Tris pH 8, 4 mM CuSO4, 100 mM sodium ascorbate, 50 µM biotin-azide) to the samples and incubating at 37 °C for 2 h. Slides were then incubated in the blocking buffer (PBS, BSA (2% w/v), glycine (0.15% w/v), Triton X-100 (0.1% v/v)) for 1 h at 37 °C, followed by incubation with primary antibodies mouse anti-MDC1 (#M2444, Sigma Aldrich, diluted 1:200) and rabbit anti-biotin (D5A7, #5597, Cell Signaling, diluted 1:1000) overnight at 4 °C.

Techniques: Knock-Out, Expressing, Immunofluorescence

Journal: bioRxiv

Article Title: MDC1 counteracts restrained replication fork restart and its loss causes chemoresistance in BRCA1/2-deficient mammary tumors

doi: 10.1101/2022.08.18.504391

Figure Lengend Snippet: A) Imaging-based analysis of EdU incorporation upon release of new origins by ATR inhibitor AZ20 or upon Mdc1 KO in KB2P1.21 cells. The scale bars represent 5 µm. B) Proliferation rate of KB2P1.21 cells expressing NT gRNA, two Mdc1 -targeting gRNAs and two KO cell lines B5 and G4. Mean ± SD of at least three biological replicates is shown. C) Analysis of MDC1 localization at active replication forks by SIRF. Representative images (left) and quantification (right) are shown. Similar results were obtain from at least two independent experiments. Scale bars represent 5 µm. D) Western blotting of DDR markers before and after 1 mM HU for 24 h in NT gRNA, B5 and G4 KB2P1.21 cells. Similar results were obtained from two independent experiments.

Article Snippet: Then, click reaction was performed by adding the click reaction buffer (100 mM Tris pH 8, 4 mM CuSO4, 100 mM sodium ascorbate, 50 µM biotin-azide) to the samples and incubating at 37 °C for 2 h. Slides were then incubated in the blocking buffer (PBS, BSA (2% w/v), glycine (0.15% w/v), Triton X-100 (0.1% v/v)) for 1 h at 37 °C, followed by incubation with primary antibodies mouse anti-MDC1 (#M2444, Sigma Aldrich, diluted 1:200) and rabbit anti-biotin (D5A7, #5597, Cell Signaling, diluted 1:1000) overnight at 4 °C.

Techniques: Imaging, Expressing, Western Blot

Journal: bioRxiv

Article Title: MDC1 counteracts restrained replication fork restart and its loss causes chemoresistance in BRCA1/2-deficient mammary tumors

doi: 10.1101/2022.08.18.504391

Figure Lengend Snippet: A) Frequency of reversed RFs in untreated WT or MDC1 knockout RPE-1 cells analyzed by transmission electron microscopy. Mean ± SD of three independent experiments is shown. An electron micrograph of a representative reversed fork. B) Scheme of RF restart experiments. C) Examples of stalled or restarted forks, the asterisks show examples of the quantified events. Analysis of the percentage of restarted forks without treatment or after RF stalling with HU and two recovery time-points. Mean ± SD of two independent experiments is shown. The numbers of counted RFs are shown in brackets. D) IdU track length analysis of the restarted forks. Median of values from two independent experiments is shown.

Article Snippet: Then, click reaction was performed by adding the click reaction buffer (100 mM Tris pH 8, 4 mM CuSO4, 100 mM sodium ascorbate, 50 µM biotin-azide) to the samples and incubating at 37 °C for 2 h. Slides were then incubated in the blocking buffer (PBS, BSA (2% w/v), glycine (0.15% w/v), Triton X-100 (0.1% v/v)) for 1 h at 37 °C, followed by incubation with primary antibodies mouse anti-MDC1 (#M2444, Sigma Aldrich, diluted 1:200) and rabbit anti-biotin (D5A7, #5597, Cell Signaling, diluted 1:1000) overnight at 4 °C.

Techniques: Knock-Out, Transmission Assay, Electron Microscopy

Journal: bioRxiv

Article Title: MDC1 counteracts restrained replication fork restart and its loss causes chemoresistance in BRCA1/2-deficient mammary tumors

doi: 10.1101/2022.08.18.504391

Figure Lengend Snippet: A) TIDE analysis showing the modification rate of the Mdc1 gene in the KB2P1.21 cells expressing non-targeting (NT) or Mdc1 -targeting gRNA. B) Immunofluorescence of MDC1 (green) in the presence or absence of irradiation. Scale bars represent 10 µm. C) Quantification of MDC1 foci formation in absence or presence of irradiation in KB2P1.21 cells. Median of measured values is shown. D) Competition assay in control and Mdc1 -targeted polyclonal cell lines with two PARP inhibitors. Representative images (left) and TIDE analysis of relative change in frequency of frame-shift modifications in Mdc1 gene compared to the KB2P1.21 cells expressing NT gRNA (right). Data represent Mean ± SD of three biological replicates. E) Western blotting of PARP1 and γ-Tubulin in NT gRNA and Mdc1 KO B5 and G4 cells. Similar results were observed in two independent experiments. F) Clonogenic survival assay with representative images (left) and quantification of cell survival (right). Mean + SEM of three independent experiments is shown. G) TIDE analysis demonstrating the Mdc1 -targeting efficiency in KB2P3.4 cells. H) Competition assay in polyclonal KB2P3.4 cell lines with representative images (left) and quantification of change in the frequency of frame-shift modifications following the treatment with PARPi (right). Mean ± SD of three independent experiments is shown. I) TIDE analysis showing the modification rate of the MDC1 gene in the RPE-1 cells expressing non-targeting (NT) or MDC1 -targeting gRNA.

Article Snippet: Incubation with the primary antibodies mouse monoclonal anti-MDC1 antibody (#M2444, Sigma Aldrich) diluted 1:500 in staining buffer and rabbit monoclonal anti-PCNA (D3H8P) XP antibody (#13110, Cell Signaling Technology) diluted 1:800 in staining buffer was carried out at 4 °C overnight.

Techniques: Modification, Expressing, Immunofluorescence, Irradiation, Competitive Binding Assay, Western Blot, Clonogenic Cell Survival Assay

Journal: bioRxiv

Article Title: MDC1 counteracts restrained replication fork restart and its loss causes chemoresistance in BRCA1/2-deficient mammary tumors

doi: 10.1101/2022.08.18.504391

Figure Lengend Snippet: A) TIDE analysis showing the frequency of WT and modified Mdc1 alleles in KB1P-G3 cells. B) Clonogenic survival assay of KB1P-G3 cells expressing NT, or Mdc1 -targeting gRNAs upon treatment with PARP inhibitors. Representative images (left) and quantification (right) are shown. Mean ± SEM of at least three independent experiments is shown. C) Clonogenic survival assay with representative images (left) and quantification of cell survival (right). Mean + SD of at least two independent experiments is shown. D) TIDE analysis showing the rate of modifications in Mdc1 gene in tumors derived from the KB2P 3D organoids. E) Kaplan-Meier curve of overall survival of mice treated with vehicle or PARPi olaparib.

Article Snippet: Incubation with the primary antibodies mouse monoclonal anti-MDC1 antibody (#M2444, Sigma Aldrich) diluted 1:500 in staining buffer and rabbit monoclonal anti-PCNA (D3H8P) XP antibody (#13110, Cell Signaling Technology) diluted 1:800 in staining buffer was carried out at 4 °C overnight.

Techniques: Modification, Clonogenic Cell Survival Assay, Expressing, Derivative Assay

Journal: bioRxiv

Article Title: MDC1 counteracts restrained replication fork restart and its loss causes chemoresistance in BRCA1/2-deficient mammary tumors

doi: 10.1101/2022.08.18.504391

Figure Lengend Snippet: A) Quantification of RAD51 foci formation in RPE-1 cells 4h upon irradiation with 10 Gy. Mean + SD of two independent experiments is shown. B) DNA fiber assay in KB2P1.21 cells with representative images of individual fibers. Median of track lengths is shown. Similar results were obtained from 2 independent experiments. C) The total replication speed assay. Median of EdU intensity measured in 500 KB2P1.21 cells using a cytometer is shown. Similar results were obtained from 2 independent experiments. D) Representative images and analysis of proximity ligation assay showing the interaction of MDC1 with PCNA (red) in EdU-positive cells (green). Median of values from three independent experiments is shown. Scale bars represent 5 µm. E) Western blotting of DDR markers before and after 1 mM HU for 24 h in WT and MDC1 knockout RPE-1 cells. Similar results were observed in two independent experiments. F) Quantification and representative images of γH2AX foci in the KB2P1.21 cells after 24 h treatment with DMSO, 300 nM mitomycin C (MMC), 1 µM olaparib or 100 nM cisplatin. Mean ± SD of four independent experiments is shown. The scale bar represents 10 µm. G) Percentage of micronuclei positive KB2P1.21 cells after 48 h treatment with DMSO or olaparib. Mean ± SD of values from two independent experiments is shown. A representative image with examples of micronuclei; the scale bar represents 10 µm.

Article Snippet: Incubation with the primary antibodies mouse monoclonal anti-MDC1 antibody (#M2444, Sigma Aldrich) diluted 1:500 in staining buffer and rabbit monoclonal anti-PCNA (D3H8P) XP antibody (#13110, Cell Signaling Technology) diluted 1:800 in staining buffer was carried out at 4 °C overnight.

Techniques: Irradiation, Cytometry, Proximity Ligation Assay, Western Blot, Knock-Out

Journal: bioRxiv

Article Title: MDC1 counteracts restrained replication fork restart and its loss causes chemoresistance in BRCA1/2-deficient mammary tumors

doi: 10.1101/2022.08.18.504391

Figure Lengend Snippet: A) DNA fiber assay showing RF degradation in KB2P1.21 cells upon HU-induced RF stalling. Minimum of 120 forks per sample were analyzed and the IdU/CldU ration was calculated. Median of values measured from two independent experiments is shown. B) DNA fiber assay showing reduced RF speed in KB2P1.21 Mdc1 knockout lines B5 and G4. Track lengths of at least 100 forks were measured. Median of track lengths is shown. Similar results were observed in at least three biological replicates. C) Track lengths analysis in KB1P-G3 cells expressing NT, or Mdc1 -targeting gRNA. Minimum of 100 forks per sample were analyzed and median of values measured from two independent experiments is shown. D) DNA fiber assay in MDC1 WT, MDC1 KO and GFP-tagged MDC1 WT complemented RPE-1 cells. The median of track lengths from two independent experiments is shown. E) Immunofluorescence analysis showing the loss of MDC1 IRIF formation in RPE-1 MDC1 knockout cell line. The scale bar represents 10 µm.

Article Snippet: Incubation with the primary antibodies mouse monoclonal anti-MDC1 antibody (#M2444, Sigma Aldrich) diluted 1:500 in staining buffer and rabbit monoclonal anti-PCNA (D3H8P) XP antibody (#13110, Cell Signaling Technology) diluted 1:800 in staining buffer was carried out at 4 °C overnight.

Techniques: Knock-Out, Expressing, Immunofluorescence

Journal: bioRxiv

Article Title: MDC1 counteracts restrained replication fork restart and its loss causes chemoresistance in BRCA1/2-deficient mammary tumors

doi: 10.1101/2022.08.18.504391

Figure Lengend Snippet: A) Imaging-based analysis of EdU incorporation upon release of new origins by ATR inhibitor AZ20 or upon Mdc1 KO in KB2P1.21 cells. The scale bars represent 5 µm. B) Proliferation rate of KB2P1.21 cells expressing NT gRNA, two Mdc1 -targeting gRNAs and two KO cell lines B5 and G4. Mean ± SD of at least three biological replicates is shown. C) Analysis of MDC1 localization at active replication forks by SIRF. Representative images (left) and quantification (right) are shown. Similar results were obtain from at least two independent experiments. Scale bars represent 5 µm. D) Western blotting of DDR markers before and after 1 mM HU for 24 h in NT gRNA, B5 and G4 KB2P1.21 cells. Similar results were obtained from two independent experiments.

Article Snippet: Incubation with the primary antibodies mouse monoclonal anti-MDC1 antibody (#M2444, Sigma Aldrich) diluted 1:500 in staining buffer and rabbit monoclonal anti-PCNA (D3H8P) XP antibody (#13110, Cell Signaling Technology) diluted 1:800 in staining buffer was carried out at 4 °C overnight.

Techniques: Imaging, Expressing, Western Blot

Journal: bioRxiv

Article Title: MDC1 counteracts restrained replication fork restart and its loss causes chemoresistance in BRCA1/2-deficient mammary tumors

doi: 10.1101/2022.08.18.504391

Figure Lengend Snippet: A) Frequency of reversed RFs in untreated WT or MDC1 knockout RPE-1 cells analyzed by transmission electron microscopy. Mean ± SD of three independent experiments is shown. An electron micrograph of a representative reversed fork. B) Scheme of RF restart experiments. C) Examples of stalled or restarted forks, the asterisks show examples of the quantified events. Analysis of the percentage of restarted forks without treatment or after RF stalling with HU and two recovery time-points. Mean ± SD of two independent experiments is shown. The numbers of counted RFs are shown in brackets. D) IdU track length analysis of the restarted forks. Median of values from two independent experiments is shown.

Article Snippet: Incubation with the primary antibodies mouse monoclonal anti-MDC1 antibody (#M2444, Sigma Aldrich) diluted 1:500 in staining buffer and rabbit monoclonal anti-PCNA (D3H8P) XP antibody (#13110, Cell Signaling Technology) diluted 1:800 in staining buffer was carried out at 4 °C overnight.

Techniques: Knock-Out, Transmission Assay, Electron Microscopy

Journal: Breast Cancer Research : BCR

Article Title: Proteogenomic analysis of Inhibitor of Differentiation 4 (ID4) in basal-like breast cancer

doi: 10.1186/s13058-020-01306-6

Figure Lengend Snippet: ID4 binds to MDC1 and interacts with the BRCA1 network. a Schematic of ID4 and IgG RIME data analysis; ID4 and IgG immunoprecipitations were conducted in technical duplicate or triplicate, and in biological triplicate. All proteins identified in the IgG controls (for each biological replicate) were removed from each of the ID4 IPs as non-specific, background proteins. The remaining proteins were compared across technical replicates to generate a list of medium-confidence proteins that were robustly identified in > 1 ID4 RIME technical replicate. The biological replicates were then compared to generate a list of targets present in > 1 biological replicate in an individual cell line (i.e., > 6 technical replicates conducted over three biological replicates). This resulted in the identification of 22 (HCC70), 21 (HCC1954), 21 (MDA-MB-468) and 30 (OVKATE) proteins. Targets from the different cell lines were compared, identifying a list of ID4 interactors present across multiple cell lines. b Venn diagram showing comparison of the high-confidence targets identified in all four cell lines: HCC70, HCC1954, MDA-MB-468 and OVKATE. Common targets across the four cell lines are indicated. Overlap generated using Venny . c Top six highest enriched gene sets identified through Gene Set Enrichment Analysis (GSEA) of the ID4 proteome (1106 proteins) identified in ( b ) (all proteins identified in ID4 RIME and not in IgG RIME), compared to C2 (curated gene sets), C4 (computational gene sets), C6 (oncogenic signatures), C7 (immunologic signatures) and H (hallmark gene sets) gene sets. Proteins identified in > 1 technical replicate of ID4 RIME (and not in IgG RIME) were considered in GSEA analysis. d , top: Immunohistochemistry analysis of ID4 protein expression across HCI001, HCI002 and HCI009 triple-negative PDX models, 100 μm scale, bottom; SWATH proteomic analysis of ID4 and IgG RIME conducted on HCI001, HCI002, HCI009 PDX models. Heatmap showing quantification of ID4 and MDC1 abundance, both significantly differentially expressed proteins in ID4 RIME compared with control IgG RIME ( p value < 0.05 and fold change > 2). RIME and SWATH were conducted on the same sample, one biological replicate per tumour. 100 μm scale. e Immunoprecipitation was conducted on MDA-MB-468 cells prepared using the RIME protocol. Top: Input control and IgG (mouse and rabbit) compared to IP with anti-ID4 antibodies and MDC1. Western blotting is shown using an independent monoclonal MDC1 antibody. Bottom: Input control and IgG rabbit compared to IP with anti-ID4 antibodies and MDC1. Western blotting is shown using an independent monoclonal ID4 antibody

Article Snippet: Protein expression was analysed using antibodies targeting ID4 (Biocheck anti-ID4 rabbit monoclonal BCH-9/82-12, 1:40,000), β-Actin (Sigma anti-Actin mouse monoclonal A5441, 1:5000) and MDC1 (Sigma anti-MDC1 mouse monoclonal M2444, 1:1000).

Techniques: Generated, Immunohistochemistry, Expressing, Immunoprecipitation, Western Blot

Journal: Breast Cancer Research : BCR

Article Title: Proteogenomic analysis of Inhibitor of Differentiation 4 (ID4) in basal-like breast cancer

doi: 10.1186/s13058-020-01306-6

Figure Lengend Snippet: ID4 interacts with DNA damage proteins at fragile chromatin sites in a DNA damage-dependent manner. a Representative proximity ligation assay (PLA) confocal images of ID4 and MDC1 interactions in MDA-MB-468 cells. siRNAs targeting MDC1 (siMDC1) and a scrambled control (siControl) used to show specificity of the assay. PLA foci (green), DAPI (blue) and phalloidin (magenta). 20 μm scale. b Graph showing quantification of PLA interactions between ID4 and MDC1 in cells treated with siControl and siMDC1. Data shown in comparison to the interaction between ID4:V5 and ID4:FLAG treated with siControl. 50–100 cells captured per condition. Quantification of interactions (number of dots/ cell). One-way ANOVA, multiple comparisons test. **** p < 0.0001. Error bars represent standard deviation. c ChIP-qPCR analysis of ID4 binding in untreated HCC70 cells compared to cells treated with ionising radiation (5 Gy with 5 h recovery time prior to fixation). ID4 binding normalised to negative control region, input DNA and IgG control. Data for each gene region is shown for both untreated (left) and ionising radiation-treated (right) cells, connected by a line. Representative of two independent experiments. Quantification of a time course of d γH2AX and e 53BP1 DNA damage foci formation following ionising radiation. ID4 was depleted from cells using the SMARTChoice inducible shRNA system following treatment with doxycycline for 72 h prior to treatment with 5 Gy ionising radiation at time 0 and allowed to recover for 0.25 and 8 h prior to analysis. Four to five images were taken for each condition; 100–200 cells in total. Number of foci per cell nucleus was calculated using FIJI by ImageJ (Schindelin et al., 2012), and samples were then collated and analysed using the Pandas package in Python 3.5. Data is normalised to 0 h, no DOX, no IR time point. n = 3–5, Student’s t test, * p < 0.05. Error bars represent standard error

Article Snippet: Protein expression was analysed using antibodies targeting ID4 (Biocheck anti-ID4 rabbit monoclonal BCH-9/82-12, 1:40,000), β-Actin (Sigma anti-Actin mouse monoclonal A5441, 1:5000) and MDC1 (Sigma anti-MDC1 mouse monoclonal M2444, 1:1000).

Techniques: Proximity Ligation Assay, Standard Deviation, Binding Assay, Negative Control, shRNA

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Mechanism for IL-15-driven B-CLL cycling: Roles for AKT and STAT5 in modulating Cyclin D2 and DNA damage response proteins 1

doi: 10.4049/jimmunol.1801142

Figure Lengend Snippet: (A) ATM, 53BP1, and MDC1 protein levels were examined in d4 lysates of ODN ± IL-15-stimulated B-CLL cultures by electrophoretic separation and Western blotting. The asterisk by CLL430 denotes this clone’s lack of ATM protein, due to del11q22 and a coding region mutation (8). MDC1 protein is manifest both as full-length MDC1 protein (~ 225 kDa) and a MDC1 cleavage fragment (~ 70 kDa) (70). Values below each lane represent relative densitometric levels adjusted on the basis of β-actin loading control. (B) Calculated values for CLL430 and CLL515 expression of ATM, 53BP1, and MDC1 (full-length or cleavage fragment) proteins in ODN+IL-15 lysates, as a percent of that seen in ODN-only lysates. (C) Fluorescence histograms representing ATM and 53BP1 protein expression in viable-gated U-CLL1953 cells stimulated with ODN (t=0) and IL-15 (t=20) and harvested at t=68, 92, or 134h. Inserted values represent RMFI (ratio of MFI in test mAb-stained cells (solid line) / MFI in isotype control cells (filled grey). (D) Bar graph of results from the U-CLL1953 experiment showing ATM and 53BP1 protein expression at the intervals following IL-15 pulse to cultures pre-stimulated with ODN for 20h. Data are plotted as a ratio of RMFI in ODN+IL15-treated cells / RMFI in ODN-treated cells (mean ± SEM of staining replicates). (E) Pooled results from CFSE-labeled B-CLL experiments monitoring pSTAT5, ATM protein, and 53BP1 protein levels as a function of division status within 5–6 day cultures stimulated with ODN+IL15 or ODN alone. (Experiments involved n=7 CLL (770, 791, 827, 1031, 1953, 1993, 2018), except for ATM analyses (n=6 CLL). Data expressed as ratio of specific fluorescence in ODN+IL15 cultures versus ODN-only cultures. Dotted line represents normalized fluorescence in ODN-only cultures. Statistical analyses by 2-sided, unpaired t-test: * indicates P=0.02 when compared to ODN only cells; ** indicates P < 0.0001. (G) Linear regression analysis of mRNA versus protein expression of ATM (left) and 53BP1 (right) within 5 B-CLL clones. Specific mRNA was assessed in 20h ODN-primed B-CLL ± additional 20 h of culture with medium or IL-15; specific protein assessed by staining and flow cytometry of day 5–6 cultures stimulated by ODN ± IL-15 (M-1031, M-2018, M-1993, U-1953, & U-1692). mRNA and protein levels are expressed as a ratio of assessed levels in ODN+IL15 cultures versus ODN only cultures.

Article Snippet: For detection of ATM, 53BP1, MDC1, and loading control β-actin protein, blots were sequentially exposed (with intervening stripping) to Abs specific for ATM (mouse anti-ATM mAb (clone 2C1; Gentex, Irvine, CA); 53BP1 (rabbit anti-53BP1 polyclonal IgG (NB100–304; Novus Bio; Littleton, CO); MDC1 (mouse anti-MDC1 mAb which recognizes the N-terminus of MDC1 (clone P2B11; LSBio, Seattle, WA; Millipore/Sigma), and β-actin (anti-actin mAb (Novus Biologicals).

Techniques: Western Blot, Clone Assay, Mutagenesis, Expressing, Fluorescence, Staining, Labeling, Flow Cytometry

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Mechanism for IL-15-driven B-CLL cycling: Roles for AKT and STAT5 in modulating Cyclin D2 and DNA damage response proteins 1

doi: 10.4049/jimmunol.1801142

Figure Lengend Snippet: Total mRNA was isolated from B-CLL cells that received 20h ODN priming and were subsequently pulsed with IL-15, or medium, for varying intervals. Quantitative RT-PCR with specific primers was performed as detailed in Materials & Methods. IL-15-induced changes in specific mRNA are shown on bar-graph ordinates as fold-increase (ratio of mRNA within ODN+IL15-treated versus ODN only-treated B-CLL), as calculated from ΔCt values by the 2^(-ΔΔCt) method (61). P values for statistical significance between IL-15-pulsed versus non-pulsed cultures were determined by 2-sided, paired t-test. (A) IL-15 influence on MYC and CCND2 mRNA. 4 h pulse = mean of 5 clones tested (CLL 693, 849, 1031, 1692, 1953); 9–16 h = mean of 3 clones tested (CLL 693, 887, 1953); 20–24 h = mean of 5 clones (693, 849, 1031, 1692, 1953). The asterisk linked to NS (not significant) for MYC mRNA indicates that when ΔCt values were used for comparisons, differences +/− IL-15 reached statistical significance (Supplementary Figure 3). (B) IL-15 influence on ATM, P53BP1, and MDC1 mRNA. Levels were significantly reduced upon IL-15 exposure (P<0.001 to 0.003), albeit the suppressive effect on MDC1 mRNA appeared to be transient. 4 h pulse = mean of 6 clones tested (CLL 693, 849, 887, 1031, 1692, 1953); 9–16 h = mean of 3 clones tested (CLL 693, 887, 1953); 20–24 h = mean of 7 clones tested (CLL 693, 849, 887, 1031, 1692, 1993, 2018). (C) Levels of ATM, TP53BP1 and MDC1 mRNA within U-CLL430 and U-CLL515 cells examined in unstimulated state (t=0) or after 4 days of culture with ODN alone or ODN+IL-15. For each mRNA species, mean ΔCt values from triplicate qRT-PCR assays of t=0 unstimulated cells, or d4 cultures stimulated with ODN+IL15, are expressed as a ratio of the mean ΔCt values from d4 cultures exposed to ODN alone, using the 2^(-ΔΔCt) method. Values for U-CLL430 mRNA in ODN and ODN+IL15 cultures represents mean ± SEM values from 3 separate experiments. Asterisks linked to the U-CLL430 experiments indicate that differences in mRNA levels between ODN+IL15 versus ODN only cultures reached statistical significance by 2-sided, paired t-test. Values for U-CLL515 are from one experiment with triplicate qRT-PCR determinations.

Article Snippet: For detection of ATM, 53BP1, MDC1, and loading control β-actin protein, blots were sequentially exposed (with intervening stripping) to Abs specific for ATM (mouse anti-ATM mAb (clone 2C1; Gentex, Irvine, CA); 53BP1 (rabbit anti-53BP1 polyclonal IgG (NB100–304; Novus Bio; Littleton, CO); MDC1 (mouse anti-MDC1 mAb which recognizes the N-terminus of MDC1 (clone P2B11; LSBio, Seattle, WA; Millipore/Sigma), and β-actin (anti-actin mAb (Novus Biologicals).

Techniques: Isolation, Quantitative RT-PCR, Clone Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Mechanism for IL-15-driven B-CLL cycling: Roles for AKT and STAT5 in modulating Cyclin D2 and DNA damage response proteins 1

doi: 10.4049/jimmunol.1801142

Figure Lengend Snippet: ENCODE Chip Seq data (materials and methods) was used for our bioinformatics evaluation of ATM, TP53BP1, and MDC1 loci. This data base provides information on DNA binding of 161 TFs, including STATs, TBP (TATA-box binding TF) and CTCF (insulator) within several established cell lines, but we here focused on STAT1, STAT3, STAT5, TBP and CTCF binding to promoter regions. Note that ENCODE provides data only for STAT5A binding; binding of closely related STAT5B which shares the same GAS specificity is not indicated. Browser tracks from which this schematic was derived are shown in Supplementary Figure 4.

Article Snippet: For detection of ATM, 53BP1, MDC1, and loading control β-actin protein, blots were sequentially exposed (with intervening stripping) to Abs specific for ATM (mouse anti-ATM mAb (clone 2C1; Gentex, Irvine, CA); 53BP1 (rabbit anti-53BP1 polyclonal IgG (NB100–304; Novus Bio; Littleton, CO); MDC1 (mouse anti-MDC1 mAb which recognizes the N-terminus of MDC1 (clone P2B11; LSBio, Seattle, WA; Millipore/Sigma), and β-actin (anti-actin mAb (Novus Biologicals).

Techniques: ChIP-sequencing, Binding Assay, Derivative Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Mechanism for IL-15-driven B-CLL cycling: Roles for AKT and STAT5 in modulating Cyclin D2 and DNA damage response proteins 1

doi: 10.4049/jimmunol.1801142

Figure Lengend Snippet: B-CLL receiving TLR-9 signals, subsequent to BCR internalization of CpG DNA-bearing microbes and/or apoptotic cell debris, upregulate CD122 mRNA/protein and thereby more effectively receive signals from IL-15/IL-15Rα complexes present on adjacent IL-15-producing stromal cells (8, 11, 17) or, alternatively, from soluble IL-15/IL-15Rα complexes cleaved from the latter (128). Earlier studies showed that ODN+IL15-driven in vitro B-CLL growth is positively influenced by del11q22 + del13q14; ATM mutations, and Trisomy 12 (8) and negatively influenced by CD122- or IL-15-specific neutralizing mAbs (17). The present study reveals that inhibitors of JAK1/3, PI-3K, and STAT5 can each block ODN+IL15-driven B-CLL growth, at least in part due to interference with IL-15-driven upregulation of cyclin D2 and IL-15-mediated repression of ATM and 53BP1. (While less clear, it seems likely that pSTAT5 also participates in IL-15-mediated repression of MDC1). Thus, treatment of B-CLL patients with agents that block IL-15 access to its signaling receptor and/or block early activation of JAK, PI-3K, or STAT5 following IL-15/CD122/γc engagement could be effective new approaches to curtailing B-CLL growth within lymphatic tissue of patients.

Article Snippet: For detection of ATM, 53BP1, MDC1, and loading control β-actin protein, blots were sequentially exposed (with intervening stripping) to Abs specific for ATM (mouse anti-ATM mAb (clone 2C1; Gentex, Irvine, CA); 53BP1 (rabbit anti-53BP1 polyclonal IgG (NB100–304; Novus Bio; Littleton, CO); MDC1 (mouse anti-MDC1 mAb which recognizes the N-terminus of MDC1 (clone P2B11; LSBio, Seattle, WA; Millipore/Sigma), and β-actin (anti-actin mAb (Novus Biologicals).

Techniques: In Vitro, Blocking Assay, Activation Assay