Journal: bioRxiv

Article Title: Single-cell transcriptional landscape of temporal neutrophil response to burn wound in larval zebrafish

doi: 10.1101/2024.04.01.587641

Figure Lengend Snippet: (A) Schematic view of validating signatures identified from zebrafish myeloid subsets in human burn patient expression profile. (B) Expression level distribution of Immature_Neuts signatures (top panel) and lgals3bpb_Mac1 (bottom panel) by patient TBSA shown as raincloud plots. Paired T-tests were performed between the Low and the Very High TBSA groups. (C) Expression distribution of GPR84 and GYG1 in patients by TBSA level. Spearman correlation (ρ) is calculated between gene expression and TBSA level. (D) Representative flow plots (left) and quantifications of frequencies shown as percentages (right) of GPR84+ neutrophils in burn (n = 6) and healthy (n = 4) blood. Paired samples t-test, p = 0.022.

Article Snippet: Protein-targeted staining was done with the following antibodies in Cell Staining Buffer: Alexa Fluor 700-conjugated anti-human CD16 at 1:400, PE/Fire-640-conjugated anti-human CD66b at 1:400, PerCP-conjugated anti-human CD3 at 1:200, PerCP-conjugated anti-human CD19 at 1:200, PerCP-conjugated anti-human CD56 at 1:400, PerCP-conjugated anti-human CD203c at 1:100 (BioLegend), BUV395-conjugated mouse anti-human CD45 at 1:100 (BD Biosciences), and PE-conjugated GPR84 at 1:200 (Alomone Labs).

Techniques: Expressing, Gene Expression

Journal: Cancer Immunology Research

Article Title: Programmed Cell Death Ligand 1 Expression in Osteosarcoma

doi: 10.1158/2326-6066.cir-13-0224

Figure Lengend Snippet: Figure 1. PDL1 expression in cancer and noncancer cell lines. A, relative expression of PDL1 from total RNA isolated from cell lines. B, representative PDL1 protein expression levels in corresponding high- and low- expressing cell lines by IHC. C, representative PDL1 protein expression levels in corresponding high- and low- expressing cell lines by flow cytometry.

Article Snippet: Unfortunately, the ProSci rabbit polyclonal failed to detect PDL1 on the positive control cell line SKOV3, and the XW mouse mAb exhibited unspecific binding on the negative control cell line MCF7 (Supplementary Fig. S1).

Techniques: Expressing, Isolation, Cytometry

Journal: Cancer Immunology Research

Article Title: Programmed Cell Death Ligand 1 Expression in Osteosarcoma

doi: 10.1158/2326-6066.cir-13-0224

Figure Lengend Snippet: Figure 2. Relative expression of PDL1 in 38 osteosarcoma specimens. PDL1 expression was evaluated from total RNA by quantitative real-time RT-PCR and showed that the expression levels range over 4 log (5,000-fold).

Article Snippet: Unfortunately, the ProSci rabbit polyclonal failed to detect PDL1 on the positive control cell line SKOV3, and the XW mouse mAb exhibited unspecific binding on the negative control cell line MCF7 (Supplementary Fig. S1).

Techniques: Expressing, Quantitative RT-PCR

Journal: Cancer Immunology Research

Article Title: Programmed Cell Death Ligand 1 Expression in Osteosarcoma

doi: 10.1158/2326-6066.cir-13-0224

Figure Lengend Snippet: Figure 3. Overall survival of 37 patients with osteosarcoma in relation to PDL1 gene expression. The median overall survival for PDL1-low patients was 89 months compared with 28 months for PDL1-high patients, which showed a trend but was not statistically significant (P ¼ 0.0544).

Article Snippet: Unfortunately, the ProSci rabbit polyclonal failed to detect PDL1 on the positive control cell line SKOV3, and the XW mouse mAb exhibited unspecific binding on the negative control cell line MCF7 (Supplementary Fig. S1).

Techniques: Gene Expression

Journal: Cancer Immunology Research

Article Title: Programmed Cell Death Ligand 1 Expression in Osteosarcoma

doi: 10.1158/2326-6066.cir-13-0224

Figure Lengend Snippet: Figure 4. Correlation between PDL1 gene expression and TILs by IHC. A, representative TILs in osteosarcoma tissues (400); score 0, no TILs; 1, rare/few TILs; 2, brisk/prominent TILs. B, significant positive correlation was shown between PDL1 gene expression and TILs in patients with osteosarcoma (P ¼ 0.0117).

Article Snippet: Unfortunately, the ProSci rabbit polyclonal failed to detect PDL1 on the positive control cell line SKOV3, and the XW mouse mAb exhibited unspecific binding on the negative control cell line MCF7 (Supplementary Fig. S1).

Techniques: Gene Expression

Journal: Cancer Immunology Research

Article Title: Programmed Cell Death Ligand 1 Expression in Osteosarcoma

doi: 10.1158/2326-6066.cir-13-0224

Figure Lengend Snippet: Figure 5. Characterization of the origins of metastases. A, PDL1 expression is significantly higher in metastatic osteosarcoma tumors that originate from lung than from other locations (P ¼ 0.0024). B, TILs also exhibit a positive correlation with pulmonary osteosarcoma metastasis compared with nonpulmonary metastasis (P ¼ 0.0443).

Article Snippet: Unfortunately, the ProSci rabbit polyclonal failed to detect PDL1 on the positive control cell line SKOV3, and the XW mouse mAb exhibited unspecific binding on the negative control cell line MCF7 (Supplementary Fig. S1).

Techniques: Expressing

Journal: Nucleic Acids Research

Article Title: A cancer persistent DNA repair circuit driven by MDM2, MDM4 (MDMX), and mutant p53 for recruitment of MDC1 and 53BP1 on chromatin

doi: 10.1093/nar/gkaf627

Figure Lengend Snippet: Reduced MDM2 protein in T47D cells causes reduced chromatin phosphoproteins 53BP1 and MDC1. ( A ) Experimental design workflow of the SILAC analysis (created in BioRender. Harmon, K. (2025) https://BioRender.com/oyd7ala ). Chromatin isolated from a mixture of T47D vector control cells (MDM2-competent) cultured in natural amino acid medium, and T47Dshmdm2 cells (MDM2-depleted) cultured in heavy isotope amino acid medium, was subjected to proteolysis followed by phospho-peptide purification and enrichment. Scatter plot represents the H/L ratio versus abundance of peptides identified by mass spectrometry, with those corresponding to TP53BP1 (magenta), TP53 (blue), MCM2 (green), and MDC1(brown) highlighted. ( B ) Chromatin (5 μg) isolated from T47D vector control, T47Dshmdm2, and T47Dshmdmx cells was subjected to SDS–PAGE/western blot analysis for 53BP1, MDC1, MCM4, lamin A/C, and mtp53. ( C and D ) IF of total 53BP1 (i), phospho-53BP1 ser25 (ii), or phospho- 53BP1 ser1778 (iii) within T47D vector control nuclei [(i) 528, (ii) 549, and (iii) 501], T47Dshmdm2 nuclei [(i) 549,(ii) 520, and (iii) 501], and T47Dshmdmx nuclei [(i) 623, (ii) 376, and (iii) 501]. Confocal images for six fields for each were acquired and the number of 53BP1 foci per nucleus from each cell population indicated above was determined. Representative data ( n = 3) with mean, 95% CI, and Kruskal–Wallis statistical significance test prepared as described in the “Materials and methods” section. **** Indicates a p-value less than or equal to 0.0001, ** indicates a p-value less than or equal to 0.01, * indicates a p-value less than or equal to 0.05, and ns is nonsignificant.

Article Snippet: Antibodies used for western blotting (WB), immunofluorescence staining (IF), immunoprecipitation (IP), and proximity ligation assay (PLA) were purchased from the following (usage denoted in parenthesis): rabbit p53 Sigma cat# A300-247A (PLA), and Proteintech cat# 10442-1- AP (WB); mouse p53 DO1 Santa Cruz Biotechnology cat# sc-126 (PLA and WB); mouse p53 DO1-HRP Santa Cruz Biotechnology cat# sc-126 HRP (WB); rabbit MDMX Proteintech cat# 17914-1-AP (WB); [ ] rabbit MDM2 R&D Systems cat# AF1244 (WB); rabbit 53BP1 Cell Signaling Technology cat# 4937 (WB and IF); rabbit phospho-Serine 177853BP1 Cell Signaling Technology cat# 2675 (WB and IF); rabbit phospho-Serine 2553BP1 Sigma cat# PLA 0126 (WB, IF, and PLA); rabbit MDC1 Sigma cat# PLA0016 (WB, IF, and PLA); rabbit MCM4 Cell Signaling Technology cat# 12973 (WB); mouse Actin-HRP Sigma cat# A3854 (WB); [ ] mouse Lamin A cat# SAB4200420 (WB); mouse PARP1 BD Biosciences cat# 51-6639GR (WB); goat 53BP1 Sigma cat# PLA0303 (PLA and IP); goat anti-mouse HRP Sigma cat# A3682 (WB); goat anti-rabbit Proteintech cat# SA00001-2 (WB); mouse Cyclin A Santa Cruz Biotechnology cat# sc-271682 (WB); rabbit Cyclin A Cell Signaling Technology cat# 67955S (IF); mouse Cyclin B Santa Cruz Biotechnology cat# sc-245 (WB); rabbit p21 Cell Signaling Technology cat# 2947S (WB); γH2AX phospho-Ser139 Cell Signaling Technology cat# 9718S (WB and IF); rabbit Poly ADP-Ribose Cell Signaling Technology cat# 83732S (WB); mouse MDM2 SMP14 Santa Cruz Biotechnology cat# sc-965 (IP); mouse IgG Santa Cruz Biotechnology cat# sc-2025 (IP); Purified mouse MDM2 4B2 [ ]; and purified mouse MDM2 2A9 [ ] were used for PLA and IP and prepared as described [ ].

Techniques: Multiplex sample analysis, Isolation, Plasmid Preparation, Control, Cell Culture, Purification, Mass Spectrometry, SDS Page, Western Blot

Journal: Nucleic Acids Research

Article Title: A cancer persistent DNA repair circuit driven by MDM2, MDM4 (MDMX), and mutant p53 for recruitment of MDC1 and 53BP1 on chromatin

doi: 10.1093/nar/gkaf627

Figure Lengend Snippet: 53BP1 in breast cancer cells interacts with both mtp53 and MDM2. ( A ) Relative abundance of mtp53, MDM2, MDMX, 53BP1, and MDC1 within whole cell extracts (20 μg) prepared from T47D (L194F) and MDA-MB-231 (R280K) cell lines determined by SDS–PAGE/western blot analysis. ( B–D ) Association of mtp53, MDM2, and 53BP1 in vivo measured using the PLA. PLA analyses of mtp53-53BP1 (panel B), MDM2-mtp53 (panel C), and MDM2-53BP1 (panel D) within T47D and MDA-MB-231 cells were performed as described in the “Materials and methods” section; primary antibodies are PLA rabbit anti-p53, PLA goat anti-53BP1, and mouse anti-MDM2 4B2. Confocal images for 4–6 fields for each were acquired and the number of PLA foci per nucleus for each cell population was determined ( n = 3 for T47D, n = 2 for MDA-MB-231). Representative data with mean, 95% CI, and Kruskal–Wallis statistical significance test prepared as described in the “Materials and methods” section from the indicated number of cells: Panel B vector (T47D = 276; MDA-MB-231 = 108), shmdmx (T47D = 239; MDA-MB-231 = 128), and shmdm2 (T47D = 310; MDA-MB-231 = 106); Panel C vector (T47D = 92; MDA-MB-231 = 104), shmdmx (T47D = 108; MDA-MB-231 = 131), and shmdm2 (T47D = 101; MDA-MB-231 = 136); Panel D vector (T47D = 761; MDA-MB-231 = 129), shmdmx (T47D = 529; MDA-MB-231 = 142), and shmdm2 (T47D = 964; MDA-MB-231 = 163). **** Indicates a p-value less than or equal to 0.0001, ** indicates a p-value less than or equal to 0.01, * indicates a p-value less than or equal to 0.05, and ns is nonsignificant.

Article Snippet: Antibodies used for western blotting (WB), immunofluorescence staining (IF), immunoprecipitation (IP), and proximity ligation assay (PLA) were purchased from the following (usage denoted in parenthesis): rabbit p53 Sigma cat# A300-247A (PLA), and Proteintech cat# 10442-1- AP (WB); mouse p53 DO1 Santa Cruz Biotechnology cat# sc-126 (PLA and WB); mouse p53 DO1-HRP Santa Cruz Biotechnology cat# sc-126 HRP (WB); rabbit MDMX Proteintech cat# 17914-1-AP (WB); [ ] rabbit MDM2 R&D Systems cat# AF1244 (WB); rabbit 53BP1 Cell Signaling Technology cat# 4937 (WB and IF); rabbit phospho-Serine 177853BP1 Cell Signaling Technology cat# 2675 (WB and IF); rabbit phospho-Serine 2553BP1 Sigma cat# PLA 0126 (WB, IF, and PLA); rabbit MDC1 Sigma cat# PLA0016 (WB, IF, and PLA); rabbit MCM4 Cell Signaling Technology cat# 12973 (WB); mouse Actin-HRP Sigma cat# A3854 (WB); [ ] mouse Lamin A cat# SAB4200420 (WB); mouse PARP1 BD Biosciences cat# 51-6639GR (WB); goat 53BP1 Sigma cat# PLA0303 (PLA and IP); goat anti-mouse HRP Sigma cat# A3682 (WB); goat anti-rabbit Proteintech cat# SA00001-2 (WB); mouse Cyclin A Santa Cruz Biotechnology cat# sc-271682 (WB); rabbit Cyclin A Cell Signaling Technology cat# 67955S (IF); mouse Cyclin B Santa Cruz Biotechnology cat# sc-245 (WB); rabbit p21 Cell Signaling Technology cat# 2947S (WB); γH2AX phospho-Ser139 Cell Signaling Technology cat# 9718S (WB and IF); rabbit Poly ADP-Ribose Cell Signaling Technology cat# 83732S (WB); mouse MDM2 SMP14 Santa Cruz Biotechnology cat# sc-965 (IP); mouse IgG Santa Cruz Biotechnology cat# sc-2025 (IP); Purified mouse MDM2 4B2 [ ]; and purified mouse MDM2 2A9 [ ] were used for PLA and IP and prepared as described [ ].

Techniques: SDS Page, Western Blot, In Vivo, Plasmid Preparation

Journal: Nucleic Acids Research

Article Title: A cancer persistent DNA repair circuit driven by MDM2, MDM4 (MDMX), and mutant p53 for recruitment of MDC1 and 53BP1 on chromatin

doi: 10.1093/nar/gkaf627

Figure Lengend Snippet: The MDM2–53BP1 interaction is promoted by the mtp53 C-terminus. ( A ) The C-terminus of mtp53 R273H within MDA-MB-468 was modified using CRISPR–Cas9 to create the cell line MDA-MB-468 R273Hfs347Δ360-393 (termed G6; mtp53 derivative R273HΔC). ( B ) Relative protein levels of 53BP1, MDM2, and mtp53 within MDA-MB-468 (25, 12.5, 6.25, and 3.125 μg) and G6 (25 μg) cell lines was examined by SDS–PAGE/western blot analysis. ( C ) Loss of mtp53 C-terminus disrupts mtp53 co-IP with MDM2. Extracts from MDA-MB-468 and G6 cell lines were incubated with either normal mouse IgG (negative control) or anti-MDM2 antibodies 4B2 (lanes 1–7) or SMP14 (lanes 8–14), and IP reactions were examined for MDM2 and mtp53 by western blot analysis. Lanes 1–7: 4B2 IP reactions from 800 μg of extract (input); lanes contain 10% of total IP and 2% of input. Lanes 8–14: SMP14 IP reactions from 1600 μg of extract; lanes contain 12.5% each IP and 0.5% of input. Lanes labeled 2× (lane 7 for the 4B2 IPs and lane 14 for the SMP14 IPs) contain twice the amount of the G6 extract MDM2 IP; a lighter exposure of mtp53 input is presented due to the vast excess of mtp53 compared to MDM2 within the MDA-MB-468 cell lines. ( D–F ) PLA analysis of mtp53–53BP1 (panel D), MDM2–mtp53 (panel E), and MDM2–53BP1 (panel F) in MDA-MB-468 and G6. PLA analysis of the indicated proteins was measured using PLA rabbit anti-p53, PLA goat anti-53BP1, and mouse anti-MDM2 2A9 antibodies. Confocal images for 3–5 fields for each were acquired and the number of PLA foci per nucleus for each cell population was determined ( n = 2). Representative data with mean, 95% CI, and Kruskal–Wallis statistical significance test prepared as described in the “Materials and methods” section from the indicated number of cells in panel: (D) R273H = 120; R273HΔC = 144; (E) R273H = 131; R273HΔC = 159; (F) R273H = 102; R273HΔC = 138. **** Indicates a p-value less than or equal to 0.0001.

Article Snippet: Antibodies used for western blotting (WB), immunofluorescence staining (IF), immunoprecipitation (IP), and proximity ligation assay (PLA) were purchased from the following (usage denoted in parenthesis): rabbit p53 Sigma cat# A300-247A (PLA), and Proteintech cat# 10442-1- AP (WB); mouse p53 DO1 Santa Cruz Biotechnology cat# sc-126 (PLA and WB); mouse p53 DO1-HRP Santa Cruz Biotechnology cat# sc-126 HRP (WB); rabbit MDMX Proteintech cat# 17914-1-AP (WB); [ ] rabbit MDM2 R&D Systems cat# AF1244 (WB); rabbit 53BP1 Cell Signaling Technology cat# 4937 (WB and IF); rabbit phospho-Serine 177853BP1 Cell Signaling Technology cat# 2675 (WB and IF); rabbit phospho-Serine 2553BP1 Sigma cat# PLA 0126 (WB, IF, and PLA); rabbit MDC1 Sigma cat# PLA0016 (WB, IF, and PLA); rabbit MCM4 Cell Signaling Technology cat# 12973 (WB); mouse Actin-HRP Sigma cat# A3854 (WB); [ ] mouse Lamin A cat# SAB4200420 (WB); mouse PARP1 BD Biosciences cat# 51-6639GR (WB); goat 53BP1 Sigma cat# PLA0303 (PLA and IP); goat anti-mouse HRP Sigma cat# A3682 (WB); goat anti-rabbit Proteintech cat# SA00001-2 (WB); mouse Cyclin A Santa Cruz Biotechnology cat# sc-271682 (WB); rabbit Cyclin A Cell Signaling Technology cat# 67955S (IF); mouse Cyclin B Santa Cruz Biotechnology cat# sc-245 (WB); rabbit p21 Cell Signaling Technology cat# 2947S (WB); γH2AX phospho-Ser139 Cell Signaling Technology cat# 9718S (WB and IF); rabbit Poly ADP-Ribose Cell Signaling Technology cat# 83732S (WB); mouse MDM2 SMP14 Santa Cruz Biotechnology cat# sc-965 (IP); mouse IgG Santa Cruz Biotechnology cat# sc-2025 (IP); Purified mouse MDM2 4B2 [ ]; and purified mouse MDM2 2A9 [ ] were used for PLA and IP and prepared as described [ ].

Techniques: Modification, CRISPR, SDS Page, Western Blot, Co-Immunoprecipitation Assay, Incubation, Negative Control, Labeling

Journal: Nucleic Acids Research

Article Title: A cancer persistent DNA repair circuit driven by MDM2, MDM4 (MDMX), and mutant p53 for recruitment of MDC1 and 53BP1 on chromatin

doi: 10.1093/nar/gkaf627

Figure Lengend Snippet: The MDM2-mtp53 and MDM2–53BP1 interactions are Nutlin 3a sensitive. ( A ) Western blot analysis of whole cell extracts (10 μg) from MCF7 and T47D cell line populations treated for the indicated time with either vehicle or 10 μM Nutlin 3a (labeled N3a or “+” in graphs) for the indicated proteins. ( B ) Nutlin 3a does not inhibit T47D cell cycle progression. Twenty-four hour post-treatment with either vehicle or 10 μM Nutlin 3a, cells were labeled with EdU for 20 min and assayed for Cyclin A2 by immunofluorescence. Confocal images from at least three fields were acquired and the number of EdU + and Cyclin A + nuclei were quantified (tabulated in S4, panel C) in each population of vehicle-treated [vector = 289; shmdmx = 278; shmdm2 = 249] and Nutlin 3a-treated [vector = 227; shmdmx = 283; shmdm2 = 245] cells. The S/G2 fraction (total Cyclin A + nuclei) for vehicle-treated: vector = 33.6%, shmdmx = 37.8%, shmdm2 = 36.8%; Nutlin 3a-treated: vector = 38.3%, shmdmx = 27.0%, shmdm2 = 29.4%. ( C and D ) Nutlin 3a disrupts mtp53–MDM2 and 53BP1–MDM2 PLA foci in T47D. Twenty-four hour post-treatment with vehicle or 10 μM Nutlin 3a cell populations PLA interactions were measured between MDM2–mtp53 (panel C) and MDM2–53BP1 (panel D) using PLA rabbit anti-p53, PLA goat anti-53BP1, and mouse anti-MDM2 4B2 primary antibodies. Confocal images for 4–6 fields for each were acquired and the number of PLA foci per nucleus from each cell population was determined ( n = 2 for panel C and n = 3 for panel D). Representative data with mean, 95% CI, and Kruskal–Wallis statistical significance test prepared as described in the materials and methods from the indicated number of cells in panel: (C) vehicle-treated [vector = 328; shmdmx = 298; shmdm2 = 249], Nutlin 3a-treated [vector = 323; shmdmx = 364; shmdm2 = 285] and (D) vehicle-treated [vector = 543; shmdmx = 548; shmdm2 = 455], Nutlin 3a-treated [vector = 610; shmdmx = 545; shmdm2 = 618]. **** Indicates a p-value less than or equal to 0.0001.

Article Snippet: Antibodies used for western blotting (WB), immunofluorescence staining (IF), immunoprecipitation (IP), and proximity ligation assay (PLA) were purchased from the following (usage denoted in parenthesis): rabbit p53 Sigma cat# A300-247A (PLA), and Proteintech cat# 10442-1- AP (WB); mouse p53 DO1 Santa Cruz Biotechnology cat# sc-126 (PLA and WB); mouse p53 DO1-HRP Santa Cruz Biotechnology cat# sc-126 HRP (WB); rabbit MDMX Proteintech cat# 17914-1-AP (WB); [ ] rabbit MDM2 R&D Systems cat# AF1244 (WB); rabbit 53BP1 Cell Signaling Technology cat# 4937 (WB and IF); rabbit phospho-Serine 177853BP1 Cell Signaling Technology cat# 2675 (WB and IF); rabbit phospho-Serine 2553BP1 Sigma cat# PLA 0126 (WB, IF, and PLA); rabbit MDC1 Sigma cat# PLA0016 (WB, IF, and PLA); rabbit MCM4 Cell Signaling Technology cat# 12973 (WB); mouse Actin-HRP Sigma cat# A3854 (WB); [ ] mouse Lamin A cat# SAB4200420 (WB); mouse PARP1 BD Biosciences cat# 51-6639GR (WB); goat 53BP1 Sigma cat# PLA0303 (PLA and IP); goat anti-mouse HRP Sigma cat# A3682 (WB); goat anti-rabbit Proteintech cat# SA00001-2 (WB); mouse Cyclin A Santa Cruz Biotechnology cat# sc-271682 (WB); rabbit Cyclin A Cell Signaling Technology cat# 67955S (IF); mouse Cyclin B Santa Cruz Biotechnology cat# sc-245 (WB); rabbit p21 Cell Signaling Technology cat# 2947S (WB); γH2AX phospho-Ser139 Cell Signaling Technology cat# 9718S (WB and IF); rabbit Poly ADP-Ribose Cell Signaling Technology cat# 83732S (WB); mouse MDM2 SMP14 Santa Cruz Biotechnology cat# sc-965 (IP); mouse IgG Santa Cruz Biotechnology cat# sc-2025 (IP); Purified mouse MDM2 4B2 [ ]; and purified mouse MDM2 2A9 [ ] were used for PLA and IP and prepared as described [ ].

Techniques: Western Blot, Labeling, Immunofluorescence, Plasmid Preparation

Journal: Nucleic Acids Research

Article Title: A cancer persistent DNA repair circuit driven by MDM2, MDM4 (MDMX), and mutant p53 for recruitment of MDC1 and 53BP1 on chromatin

doi: 10.1093/nar/gkaf627

Figure Lengend Snippet: MDM2 promotes 53BP1–MDC1 complex formation. ( A ) Reduced 53BP1–MDC1 PLA foci in T47D lacking MDM2. EdU-labeled T47D vector and shmdm2 cells were assayed for 53BP1–MDC1 PLA foci using PLA goat anti-53BP1 and PLA rabbit anti-MDC1 antibodies. Shown under representative images ( n = 3) is the number of EdU + nuclei identified within each cell population and graphed is the number of PLA foci per nucleus. For the PLA analysis representative data with mean, 95% CI, and Kruskal–Wallis statistical significance test prepared as described in the “Materials and methods” section from the indicated number of cells: vector (total = 329, S-phase = 100, G1-G2 = 229); shmdm2 (total = 251, S-phase = 79, G1-G2 = 172). ( B ) Activation of the DDR in T47D cell lines by Etoposide but not Nutlin-3a. Whole cell extracts (20 μg) from T47D cell lines treated with either 50 μM Etoposide for 5 h (Etop), or 10 μM Nutlin 3a for 24 h (N3a) were analyzed for the indicated proteins by WB. ( C and D ) MDC1–53BP1 PLA foci are disrupted by both DDR activation and Nutlin 3a. MDC1–53BP1 PLA analyses were performed within each T47D cell line at the indicated time points post Etoposide treatment (panel C) or 24 h-post Nutlin 3a treatment (panel D) using PLA goat anti-53BP1 and PLA rabbit anti-MDC1. Confocal images for 3–6 fields for each were acquired and the number of PLA per nucleus from each cell population was determined ( n = 3 for panel C and n = 3 for panel D). Representative data with mean, 95% CI, and Kruskal–Wallis statistical significance test prepared as described in the “Materials and methods” section from the indicated number of cells in panel: (C) vehicle-treated (0 h Etop) [vector = 208; shmdmx = 198; shmdm2 = 247], 2 h Etoposide [vector = 205; shmdmx = 184; shmdm2 = 231], 5 h Etoposide [vector = 189; shmdmx = 224; shmdm2 = 176]; (D) vehicle-treated [vector = 198; shmdmx = 270; shmdm2 = 336] and Nutlin 3a-treated [vector = 380; shmdmx = 339; shmdm2 = 252]. **** Indicates a p-value less than or equal to 0.0001, ** indicates a p-value less than or equal to 0.01, * indicates a p-value less than or equal to 0.05, and ns is nonsignificant.

Article Snippet: Antibodies used for western blotting (WB), immunofluorescence staining (IF), immunoprecipitation (IP), and proximity ligation assay (PLA) were purchased from the following (usage denoted in parenthesis): rabbit p53 Sigma cat# A300-247A (PLA), and Proteintech cat# 10442-1- AP (WB); mouse p53 DO1 Santa Cruz Biotechnology cat# sc-126 (PLA and WB); mouse p53 DO1-HRP Santa Cruz Biotechnology cat# sc-126 HRP (WB); rabbit MDMX Proteintech cat# 17914-1-AP (WB); [ ] rabbit MDM2 R&D Systems cat# AF1244 (WB); rabbit 53BP1 Cell Signaling Technology cat# 4937 (WB and IF); rabbit phospho-Serine 177853BP1 Cell Signaling Technology cat# 2675 (WB and IF); rabbit phospho-Serine 2553BP1 Sigma cat# PLA 0126 (WB, IF, and PLA); rabbit MDC1 Sigma cat# PLA0016 (WB, IF, and PLA); rabbit MCM4 Cell Signaling Technology cat# 12973 (WB); mouse Actin-HRP Sigma cat# A3854 (WB); [ ] mouse Lamin A cat# SAB4200420 (WB); mouse PARP1 BD Biosciences cat# 51-6639GR (WB); goat 53BP1 Sigma cat# PLA0303 (PLA and IP); goat anti-mouse HRP Sigma cat# A3682 (WB); goat anti-rabbit Proteintech cat# SA00001-2 (WB); mouse Cyclin A Santa Cruz Biotechnology cat# sc-271682 (WB); rabbit Cyclin A Cell Signaling Technology cat# 67955S (IF); mouse Cyclin B Santa Cruz Biotechnology cat# sc-245 (WB); rabbit p21 Cell Signaling Technology cat# 2947S (WB); γH2AX phospho-Ser139 Cell Signaling Technology cat# 9718S (WB and IF); rabbit Poly ADP-Ribose Cell Signaling Technology cat# 83732S (WB); mouse MDM2 SMP14 Santa Cruz Biotechnology cat# sc-965 (IP); mouse IgG Santa Cruz Biotechnology cat# sc-2025 (IP); Purified mouse MDM2 4B2 [ ]; and purified mouse MDM2 2A9 [ ] were used for PLA and IP and prepared as described [ ].

Techniques: Labeling, Plasmid Preparation, Activation Assay

Journal: Nucleic Acids Research

Article Title: A cancer persistent DNA repair circuit driven by MDM2, MDM4 (MDMX), and mutant p53 for recruitment of MDC1 and 53BP1 on chromatin

doi: 10.1093/nar/gkaf627

Figure Lengend Snippet: Co-IP demonstrates a 53BP1–MDC1–MDM2 multiprotein complex. ( A ) Co-IP demonstrates a 53BP1–MDC1–MDM2 multiprotein complex. 53BP1 was immunoprecipitated from T47D CES as described in the “Materials and methods” section subjected to western blot analysis for 53BP1, MDC1, MDM2, and p53. Lanes are as follows: (1) cytoplasmic extract, (2) CES IP input, (3) IP with IgG, and (4) IP for 53BP1. ( B ) Inhibition of ATM promotes MDM2 activity. Western blot analysis for the indicated proteins within extracts (10 μg) from T47D populations treated for 24 h with either vehicle,10 μM ALRN-6924 (MDM2/X dual inhibitor), 10 μM KU-55933 (ATMi; ATM inhibitor) or both. ( C ) The MDM2 inhibitor ALRN-6924 reduces whereas the ATM inhibitor increases MDC1–53BP1 PLA foci in T47D. Twenty-four hour post treatment with either vehicle,10 μM ALRN-6924, 10 μM KU-55933 (ATMi) or both T47D vector and shmdm2 cells were labeled with EdU for 20 min and then assayed for 53BP1–MDC1 PLA foci using PLA goat anti-53BP1 and PLA rabbit anti-MDC1 antibodies. Confocal images for several fields were acquired and the number of PLA foci/nucleus from each cell population was determined. Representative data with mean, 95% CI, and Kruskal–Wallis statistical significance test prepared as described in the “Materials and methods” section from the indicated number of cells in: vehicle-treated [vector = 186; shmdm2 = 87], ATMi-treated [vector = 163; shmdm2 = 188], ALRN-6924-treated [vector = 196; shmdm2 = 176], ALRN-6924 + ATMi-treated [vector = 154; shmdm2 = 159]. **** Indicates a p-value less than or equal to 0.0001, ** indicates a p-value less than or equal to 0.01, * indicates a p-value less than or equal to 0.05, and ns is nonsignificant.

Article Snippet: Antibodies used for western blotting (WB), immunofluorescence staining (IF), immunoprecipitation (IP), and proximity ligation assay (PLA) were purchased from the following (usage denoted in parenthesis): rabbit p53 Sigma cat# A300-247A (PLA), and Proteintech cat# 10442-1- AP (WB); mouse p53 DO1 Santa Cruz Biotechnology cat# sc-126 (PLA and WB); mouse p53 DO1-HRP Santa Cruz Biotechnology cat# sc-126 HRP (WB); rabbit MDMX Proteintech cat# 17914-1-AP (WB); [ ] rabbit MDM2 R&D Systems cat# AF1244 (WB); rabbit 53BP1 Cell Signaling Technology cat# 4937 (WB and IF); rabbit phospho-Serine 177853BP1 Cell Signaling Technology cat# 2675 (WB and IF); rabbit phospho-Serine 2553BP1 Sigma cat# PLA 0126 (WB, IF, and PLA); rabbit MDC1 Sigma cat# PLA0016 (WB, IF, and PLA); rabbit MCM4 Cell Signaling Technology cat# 12973 (WB); mouse Actin-HRP Sigma cat# A3854 (WB); [ ] mouse Lamin A cat# SAB4200420 (WB); mouse PARP1 BD Biosciences cat# 51-6639GR (WB); goat 53BP1 Sigma cat# PLA0303 (PLA and IP); goat anti-mouse HRP Sigma cat# A3682 (WB); goat anti-rabbit Proteintech cat# SA00001-2 (WB); mouse Cyclin A Santa Cruz Biotechnology cat# sc-271682 (WB); rabbit Cyclin A Cell Signaling Technology cat# 67955S (IF); mouse Cyclin B Santa Cruz Biotechnology cat# sc-245 (WB); rabbit p21 Cell Signaling Technology cat# 2947S (WB); γH2AX phospho-Ser139 Cell Signaling Technology cat# 9718S (WB and IF); rabbit Poly ADP-Ribose Cell Signaling Technology cat# 83732S (WB); mouse MDM2 SMP14 Santa Cruz Biotechnology cat# sc-965 (IP); mouse IgG Santa Cruz Biotechnology cat# sc-2025 (IP); Purified mouse MDM2 4B2 [ ]; and purified mouse MDM2 2A9 [ ] were used for PLA and IP and prepared as described [ ].

Techniques: Co-Immunoprecipitation Assay, Immunoprecipitation, Western Blot, Inhibition, Activity Assay, Plasmid Preparation, Labeling

Journal: Nucleic Acids Research

Article Title: A cancer persistent DNA repair circuit driven by MDM2, MDM4 (MDMX), and mutant p53 for recruitment of MDC1 and 53BP1 on chromatin

doi: 10.1093/nar/gkaf627

Figure Lengend Snippet: Depletion of MDM2 increases poly(ADP-ribose) modification (PARylation) levels of chromatin bound proteins. Cytosolic ( A ) and chromatin ( B ) fractions were prepared from T47D cells with constitutive shmdm2, shmdmx, or mir30 shRNA-expressing vector cells treated with either vehicle (DMSO), or a combination of 1 mM temozolomide plus10 μM talazoparib (Temo + Tal) for 4 h, or combination of 1 mM temozolomide plus10 μM talazoparib (Temo + Tal) for 4 h and then replaced with fresh media for an additional 24 h. Ten micrograms of cytosolic or chromatin protein was loaded on a SDS–PAGE and protein levels were determined by western blot analysis using the indicated antibodies.

Article Snippet: Antibodies used for western blotting (WB), immunofluorescence staining (IF), immunoprecipitation (IP), and proximity ligation assay (PLA) were purchased from the following (usage denoted in parenthesis): rabbit p53 Sigma cat# A300-247A (PLA), and Proteintech cat# 10442-1- AP (WB); mouse p53 DO1 Santa Cruz Biotechnology cat# sc-126 (PLA and WB); mouse p53 DO1-HRP Santa Cruz Biotechnology cat# sc-126 HRP (WB); rabbit MDMX Proteintech cat# 17914-1-AP (WB); [ ] rabbit MDM2 R&D Systems cat# AF1244 (WB); rabbit 53BP1 Cell Signaling Technology cat# 4937 (WB and IF); rabbit phospho-Serine 177853BP1 Cell Signaling Technology cat# 2675 (WB and IF); rabbit phospho-Serine 2553BP1 Sigma cat# PLA 0126 (WB, IF, and PLA); rabbit MDC1 Sigma cat# PLA0016 (WB, IF, and PLA); rabbit MCM4 Cell Signaling Technology cat# 12973 (WB); mouse Actin-HRP Sigma cat# A3854 (WB); [ ] mouse Lamin A cat# SAB4200420 (WB); mouse PARP1 BD Biosciences cat# 51-6639GR (WB); goat 53BP1 Sigma cat# PLA0303 (PLA and IP); goat anti-mouse HRP Sigma cat# A3682 (WB); goat anti-rabbit Proteintech cat# SA00001-2 (WB); mouse Cyclin A Santa Cruz Biotechnology cat# sc-271682 (WB); rabbit Cyclin A Cell Signaling Technology cat# 67955S (IF); mouse Cyclin B Santa Cruz Biotechnology cat# sc-245 (WB); rabbit p21 Cell Signaling Technology cat# 2947S (WB); γH2AX phospho-Ser139 Cell Signaling Technology cat# 9718S (WB and IF); rabbit Poly ADP-Ribose Cell Signaling Technology cat# 83732S (WB); mouse MDM2 SMP14 Santa Cruz Biotechnology cat# sc-965 (IP); mouse IgG Santa Cruz Biotechnology cat# sc-2025 (IP); Purified mouse MDM2 4B2 [ ]; and purified mouse MDM2 2A9 [ ] were used for PLA and IP and prepared as described [ ].

Techniques: Modification, shRNA, Expressing, Plasmid Preparation, SDS Page, Western Blot

Journal: Cancers

Article Title: Molecular Interplay between Dormant Bone Marrow-Resident Cells (BMRCs) and CTCs in Breast Cancer

doi: 10.3390/cancers12061626

Figure Lengend Snippet: Validation of ex vivo BMRC/CTC using complementary imaging platforms. ( A ) Detection of ex vivo human CTCs by CellSearch CTC (epithelial) kit. The kit enumerates CTCs using DAPI, Pan-cytokeratin-PE (CK-PE), and CD45-APC staining. Upper panel shows a CTC clustering with a CD45+ cell (lymphocyte); lower panels show an individual CTC. Image resolution achieved in accordance with the USFDA-approved CellSearch platform. ( B ) Immunofluorescent (IF) staining of ex vivo BMRC using GCDFP15/MG cocktail. Image of a BMRC showing red IF staining against mouse primary antibody clones 23A3 (GCDFP-15) and 304-1A5 (MG), and green IF staining for rabbit clone 31A5 (MG). ( C ) IF images of single ex vivo epithelial and stem-like BMRCs and CTCs. ( D ) DEPArray images of ex vivo epithelial and stem-like BMRCs and CTCs. Image magnification: 10×. The DEPArray platform allows the capture, visualization, and downstream interrogation of single CTCs. Since the image resolution and magnification obtained from FDA-cleared CellSearch and DEPArray platforms cannot be altered by the user, we used IF to provide a high-resolution image of captured ex vivo BMRC/CTCs.

Article Snippet: IHC on mouse tissue using antibodies of mouse origin were performed using M.O.M. elite peroxidase kit; dual IHCs were performed using ImmPRESS Duet Double Staining HRP/AP Polymer Kit, and triple IHCs were performed by multiplexing with ImmPRESS-AP Anti-Rat IgG, Mouse Adsorbed Polymer Detection Kit (Vector Labs, Burlingame, CA, USA).

Techniques: Ex Vivo, Imaging, Staining, Clone Assay