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: Nature Communications

Article Title: A common MET polymorphism harnesses HER2 signaling to drive aggressive squamous cell carcinoma

doi: 10.1038/s41467-020-15318-5

Figure Lengend Snippet: a , b Stable isotope labeling with amino acids in cell culture (SILAC) was performed to identify novel binding partners. MET wt-tGFP and MET N375S-tGFP cells were labeled with heavy (H) and light (L) amino acids. The cutoff values for SILAC ratios, after normalizing with MET expression, were set at (>2, <0.5) respectively. a Scatter plot of transformed MET wt /MET N375S ratios of membrane-bound proteins. Both axes represent MET wt (H)/MET N375S (L) and MET wt (L)/MET N375S (H) ratios, respectively. b List of various membranous proteins identified in SILAC analysis found to be associated with MET N375S . c Interaction of ectopic MET and endogenous HER2 in H2170 MET wt-tGFP and MET N375S-tGFP cells was detected with immunoprecipitation and immunoblotting. Left, input controls. Total MET and HER2 band intensities, normalized to input controls and relative to MET wt , are shown below ( n = 5). d , e Detection of MET/HER2 co-localization (red) in EV, MET wt-tGFP , and MET N375S-tGFP cells with proximity ligation assay (PLA). Representative images are shown ( d ), with the PLA signals quantified ( e ) and expressed as the number of signals/cell ± SD ( n > 3). Scale bar, 20 µm. f Representative confocal microscopy images of MET (Alexa-488; green) and HER2 (Alexa-594; red) in isogenic H2170 clones ( n = 2). DAPI (blue) was used as nuclear counter stain. Co-localized proteins appeared in yellow. The smaller panels are detailed views of the outlined (white) squares in the respective images. Scale bar, 20 µm. The total MET fluorescence that co-localized with HER2 signal in each variant was tabulated on the right, data presented as mean ± SD (five fields in one representative experiment). g MET/HER2 interaction in H2170 MET wt-tGFP and MET N375S-tGFP tumors shown in Fig. was detected with immunoprecipitation and immunoblotting. IgG was used as a loading control. h – k The role of Sema domain in MET N375S-tGFP cells was examined with recombinant Sema proteins, wild-type, rSema wt ; N375S mutant, rSema N375S . Cell viability of MET wt-tGFP ( h ) and MET N375S-tGFP ( i ) cells after treatment with 1, 5, 10 µg/ml of rSema wt or rSema N375S for 72 h, presented as mean ± SD ( n = 3). Two-tailed Student’s t test; * P < 0.05, ** P < 0.01, *** P < 0.001. j Immunoblots showing the total and phosphorylated expressions of MET, HER2, Src, Akt, and ERK1/2 in lysates of the indicated cell lines after treatment with 10 µg/ml rSema. β-Actin was used as a loading control. k MET/HER2 interaction in H2170 MET N375S-tGFP cells was detected with immunoprecipitation and immunoblotting after treatment with 10 µg/ml rSema. Left, input controls. l MET/HER2 interaction in H2170 MET N375S-tGFP cells was detected after serum starvation (0.1% FBS), or co-incubated with HGF (0.1% FBS + 10 ng/ml HGF). Left, input controls. m HEK293 cells were transfected with 1 µg of either pCMV6-EV-tGFP vector, MET-wt, N375S, ∆Sema, N375Q, M1268T, or Y1248H plasmid, together with pCMV6-ERBB2-DDK plasmid, for 24 h. MET/HER2 interaction in HEK293 cells was detected with immunoprecipitation and immunoblotting. Total MET and HER2 band intensities, relative to MET wt , are shown below ( n = 3). Left, input controls and phosphorylated proteins. β-Actin was used as a loading control.

Article Snippet: For immunoblotting, p-MET (Tyr1234/1235, #3077), total MET (#8198), p-HER2 (Tyr1221/1222, #2243), total HER2 (#2165), p-p38 (Thr180/Tyr182, #4511), total p38 (#8690), p-Src (Tyr416, #2101), total Src (#2108), p-ERK1/2 (Thr202/Tyr204, #4377), total ERK1/2 (#4695), p-mTOR (Ser2448, #2971), total mTOR (#2172), p-Stat3 (Tyr705, #9131), total Stat3 (#4904), p-EGFR (Tyr1068, #2234), total EGFR (#2646), p-Akt (Ser473, #4060), total Akt (#9272), α-tubulin (#2125), horseradish peroxidase (HRP)-conjugated MET (#24294), HER2 (#60388), HSP90 (#4874), and β-actin (#5125) antibodies were purchased from Cell Signaling Technology; turboGFP antibody was from Origene (#TA150041); pan-Cadherin antibody (#ab22744) from Abcam.

Techniques: Quantitative Proteomics, Cell Culture, Multiplex sample analysis, Binding Assay, Labeling, Expressing, Transformation Assay, Membrane, Immunoprecipitation, Western Blot, Proximity Ligation Assay, Confocal Microscopy, Clone Assay, Staining, Fluorescence, Variant Assay, Control, Recombinant, Mutagenesis, Two Tailed Test, Incubation, Transfection, Plasmid Preparation

Journal: Nature Communications

Article Title: A common MET polymorphism harnesses HER2 signaling to drive aggressive squamous cell carcinoma

doi: 10.1038/s41467-020-15318-5

Figure Lengend Snippet: a – c Isogenic H2170 MET N375S-tGFP cells were treated with vehicle (0.1% DMSO), MET inhibitor crizotinib (1 µM), HER2 inhibitor lapatinib (0.3 µM), or crizotinib/lapatinib combination. a Interaction of ectopic MET and endogenous HER2 was detected with immunoprecipitation and immunoblotting. Total MET and HER2 band intensities, relative to vehicle treated group, are shown below. Values represent average of three independent experiments. Left, input controls. b Detection of MET/HER2 co-localization (red) in MET N375S-tGFP cells with proximity ligation assay (PLA). Representative images are shown. Scale bar, 20 µm. c The PLA signals were quantified and expressed as number of signals/cell ± SD ( n > 3). Crizo, crizotinib; Lapa, lapatinib. d MET/HER2 interaction in H2170 MET N375S-tGFP tumors from Supplementary Fig. was detected with immunoprecipitation and immunoblotting after crizotinib treatment. Left, input controls. Cells were harvested 48 h after treatment, β-Actin was used as a loading control. e – h Immunohistochemistry staining of H2170 MET wt-tGFP and MET N375S-tGFP tumors from Supplementary Fig. . Representative images for p-MET ( e ) and p-HER2 ( g ) staining are shown. Expression of p-MET ( f ) and p-HER2 ( h ) were quantified and expressed as mean of positive-staining/100 cells ± SD ( n = 5). Scale bar, 50 µm. Two-tailed Student’s t test; * P < 0.05.

Article Snippet: For immunoblotting, p-MET (Tyr1234/1235, #3077), total MET (#8198), p-HER2 (Tyr1221/1222, #2243), total HER2 (#2165), p-p38 (Thr180/Tyr182, #4511), total p38 (#8690), p-Src (Tyr416, #2101), total Src (#2108), p-ERK1/2 (Thr202/Tyr204, #4377), total ERK1/2 (#4695), p-mTOR (Ser2448, #2971), total mTOR (#2172), p-Stat3 (Tyr705, #9131), total Stat3 (#4904), p-EGFR (Tyr1068, #2234), total EGFR (#2646), p-Akt (Ser473, #4060), total Akt (#9272), α-tubulin (#2125), horseradish peroxidase (HRP)-conjugated MET (#24294), HER2 (#60388), HSP90 (#4874), and β-actin (#5125) antibodies were purchased from Cell Signaling Technology; turboGFP antibody was from Origene (#TA150041); pan-Cadherin antibody (#ab22744) from Abcam.

Techniques: Immunoprecipitation, Western Blot, Proximity Ligation Assay, Control, Immunohistochemistry, Staining, Expressing, Two Tailed Test

Journal: Nature Communications

Article Title: A common MET polymorphism harnesses HER2 signaling to drive aggressive squamous cell carcinoma

doi: 10.1038/s41467-020-15318-5

Figure Lengend Snippet: a – c Cellular invasion on isogenic wild-type (MET wt-tGFP ) and N375S mutant (MET N375S-tGFP ) clones, treated with kinase inhibitors or siRNA silencing of MET or HER2, were being evaluated. a H2170 MET N375S-tGFP cells were treated with crizotinib (10 µM), lapatinib (0.3 µM) or a crizotinib/lapatinib combination for 36 h. Representative images of cell invasion are shown. b H2170 MET N375S-tGFP cells were co-incubated with 10 nM of the indicated siRNA overnight, and seeded in Matrigel invasion chambers for 36 h. Representative images are shown. Scale bar: 200 μm. c Percentage of invaded cells relative to vehicle control (0.1% DMSO) or Scr siRNA control were expressed as mean ± SD ( n = 3). One-way ANOVA; * P < 0.05, ** P < 0.01, *** P < 0.001. Left, immunoblots of siRNA-treated H2170 MET N375S-tGFP cells. For immunoblots, cells were harvested 48 h after siRNA treatment, and β-actin was used as a loading control. d – f Calu-1 MET wt-tGFP and MET N375S-tGFP cells were co-incubated with 10 nM of the indicated siRNA overnight, and seeded in Matrigel invasion chambers for 24 h. Representative images of cell invasion for MET wt-tGFP ( d ) and MET N375S-tGFP cells ( e ) are shown. Scale bar: 200 μm. f Percentage of invaded cells relative to Scr siRNA control in Calu-1 MET wt-tGFP and MET N375S-tGFP cells were expressed as mean ± SD ( n = 3). Left, immunoblots of siRNA-treated Calu-1 MET N375S-tGFP cells. For immunoblots, cells were harvested 48 h after siRNA treatment, and β-actin was used as a loading control. One-way ANOVA; * P < 0.05, ** P < 0.01, *** P < 0.001. g , h Anchorage-independent colony formation on isogenic H2170 MET N375S-tGFP cells, treated with kinase inhibitors or siRNA silencing of MET or HER2, were being evaluated. g H2170 MET N375S-tGFP cells were co-incubated with 10 nM of the indicated siRNA overnight, and seeded in soft agar for 4 weeks. Representative images are shown. h The number of colonies were quantified after treatment with crizotinib, lapatinib, trastuzumab, or in combination. Data were presented as percentage of colonies relative to vehicle control (0.1% DMSO) ± SD ( n = 3). i – l Efficacies of HER2 inhibitors were evaluated in xenograft models. Tumor growth of MET wt-tGFP ( i ) and MET N375S-tGFP ( j ) xenografts after treatment with trastuzumab, pertuzumab, lapatinib, and ASLAN001 were expressed at mean ± SEM ( n = 5). Two-way ANOVA; * P < 0.05, ** P < 0.01, *** P < 0.001. k Immunohistochemistry staining showing the changes in p-MET after treatment with trastuzumab in MET wt-tGFP and MET N375S-tGFP tumors. Representative images are shown. Scale bar, 50 µm. l Expression of p-MET was quantified and expressed at mean of positive-staining/100 cells ± SD ( n = 5). Two-tailed Student’s t test; * P < 0.05, ** P < 0.01, *** P < 0.001. m HEK293 cells were transfected with 1 µg of either pCMV6-ERBB2-wt, ∆D1, ∆D2, ∆D3, ∆D4, or ∆TK plasmid, together with pCMV6-MET-N375S plasmid, for 24 h. MET/HER2 interaction in HEK293 cells was detected with immunoprecipitation and immunoblotting. Left, input controls and phosphorylated proteins. β-Actin was used as a loading control. n , o Isogenic Calu-1 MET wt-tGFP ( n ) and MET N375S-tGFP ( o ) cells were engrafted into SCID mice, and treated daily with vehicle ( n = 6) or 15 mg/kg afatinib ( n = 5) starting 14 days after inoculation. Arrows indicate treatment start date. Kaplan–Meier analyses of the mice are shown.

Article Snippet: For immunoblotting, p-MET (Tyr1234/1235, #3077), total MET (#8198), p-HER2 (Tyr1221/1222, #2243), total HER2 (#2165), p-p38 (Thr180/Tyr182, #4511), total p38 (#8690), p-Src (Tyr416, #2101), total Src (#2108), p-ERK1/2 (Thr202/Tyr204, #4377), total ERK1/2 (#4695), p-mTOR (Ser2448, #2971), total mTOR (#2172), p-Stat3 (Tyr705, #9131), total Stat3 (#4904), p-EGFR (Tyr1068, #2234), total EGFR (#2646), p-Akt (Ser473, #4060), total Akt (#9272), α-tubulin (#2125), horseradish peroxidase (HRP)-conjugated MET (#24294), HER2 (#60388), HSP90 (#4874), and β-actin (#5125) antibodies were purchased from Cell Signaling Technology; turboGFP antibody was from Origene (#TA150041); pan-Cadherin antibody (#ab22744) from Abcam.

Techniques: Mutagenesis, Clone Assay, Incubation, Control, Western Blot, Immunohistochemistry, Staining, Expressing, Two Tailed Test, Transfection, Plasmid Preparation, Immunoprecipitation

Journal: Nucleic Acids Research

Article Title: Selection-free precise gene repair using high-capacity adenovector delivery of advanced prime editing systems rescues dystrophin synthesis in DMD muscle cells

doi: 10.1093/nar/gkae057

Figure Lengend Snippet: Gene correction through AdVP-based prime editing in DMD defective myoblasts. ( A ) Genome structures of AdVPs assembled for DMD prime editing. DMD target sequences before and after prime editing are depicted. The hybrid CAG promoter drives PEmax expression whilst the human U6 promoter controls the synthesis of the indicated epegRNAs and gRNAs. Spacer, primer binding site (PBS) and reverse transcriptase template (RTT) sequences of epegRNAs are marked in cyan, orange and magenta, respectively, with encoded and installed edits labelled in green. Protospacer adjacent motifs (NGG) are boxed, and nicking positions are marked by open arrowheads. ITR and Ψ, adenovirus type-5 cis -acting inverted terminal repeats and packaging signal, respectively. ( B ) AdVP-based prime editing in DMD myoblasts. Human myoblasts with a Δ48-50 genotype (DMD.Δ48-50) were transduced with different multiplicities-of-infection (MOI) of AdVP.PE2 DMD.INS+1 , AdVP.PE3 DMD.DEL-2 and AdVP.PE3 DMD.INS+1 ; TU cell −1 , transducing units per cell. Prime edits and unwarranted byproducts (i.e. indels and scaffold-derived insertions) were quantified by next-generation deep sequencing at 10 days post-transduction (50,000 paired-end reads per sample). Bars and error bars denote mean ± SEM, respectively, of three biological replicates. ( C ) Prime-editing precision indexes upon AdVP transduction. Precision indexes corresponding to the cumulative ratios of precise edits to byproduct frequencies measured in AdVP-transduced myoblasts DMD.Δ48-50 (AB1098) are plotted as mean ± SEM of the independent datapoints. Significances were calculated with one-way ANOVA followed by Dunnett's multiple comparison tests; ***0.0001 < P < 0.001, **0.001 < P < 0.01. ( D ) Detection of dystrophin and β-dystroglycan in DMD.Δ48-50 (AB1098) muscle cells prime-edited using AdVPs. Dual-color immunofluorescence microscopy for dystrophin and β-dystroglycan was done on myotubes differentiated from DMD.Δ48–50 myoblasts transduced with the indicated DMD prime-editing AdVPs. Co-localization of dystrophin and β-dystroglycan at the plasma membrane of prime-edited DMD myotubes was assessed by image merging and dystrophin- plus β-dystroglycan-specific fluorescence signal measurements (boxed areas). Nuclei are labeled with DAPI in the merged images. ( E ) Dystrophin-β-dystroglycan interaction analysis in DMD muscle cells prime-edited using AdVPs. Proximity ligation assay detection of endogenous dystrophin-β-dystroglycan interactions was carried out on myotubes differentiated from DMD.Δ48–50 myoblasts transduced with the indicated DMD prime-editing AdVPs (red foci). Healthy donor (wild-type) and untreated DMD patient-derived myotubes served as positive and negative controls, respectively. Nuclei were labelled by DAPI staining.

Article Snippet: Subsequently, the specimens were blocked with Duolink ® Blocking Solution (Sigma-Aldrich; Cat. No.: DUO82007) for 1 h at 37°C in a heated humidity chamber and were then incubated overnight at 4°C with primary antibodies against the C-terminus of dystrophin (1:100 dilution; Abcam; Cat. No.: ab15277) and β-dystroglycan (1:100 dilution; Santa Cruz Biotechnologies; Cat. No.: sc-33702) diluted in Duolink ® Antibody Diluent (Sigma-Aldrich; Cat. No.: DUO82008).

Techniques: Expressing, Binding Assay, Reverse Transcription, Transduction, Infection, Derivative Assay, Sequencing, Comparison, Immunofluorescence, Microscopy, Clinical Proteomics, Membrane, Fluorescence, Labeling, Proximity Ligation Assay, Staining

Journal: Nucleic Acids Research

Article Title: Selection-free precise gene repair using high-capacity adenovector delivery of advanced prime editing systems rescues dystrophin synthesis in DMD muscle cells

doi: 10.1093/nar/gkae057

Figure Lengend Snippet: Gene correction through AdVP-based multiplexing prime editing in DMD defective myoblasts. ( A ) Genome structure of AdVP assembled for DMD gene correction using multiplexing prime editing complexes. ITR and Ψ, adenovirus type-5 cis -acting inverted terminal repeats and packaging signal, respectively. The hybrid CAG promoter drives PEmax synthesis whilst human U6 promoters drive the expression of an epegRNA pair (i.e. epegRNA IN50 and epegRNA IN51 ) for DMD reading frame repair in muscle cells amenable to exon 51 excision (e.g. DMD.Δ48-50). ( B ) Schematics of DMD exon 51 excision through twin prime editing. Spacer, primer binding site (PBS) and reverse transcriptase template (RTT) sequences of epegRNA IN50 and epegRNA IN51 are highlighted in green, orange and magenta, respectively. The latter sequence encodes exogenous genetic information in the form of the serine recombinase Bxb1 attB recognition site. Protospacer adjacent motifs (NGG) are boxed, and nicking positions are marked by open arrowheads. Twin prime editors engage offset protospacer sequences on opposite DNA strands generating nicks that lead to the hybridization of the released single-stranded DNA strands to each PBS. The resulting free 3’ hydroxyl groups prime the synthesis of 3’ DNA flaps over RTT sequences by the reverse transcriptases. After the annealing of 3’ and 5’ DNA flaps containing edited and original DNA sequences (not shown), respectively, removal of the 5’ flaps followed by ligation of the 3’ flaps to the respective DNA excising nicks yields the intended gene-editing product, i.e. replacement of genomic DNA encompassing DMD exon 51 by the Bxb1 attB recognition site. ( C ) Testing AdVP delivery of functional prime-editing multiplexes. Human myoblasts with a Δ48-50 genotype (AB1098) were transduced with AdVP.TwinPE ΔEX51 or AdVP.PE3 DMD.DEL-2 at 50, 100, 200 and 400 TU cell −1 . Twin PE- and PE3-derived prime edits were traced at 3 days post-transduction by DNA densitometry and sequencing of target amplicons, respectively. ( D ) Dystrophin detection in DMD muscle cells corrected via AdVP delivery of prime-editing multiplexes. Western blotting was performed on myotubes differentiated from DMD.Δ48-50 myoblasts previously transduced with the indicated DMD prime-editing AdVPs (sixty micrograms of total protein loaded per lane; 6% SDS-PAGE gel). Detection of vinculin and tubulin provided for independent protein loading controls. ( E ) Dystrophin-β-dystroglycan interaction analysis in DMD muscle cells after AdVP transfer of single and dual prime-editing complexes. Detection of endogenous dystrophin-β-dystroglycan interactions by proximity ligation assays on myotubes differentiated from DMD.Δ48–50 myoblasts transduced with the indicated DMD prime-editing AdVPs (red foci). Parallel cultures of untreated DMD.Δ48–50 myotubes (Mock) served as negative controls. Nuclei were labelled by DAPI staining.

Article Snippet: Subsequently, the specimens were blocked with Duolink ® Blocking Solution (Sigma-Aldrich; Cat. No.: DUO82007) for 1 h at 37°C in a heated humidity chamber and were then incubated overnight at 4°C with primary antibodies against the C-terminus of dystrophin (1:100 dilution; Abcam; Cat. No.: ab15277) and β-dystroglycan (1:100 dilution; Santa Cruz Biotechnologies; Cat. No.: sc-33702) diluted in Duolink ® Antibody Diluent (Sigma-Aldrich; Cat. No.: DUO82008).

Techniques: Multiplexing, Expressing, Binding Assay, Reverse Transcription, Sequencing, Hybridization, Ligation, Functional Assay, Transduction, Derivative Assay, Western Blot, SDS Page, Staining

Journal: Developmental cell

Article Title: Loss of Sirtuin 1 Alters the Secretome of Breast Cancer Cells by Impairing Lysosomal Integrity

doi: 10.1016/j.devcel.2019.03.011

Figure Lengend Snippet: KEY RESOURCE TABLE

Article Snippet: Human: HK-2 , ATCC , CRL2190.

Techniques: Virus, Recombinant, Multiplex sample analysis, Isolation, Luciferase, Cloning, Reverse Transcription, Activity Assay, Control, shRNA, Sequencing, Plasmid Preparation, Software, SYBR Green Assay, Transfection