Journal: Cell reports

Article Title: Functional genomics identifies N -acetyllactosamine extension of complex N -glycans as a mechanism to evade lysis by natural killer cells

doi: 10.1016/j.celrep.2024.114105

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Dead Cell Removal Kit , Miltenyi Biotec , 130-090-101.

Techniques: Virus, Cell Isolation, Recombinant, Plasmid Preparation, Selection, CRISPR, Glycoproteomics, Knock-Out, Software

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: Molecular Cell

Article Title: Bromodomain Protein BRD4 Is a Transcriptional Repressor of Autophagy and Lysosomal Function

doi: 10.1016/j.molcel.2017.04.027

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-BRD3 , Bethyl Laboratories , Cat#: A302-368A; RRID: AB_1907251.

Techniques: Control, Membrane, Virus, Recombinant, Sample Prep, Flow Cytometry, Western Blot, Microscopy, Plasmid Preparation, Phospho-proteomics, Variant Assay, shRNA, Software, CRISPR

Journal: International journal of oncology

Article Title: Expression of ribophorine II is a promising prognostic factor in human gastric adenocarcinoma.

doi: 10.3892/ijo.2016.3822

Figure Lengend Snippet: Figure 4. Detection of genome editing at RPN2 gene. (A) Cloning target RPN2 sequence using the Guide-it CRISPR/Cas9 system. A red line under AGG indicates protospacer-adjacent motif. (B) Fluorescence analysis shows that transfection with RPN2-Cas9-GFP plasmid increased GFP-expressing cells in MKN74 and KATO III cell lines. Fluorescence cell imaging were visualized 96 h following transfection. (C) Indel frequency was measured using the T7E1 assay and the percentages are shown at the bottom of each lane. (D) Protein extracted from fluorescence positive cells was analyzed for RPN2 and GAPDH by western blot assay. (E) Sanger sequencing of PCR products around gRNA binding site (red letters) and protospacer adjacent motif (PAM) site (blue letters). Wild-type reference sequences are given on the top.

Article Snippet: Anti-RPN2 Ab was from Aviva Systems and anti-GAPDH Ab was from IMGENEX.

Techniques: Cloning, Sequencing, CRISPR, Fluorescence, Transfection, Plasmid Preparation, Expressing, Imaging, Western Blot, Binding Assay

Journal: International journal of oncology

Article Title: Expression of ribophorine II is a promising prognostic factor in human gastric adenocarcinoma.

doi: 10.3892/ijo.2016.3822

Figure Lengend Snippet: Figure 6. Relationship between RPN2 and p53 in gastric adenocarcinoma. (A) p53 protein stained in brown was detected in the nucleus of primary gastric adenocarcinoma lesions. (B) Evidence for knockdown of RPN2 caused by transfection with siRNA. MKN45 cells were transfected with scrambled siRNA (control); GAPDH was used as loading control. RPN2 siRNA followed by western blot assays using anti-RPN2 antibodies. (C) The knockout of RPN2 expression in MKN45 cell line did not reduce the ability of invasion. Data are expressed as mean values ± SD from triplicate experiments.

Article Snippet: Anti-RPN2 Ab was from Aviva Systems and anti-GAPDH Ab was from IMGENEX.

Techniques: Staining, Knockdown, Transfection, Control, Western Blot, Knock-Out, Expressing

Journal: eLife

Article Title: LRG1 is an adipokine that promotes insulin sensitivity and suppresses inflammation

doi: 10.7554/eLife.81559

Figure Lengend Snippet: ( A ) Search strategy for identification of novel adipokines with a potential role in whole-body metabolism. ( B ) Relative Lrg1 mRNA levels across a panel of tissues from 8-week-old male B6 mice. Visc, visceral fat depots; SubQ, subcutaneous fat depots. n=4–5 biological replicates per group. ( C ) Relative Lrg1 mRNA levels of indicated adipose tissues fractionated into mature adipocytes and SVF. ****p<0.0001 from Šídák post hoc test following two-way ANOVA. n=4 biological replicates per group. ( D ) Relative Lrg1 mRNA levels during in vitro differentiation of primary SVF into adipocytes. n=3 technical replicates per group. ( E ) Western blot of LRG1 and CFD in CM before (day 0) and after (day 7) in vitro adipogenic differentiation, in technical replicates. ( F ) Serum western blot of LRG1 from male B6 mice on standard chow or HFD for indicated weeks. HFD was started at 6 weeks of age. Each lane represents a biological replicate. ( G ) Relative Lrg1 mRNA levels of indicated tissues from mice on standard chow or HFD for 15 weeks. HFD was started at 6 weeks of age. ***p<0.001 from two-sided Welch’s t -test. n=6 biological replicates per group. ( H ) Relative Lrg1 mRNA levels of primary SubQ adipocytes treated with recombinant TNFα and 850 nM insulin for 6 hr. **p<0.01, ***p<0.001 from Dunnett post hoc test following one-way ANOVA. n=3 technical replicates per group. ( I ) Western blot of LRG1 in CM of primary SubQ adipocytes treated with PBS or 100 ng/mL recombinant TNFα without insulin for 24 hr. Each lane represents a technical replicate. Data are presented as mean ± SEM. Figure 3—source data 1. Uncropped gel and western blot images in and . Figure 3—source data 2. Raw gel and western blot images in and .

Article Snippet: Peptide, recombinant protein , Recombinant human TNFα , R and D Systems , Cat# 210-TA-020/CF , .

Techniques: In Vitro, Western Blot, Recombinant

Journal: eLife

Article Title: LRG1 is an adipokine that promotes insulin sensitivity and suppresses inflammation

doi: 10.7554/eLife.81559

Figure Lengend Snippet: ( A ) α-FLAG co-IP of C-terminally FLAG-tagged LRG1 and Cyt c from the serum of AAV-transduced db/db mice at 7 weeks of age. ( B ) Western blot of Cyt c in the serum of db/db or littermate m/m mice at 7 or 10 weeks of age. ( C ) Western blot of Cyt c in the serum of B6 mice on standard chow or HFD for indicated weeks. HFD was started at 6 weeks of age. Developed from the same membrane in . ( D ) Western blot of Cyt c in the CM of primary SubQ adipocytes under conditions promoting cell death. SubQ cells were treated with 1 µM staurosporine (STS) or DMSO for 24 hr (top); placed in normoxic or hypoxic chambers for 24 hr (middle); or treated with 100 ng/mL recombinant TNFα or PBS without insulin for 24 hr. Developed from the same membrane in (bottom). ( E ) Western blot of Cyt c in the insterstitial fluid (ISF) of eWAT and iWAT from B6 mice on standard chow or HFD for indicated weeks. ( F ) Relative mRNA levels of a panel of pro-inflammatory genes in BMDMs in response to 30 µg/mL equine Cyt c or 10 µM TAK-242, a TLR4 inhibitor. BMDMs were treated with Cyt c or PBS for 6 hr following 1 hr pre-incubation with TAK-242 or DMSO. ****p<0.0001 from Tukey post hoc test results following two-way ANOVA. n=3 technical replicates per group. ( G ) Relative mRNA levels of a panel of pro-inflammatory genes in BMDMs in response to 20 µg/mL equine Cyt c and 50 µg/mL recombinant human LRG1. Indicated reagents or PBS were rotated for 1 hr at RT prior to BMDM treatment for 6 hr. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 from Tukey post hoc test results following two-way ANOVA. n=3–4 technical replicates per group. ( H ) Western blot of intact and cleaved Caspase-3 in eWAT of AAV-transduced db/db mice. ( I ) Western blot of LRG1 and Cyt c in the serum and ISF of db/db mice at 10 weeks of age. Vinculin was used to assess tissue leakage during sample preparation. ( J ) Schematic depicting proposed mechanism. Data are presented as mean ± SEM. Figure 7—source data 1. Labeled uncropped western blot images in and . Figure 7—source data 2. Raw western blot images in and .

Article Snippet: Peptide, recombinant protein , Recombinant human TNFα , R and D Systems , Cat# 210-TA-020/CF , .

Techniques: Co-Immunoprecipitation Assay, Western Blot, Membrane, Recombinant, Incubation, Sample Prep, Labeling

Journal: eLife

Article Title: LRG1 is an adipokine that promotes insulin sensitivity and suppresses inflammation

doi: 10.7554/eLife.81559

Figure Lengend Snippet:

Article Snippet: Peptide, recombinant protein , Recombinant human TNFα , R and D Systems , Cat# 210-TA-020/CF , .

Techniques: Produced, CRISPR, Electroporation, Plasmid Preparation, Western Blot, Magnetic Beads, Immunoprecipitation, Recombinant, Protein Enrichment, Sequencing

Journal: Nature communications

Article Title: Mutations in SLC39A14 disrupt manganese homeostasis and cause childhood-onset parkinsonism-dystonia.

doi: 10.1038/ncomms11601

Figure Lengend Snippet: Figure 2 | SLC39A14 deficiency causes hypermanganesemia and neurodegeneration that responds to chelation treatment with Na2CaEDTA. (a) Liver MRIs of a patient with SLC30A10 deficiency, individual E-II-2 with SLC39A14 mutations and a control subject. The extensive signal hyperintensity on T1-weighted imaging caused by hepatic Mn deposition in SLC30A10 deficiency is absent in individual E-II-2. There is only a subtle degree of T1- hyperintensity when compared with the control subject. Signal intensity of the liver (yellow arrow) was compared with that of the spleen (blue arrow). (b) Brain histology from post-mortem examination of subject D-II-1. Sections of globus pallidus and dentate nucleus stained with hematoxylin and eosin (H&E) show marked neuronal loss with only occasional remaining neurons (arrow) accompanied by reactive astrocytosis (shown within the ribbon of the dentate nucleus (between arrows)). Scale bar, 100 mm. Luxol fast blue/cresyl violet stain of a section of the cerebral white matter demonstrates patchy loss of myelin associated with coarse vacuoles (arrow). Scale bar 200 mm. (c) Graph showing whole-blood Mn levels and urinary Mn excretion over four courses of Na2CaEDTA treatment in individual E-II-2. Arrows indicate timing of Na2CaEDTA courses (day 1, 34, 52 and 84). Administration of Na2CaEDTA causes a significant increase in urinary Mn excretion (red) accompanied by a drop in whole-blood Mn levels (blue).

Article Snippet: Immunohistochemistry on normal human brain tissue from the Oregon Brain Bank was performed by hand using the rabbit polyclonal anti-SLC39A14 antibody (1:1,000; NBP1-81551, Novus) with DAB/HRP development as above.

Techniques: Control, Imaging, Staining

Journal: Nature communications

Article Title: Mutations in SLC39A14 disrupt manganese homeostasis and cause childhood-onset parkinsonism-dystonia.

doi: 10.1038/ncomms11601

Figure Lengend Snippet: Figure 3 | SLC39A14 isoforms 1 and 2 show differences in tissue expression, Mn uptake and transcriptional regulation. (a) Immunostaining for SLC39A14 (all isoforms) in healthy control liver shows cell membrane expression (yellow arrow) and punctate cytoplasmic staining (black arrow); scale bar, 50mm; Abcam anti-SLC39A14 antibody (ab106568, 1:100); and in globus pallidus (GP) from a healthy control shows positively stained large neurons (black arrow); scale bar, 100 mm; Novus anti-SLC39A14 antibody (NBP1-81551, 1:1,000). (b,c) Confocal images demonstrating the subcellular localization of fluorescently tagged human SLC39A14 isoform 1 (b) and isoform 2 (c) expressed in zebrafish embryos. Immunostaining for EGFP and mCherry at 6 hpf shows that both isoforms are expressed at the cell membrane (co-localization with membrane mCherry) and in the cytoplasm. 40,6-diamidino-2-phenylindole (DAPI) was used as a nuclear stain. Scale bar, 50mm. (d) RT–PCR of adult and fetal human tissues showing differences in mRNA expression between isoform 1 (ubiquitous expression in the tissues examined) and isoform 2 (* absent expression in brain, heart, skeletal muscle and skin). Amplicons for isoform 1 and 2 span 139 bp and 109 bp, respectively. Hypoxanthine-guanine phosphoribosyltransferase (HPRT) was used as a housekeeping gene. L, 100 bp ladder (Promega). (e) Graph showing Mn uptake in HEK-293 cells transiently transfected with wild-type SLC39A14 isoform 1 and 2, and empty pCS2þ vector following 15 and 30min of MnCl2 (1mM) exposure. Both isoforms facilitate Mn uptake. Cells transfected with isoform 2 have significantly higher Mn levels (P ¼ 0.009). Data are presented as means±s.d. from two independent experiments. Statistical analysis was performed using one-way ANOVA (P ¼ 0.0002 (15 min), P ¼ 0.0002 (30 min)) and Tukey’s multiple comparison test (***Po0.001). (f) Graph showing slc39a14 transcript levels assessed by qRT–PCR in 5 dpf zebrafish larvae after exposure to 500 mM MnCl2 for 24h. Overall transcript levels are increased (P ¼ 0.035). Transcript levels of isoform 1 are unchanged (P ¼ 0.41) while those of isoform 2 show a 4.6-fold increase (P ¼ 0.005). Data are presented as means±s.d. from three independent experiments. Statistical analysis was performed using Student’s two-tailed t-test on individual DCt values (*Po0.05, **Po0.01). ANOVA, analysis of variance.

Article Snippet: Immunohistochemistry on normal human brain tissue from the Oregon Brain Bank was performed by hand using the rabbit polyclonal anti-SLC39A14 antibody (1:1,000; NBP1-81551, Novus) with DAB/HRP development as above.

Techniques: Expressing, Immunostaining, Control, Membrane, Staining, Reverse Transcription Polymerase Chain Reaction, Transfection, Plasmid Preparation, Comparison, Quantitative RT-PCR, Two Tailed Test

Journal: Nature communications

Article Title: Mutations in SLC39A14 disrupt manganese homeostasis and cause childhood-onset parkinsonism-dystonia.

doi: 10.1038/ncomms11601

Figure Lengend Snippet: Figure 4 | In vitro expressed mutant SLC39A14 shows compromised Mn uptake despite normal subcellular localization. (a) Immunoblot of whole-cell lysates of stably transfected HEK-293 cells showing expression of the wild-type and three mutant SLC39A14 proteins. Actin was used as a loading control. (b) Quantification of SLC39A14 protein levels relative to actin expression revealed no significant difference between wild-type and mutant SLC39A14. Data are presented as means±s.d. from three repeat experiments. Statistical analysis was performed using one-way ANOVA (P ¼ 0.071). (c,d) Confocal images of stably transfected HEK-293 cells expressing FLAG-tagged SLC39A14 (green) showing that wild-type and mutant transporters co-localize with wheat germ agglutinin (WGA)-labelled plasma membrane (red) in (c) non-permeabilized HEK-293 cells and show additional intracellular localization in (d) permeabilized HEK-293 cells. Nuclei are stained with DAPI. Scale bars, 10 mm. (e) Mn influx studies in HEK-293 cells stably expressing wild-type (WT) and mutant forms of SLC39A14 show a decrease in Mn uptake for all SLC39A14 mutants (P ¼ 0.000 [F98V], P ¼ 0.000 [G383R], P ¼ 0.02 [N469K]. Data are presented as means±s.d. from three technical replicates. Statistical analysis was performed using one-way ANOVA (P ¼ 0.000) and Tukey’s multiple comparison test (*Po0.05, ***Po0.001). ANOVA, analysis of variance.

Article Snippet: Immunohistochemistry on normal human brain tissue from the Oregon Brain Bank was performed by hand using the rabbit polyclonal anti-SLC39A14 antibody (1:1,000; NBP1-81551, Novus) with DAB/HRP development as above.

Techniques: In Vitro, Mutagenesis, Western Blot, Stable Transfection, Transfection, Expressing, Control, Clinical Proteomics, Membrane, Staining, Comparison

Journal: Nature communications

Article Title: Mutations in SLC39A14 disrupt manganese homeostasis and cause childhood-onset parkinsonism-dystonia.

doi: 10.1038/ncomms11601

Figure Lengend Snippet: Figure 5 | Zebrafish slc39a14 is expressed during early zebrafish development and significantly reduced in slc39a14U801 mutants. (a) RT–PCR showing slc39a14 expression between 3 and 120 hpf in zebrafish. L, 100 bp ladder (Promega). (b) Whole-mount in situ hybridization using a DIG-labelled antisense RNA probe showing slc39a14 expression in the proximal convoluted (black arrows) and straight (red arrows) pronephric tubules in zebrafish larvae at 4 dpf. Top, lateral view; bottom, dorsal view. Scale bar, 200 mm. (c) DNA sequence of the region within exon 5 of slc39a14 targeted by a CRISPR guide RNA is highlighted in yellow and the 2-bp deletion introduced in the slc39a14U801 mutant indicated by dashes. Pam sequence underlined. (d) qRT–PCR demonstrates a 2.2-fold reduction in slc39a14 expression in homozygous slc39a14U801 mutants (*P ¼ 0.0117). Primers were designed to detect all slc39a14 transcripts (Supplementary Table 4). Ef1a was used as a reference gene. Data are presented as means±s.d. from three independent experiments. Statistical analysis was performed using Student’s two-tailed t-test on individual DCt values (*Po00.5).

Article Snippet: Immunohistochemistry on normal human brain tissue from the Oregon Brain Bank was performed by hand using the rabbit polyclonal anti-SLC39A14 antibody (1:1,000; NBP1-81551, Novus) with DAB/HRP development as above.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, In Situ Hybridization, Sequencing, CRISPR, Mutagenesis, Quantitative RT-PCR, Two Tailed Test

Journal: Nature communications

Article Title: Mutations in SLC39A14 disrupt manganese homeostasis and cause childhood-onset parkinsonism-dystonia.

doi: 10.1038/ncomms11601

Figure Lengend Snippet: Figure 6 | Loss of slc39a14 function in zebrafish leads to increased Mn accumulation and sensitivity as well as impaired locomotor behaviour. (a) Mn levels assessed in homozygous slc39a14U801 and wild-type (WT) larvae show that mutant larvae have significantly raised Mn levels at 5 dpf (P ¼ 0.001) and 14 dpf (P ¼ 0.0002), and Mn accumulation on MnCl2 exposure (50 mM from 2 dpf) is significantly higher in mutant compared with WT larvae (P ¼ 0.000) at 5 dpf. Measurements were taken from pools of 10 larvae. Data are presented as means±s.d. from a minimum of five independent experiments. Statistical analysis was performed using Student’s two-tailed t-test (***Po0.001). (b) Graph showing Fe, Zn and Cd levels in 14 dpf mutant and WT larvae. Levels of all three metals are not significantly different between the two groups (P ¼ 0.906 [Fe], P ¼ 0.257 [Zn], P ¼ 0.834 [Cd]). Measurements were taken from pools of 10 larvae. Data are presented as means±s.d. from five independent experiments. Statistical analysis was performed using Student’s two-tailed t-test (NS, not significant). (c) Graph presenting the lethality in homozygous slc39a14U801 and WT larvae at 5 dpf on MnCl2 exposure between 2 and 5 dpf. Median lethal concentration (LC50) of MnCl2 determined by Probit regression analysis was 661 mM for WT (95% confidence interval (CI) 548–808 mM) and 377 mM (95% CI 313–455 mM) for mutant fish. Data are presented as means±s.e.m. from nine independent experiments. (d) Locomotor behaviour studies of homozygous slc39a14U801 and WT larvae show that in unexposed conditions there is no significant difference in locomotor activity; and on MnCl2 exposure, locomotor activity is markedly reduced in mutant larvae compared with WT. The locomotor behaviour was tracked during 4 and 7 dpf using automated analysis software. s/min, movement in seconds per minute. Data are presented as means±s.e.m. 12 larvae were analysed per condition. Statistical analysis was performed using two way ANOVA (i, P ¼ 0.18; ii, P ¼ 0.000) (***Po0.001; NS, not significant). ANOVA, analysis of variance.

Article Snippet: Immunohistochemistry on normal human brain tissue from the Oregon Brain Bank was performed by hand using the rabbit polyclonal anti-SLC39A14 antibody (1:1,000; NBP1-81551, Novus) with DAB/HRP development as above.

Techniques: Mutagenesis, Two Tailed Test, Concentration Assay, Activity Assay, Software

Journal: Nature communications

Article Title: Mutations in SLC39A14 disrupt manganese homeostasis and cause childhood-onset parkinsonism-dystonia.

doi: 10.1038/ncomms11601

Figure Lengend Snippet: Figure 8 | Proposed disease mechanism in patients with SLC39A14 mutations. (a) Under normal conditions nutritional Mn (red) is absorbed in the duodenum and enters the enterohepatic circulation via the portal vein from which it is transported into the liver via SLC39A14, a Mn uptake transporter (orange). Any excess Mn is rapidly removed from the systemic circulation by uptake into the liver via SLC39A14 and excreted into the bile via SLC30A10 (brown), a Mn efflux protein. (b) Dysfunction of SLC39A14 (indicated by a black X) impairs hepatic uptake of Mn for subsequent biliary excretion. Consequently, Mn accumulates in the blood and brain leading to hypermanganesemia and neurotoxicity, respectively. IVC, inferior vena cava.

Article Snippet: Immunohistochemistry on normal human brain tissue from the Oregon Brain Bank was performed by hand using the rabbit polyclonal anti-SLC39A14 antibody (1:1,000; NBP1-81551, Novus) with DAB/HRP development as above.

Techniques:

Journal: Nature Communications

Article Title: Interplay between Notch1 and Notch3 promotes EMT and tumor initiation in squamous cell carcinoma

doi: 10.1038/s41467-017-01500-9

Figure Lengend Snippet: Lineage tracing reveals mesenchymal traits and Notch1 activation in ESCC lesions. a Schematic of cell-lineage tracing experiments. b Macroscopic and fluorescent images of representative tdTomato-labeled esophagus bearing 4NQO-induced tumors (arrows). Scale bar, 1 mm. c qRT-PCR analysis for indicated genes comparing EpCAM neg to EpCAM pos cells from a representative tdTomato-labeled tumor. * P < 0.0001 and # P < 0.05 vs. EpCAM pos , n = 3. d Representative IF images for Zeb1 and E-cadherin (E-cad) in normal mucosa (top) and ESCC (bottom). Dashed line denotes interface between stroma and basal keratinocytes (top) or invasive ESCC cells (bottom). Note E-cadherin downregulation in ESCC cells with nuclear Zeb1 (arrowheads) at the tumor invasive front. Scale bar, 50 µm. e H&E, multicolor IF for E-cadherin and Zeb1, and IHC for ICN1 (ICN1 Val1744 ) in representative single-cell-derived organoids from normal mucosa or 4NQO-induced tumors. Note that spherical organoids from 4NQO-untreated control mice exhibit a differentiation gradient with predominant luminal keratinization, whereas tumor-derived organoids (neoplastic) display irregular morphology with increased cellularity and diminished keratinization. Zeb1 expression was robust in tumor organoids, particularly at invasive protrusions with decreased E-cadherin expression and detectable ICN1 expression. Box denotes area magnified in panel below. Scale bars, 20 µm. f Representative IHC image for ICN1 4NQO-induced IEN (dysplasia) containing spindle-shaped cells (arrowheads). Scale bar, 50 µm. g Quantification of ICN1 IHC scoring in normal mucosa and 4NQO-induced lesions in mice of indicated genotype. * P < 0.05 vs. p53 +/+ ( K5Cre ERT2 ;R26tdTomato lsl/lsl ) normal, # P < 0.0005 vs. p53 −/− ( K5Cre ERT2 ;R26tdTomato lsl/lsl ; p53 loxP/loxP ) normal; † P < 0.05 vs. p53 −/− IEN; ns not significant vs. p53 +/+ normal. Data in b – f represent at least three independent 4NQO-induced lesions and >20 organoids from at least two independent experimental replicates. In g , n = 5 normal, n = 8 IEN, and n = 5 invasive ESCC in p53 +/+ esophagi. N.D. not detectable. n = 3 normal, n = 7 IEN, n = 5 invasive ESCC, and n = 4 metastatic tumors in p53 −/− esophagi. Two independent experimental replicates were carried out. Bar diagrams represent mean ± s.d. in c and mean ± s.e.m. in g . Student’s t -test was used for paired data comparisons in c , g . ANOVA with Tukey’s post hoc test was used for multiple comparisons in g

Article Snippet: RNA isolation, cDNA synthesis, and qRT-PCR were done using StepOnePlusTM Real-Time PCR System (Applied Biosystems) by TaqMan ® Gene Expression Assays (Applied Biosystems) for NOTCH1 (Hs01062014_m1), Notch1 (Mm00435249_m1), NOTCH3 (Hs00166432_m1), Notch3 (Mm00435270_m1), IVL (Hs00846307_s1), CK13 (s00999762_m1), CDH1 (Hs00170423_m1), CDH2 (Hs00983062 _m1), ZEB1 (Hs00232783_m1), SNAI1 (Hs00195591_m1), and PAI1 (Hs01126606_m1), and SYBR ® Green PCR for human ACTB (β-Actin) as well as murine Cdh1 (5′-TCAAGCTCGCGGATAACCAGAACA-3′ and 5′-ATTCCCGCCTTCATGCAGTTGTTG-3′), Cdh2 (5′-ATGGCCTTTCAAACACAGCCACAG-3′ and 5′-ACAATGACGTCCACCCTGTTCTCA-3′), Zeb1 (5′-TGAGCACACAGGTAAGAGGCC-3′ and 5′-GGCTTTTCCCCAGAGTGCA-3′), Zeb2 (5′-TGATAGCCTTGCAAACCCTCTGGA-3′ and 5′-TCCTTCATTTCTTCTGGACCGGCT-3′), Twist (5′-AGCTGAGCAAGATTCAGACCCTCA-3′ and 5′-TGCAGCTTGCCATCTTGGAGT-3′), and Gapdh (5′-GGTGGTCTCCTCTGACTTCAACA-3′ and 5′-GTTGCTGTAGCCAAATTCGTTGT-3′) and as described , , .

Techniques: Activation Assay, Labeling, Quantitative RT-PCR, Derivative Assay, Control, Expressing

Journal: Nature Communications

Article Title: Interplay between Notch1 and Notch3 promotes EMT and tumor initiation in squamous cell carcinoma

doi: 10.1038/s41467-017-01500-9

Figure Lengend Snippet: Notch1 promotes ESCC tumorigenesis. a Schematic of 8×CSL-GFP reporter. Notch activation permits GFP reporter expression via concatemeric CSL-binding sites. Constitutively active UBC promoter drives tRFP expression concurrently. Cells without tRFP represent host-derived stromal cells. b Representative flow cytometry scatter plots determining 8×CSL -mediated GFP reporter activation, indicating Notch active population of tRFP-labeled ESCC cells in TE11 and EN60 xenograft tumors. On average, 43.5 ± 0.5% s.d. of EN60 and 11.2 ± 6.5% s.d. of TE11 cells comprised the live GFP Pos /tRFP Pos fraction across three independent tumors. c , d Tumor growth curves in immunodeficient mice carrying TE11 tumors of indicated genotypes. Upon xenograft transplantation, mice were treated with DOX to induce ICN1 or NOTCH 1-directed shRNA. Tumor growth was monitored for indicated time periods. In c , * P < 0.01 vs. zeo and DOX (−), # P < 0.05 vs. zeo and DOX (+), n = 6–8 per group. In d , * P < 0.05 vs. DOX (−); n = 6 per group. e TE11 cells with CRISPR/Cas9-mediated NOTCH1 deletion. Immunoblotting confirms ICN1 loss in NOTCH1 -deleted TE11 (N1 Δ ), but not non-targeted control TE11 (N1 WT ) cells. Bar graph shows tumor formation rate in immunodeficient mice 7 weeks after xenograft transplantation. * P < 0.05 vs. N1 WT , n = 8 per group. f Esophageal keratinocytes isolated from 4NQO-induced IEN (dysplasia) lesions or ESCC tumors of Notch1 loxP/loxP mice were utilized to generate 3D esophageal organoids in the presence or absence of ex vivo Cre-mediated recombination. qRT-PCR analysis confirms inhibition of Notch1 gene expression upon ex vivo Cre-mediated recombination in ESCC organoids. * P < 0.000 vs. Cre (−), n = 3. Organoid formation rate was evaluated at day 14 post-plating and is represented as relative to Cre (−) for IEN and ESCC. * P < 0.000 vs. Cre (−) IEN; # P < 0.001 vs. Cre (−) ESCC. Line graphs represent mean ± s.e.m. in c , d . Bar diagrams represent mean ± s.d. in f . At least two independent replicates were performed for all experiments. Repeated measures ANOVA with Tukey’s post hoc test were used for multiple comparisons in c , d . Chi square with Fisher’s exact test was used for percentage comparisons in e . Student’s t -test was used for paired data comparisons in f

Article Snippet: RNA isolation, cDNA synthesis, and qRT-PCR were done using StepOnePlusTM Real-Time PCR System (Applied Biosystems) by TaqMan ® Gene Expression Assays (Applied Biosystems) for NOTCH1 (Hs01062014_m1), Notch1 (Mm00435249_m1), NOTCH3 (Hs00166432_m1), Notch3 (Mm00435270_m1), IVL (Hs00846307_s1), CK13 (s00999762_m1), CDH1 (Hs00170423_m1), CDH2 (Hs00983062 _m1), ZEB1 (Hs00232783_m1), SNAI1 (Hs00195591_m1), and PAI1 (Hs01126606_m1), and SYBR ® Green PCR for human ACTB (β-Actin) as well as murine Cdh1 (5′-TCAAGCTCGCGGATAACCAGAACA-3′ and 5′-ATTCCCGCCTTCATGCAGTTGTTG-3′), Cdh2 (5′-ATGGCCTTTCAAACACAGCCACAG-3′ and 5′-ACAATGACGTCCACCCTGTTCTCA-3′), Zeb1 (5′-TGAGCACACAGGTAAGAGGCC-3′ and 5′-GGCTTTTCCCCAGAGTGCA-3′), Zeb2 (5′-TGATAGCCTTGCAAACCCTCTGGA-3′ and 5′-TCCTTCATTTCTTCTGGACCGGCT-3′), Twist (5′-AGCTGAGCAAGATTCAGACCCTCA-3′ and 5′-TGCAGCTTGCCATCTTGGAGT-3′), and Gapdh (5′-GGTGGTCTCCTCTGACTTCAACA-3′ and 5′-GTTGCTGTAGCCAAATTCGTTGT-3′) and as described , , .

Techniques: Activation Assay, Expressing, Binding Assay, Derivative Assay, Flow Cytometry, Labeling, Transplantation Assay, shRNA, CRISPR, Western Blot, Control, Isolation, Ex Vivo, Quantitative RT-PCR, Inhibition, Gene Expression

Journal: Nature Communications

Article Title: Interplay between Notch1 and Notch3 promotes EMT and tumor initiation in squamous cell carcinoma

doi: 10.1038/s41467-017-01500-9

Figure Lengend Snippet: Notch1 facilitates expansion of CD44H cells with mesenchymal properties. a qRT-PCR analysis for indicated genes comparing EN60 intratumoral CD44L and CD44H cells. * P < 0.05 vs. CD44L; # P < 0.0001 vs. CD44L; n = 3 per group. b Representative flow cytometry scatter plot determining CD44H cells in EN60 tumors grown for 4 weeks with or without DOX-induced ICN1 (EN60- ICN1 TetOn ). c Experimental design of serial transplantation experiments and tumor formation rates by CD44L and CD44H cells purified from parental xenograft tumors carrying DOX-inducible ICN1 (EN60- ICN1 TetOn and TE11- ICN1 TetOn ). Parental tumors were grown in mice without DOX treatment and dissociated for FACS-purification of CD44L and CD44H cells. Purified cells were serially transplanted (10 3 cells per injection site) into recipient mice to monitor tumor formation. Recipient mice were treated with or without DOX. TE11 carried DNMAML1 or zeo (empty vector control). * P < 0.05 for CD44L and DOX (+) vs. CD44L and DOX (−), n = 10–12 per group; ns not significant for CD44L and DOX (+) vs. CD44H (with or without DOX treatment), n = 10 per group; # P < 0.05 vs. TE11- ICN1 TetOn -zeo CD44L and DOX (+), n = 10 per group. d Flow cytometry analysis for EpCAM-negative cells in TE11 with NOTCH1 deletion (N1 Δ ) or wild-type control (N1 WT ). Cells were treated with or without 5 ng/ml TGFβ for 72 h. * P < 0.05 vs. TGFβ (−); # P < 0.0001 vs. N1 WT and TGFβ (+), n = 3. Data are presented as mean ± s.e.m. e Multicolor IF for E-cadherin and Zeb1 in representative single-cell-derived organoids from 4NQO-induced ESCC tumors of Notch1 loxP/loxP mice with or without ex vivo Cre-mediated recombination. Zeb1 expression diminished in organoids upon Cre-mediated Notch1 deletion (arrowheads). Box denotes area that is magnified in panel below. KC keratinized core of organoids. Scale bars, 20 µm. Bar diagrams represent mean ± s.d. in a , d . At least two independent replicates were performed for all experiments. Student’s t -test was used for paired data comparisons in a , d . Chi square with Fisher’s exact test was used for percentage comparisons in c

Article Snippet: RNA isolation, cDNA synthesis, and qRT-PCR were done using StepOnePlusTM Real-Time PCR System (Applied Biosystems) by TaqMan ® Gene Expression Assays (Applied Biosystems) for NOTCH1 (Hs01062014_m1), Notch1 (Mm00435249_m1), NOTCH3 (Hs00166432_m1), Notch3 (Mm00435270_m1), IVL (Hs00846307_s1), CK13 (s00999762_m1), CDH1 (Hs00170423_m1), CDH2 (Hs00983062 _m1), ZEB1 (Hs00232783_m1), SNAI1 (Hs00195591_m1), and PAI1 (Hs01126606_m1), and SYBR ® Green PCR for human ACTB (β-Actin) as well as murine Cdh1 (5′-TCAAGCTCGCGGATAACCAGAACA-3′ and 5′-ATTCCCGCCTTCATGCAGTTGTTG-3′), Cdh2 (5′-ATGGCCTTTCAAACACAGCCACAG-3′ and 5′-ACAATGACGTCCACCCTGTTCTCA-3′), Zeb1 (5′-TGAGCACACAGGTAAGAGGCC-3′ and 5′-GGCTTTTCCCCAGAGTGCA-3′), Zeb2 (5′-TGATAGCCTTGCAAACCCTCTGGA-3′ and 5′-TCCTTCATTTCTTCTGGACCGGCT-3′), Twist (5′-AGCTGAGCAAGATTCAGACCCTCA-3′ and 5′-TGCAGCTTGCCATCTTGGAGT-3′), and Gapdh (5′-GGTGGTCTCCTCTGACTTCAACA-3′ and 5′-GTTGCTGTAGCCAAATTCGTTGT-3′) and as described , , .

Techniques: Quantitative RT-PCR, Flow Cytometry, Transplantation Assay, Purification, Injection, Plasmid Preparation, Control, Derivative Assay, Ex Vivo, Expressing

Journal: Nature Communications

Article Title: Interplay between Notch1 and Notch3 promotes EMT and tumor initiation in squamous cell carcinoma

doi: 10.1038/s41467-017-01500-9

Figure Lengend Snippet: TGFβ signaling facilitates ESCC tumor growth, Notch1 signaling, and EMT. a Growth curve for mice bearing TE11 or HNSCC PDX tumors receiving either ID11 anti-TGFβ blocking antibody or control IgG via intraperitoneal injection at indicated time points (arrows). ID11 neutralizes all TGFβ isoforms. * P < 0.05 vs. IgG, n = 5–8 per group. b , c p53 +/+ mice were treated with 4NQO for 16 weeks. Six weeks following 4NQO withdrawal, mice were treated with ID11 anti-TGFβ blocking antibody or control IgG via intraperitoneal injection three times weekly for a period of 2 weeks. In b , quantification of ICN1 IHC scoring in esophageal epithelium with representative IHC images for IgG-treated and anti-TGFβ blocking antibody-treated animals. * P < 0.05; n = 4–5 per group. Scale bar, 50 µm. In c , multicolor IF for E-cadherin and Zeb1 in representative 4NQO-induced IEN lesions from IgG-treated and anti-TGFβ blocking antibody-treated animals. Data indicate mean ± s.e.m. in a , b . At least two independent replicates were performed for all experiments. Repeated measures ANOVA with Tukey’s post hoc test were used for multiple comparisons in a . Student’s t -test was used for paired data comparisons in b

Article Snippet: RNA isolation, cDNA synthesis, and qRT-PCR were done using StepOnePlusTM Real-Time PCR System (Applied Biosystems) by TaqMan ® Gene Expression Assays (Applied Biosystems) for NOTCH1 (Hs01062014_m1), Notch1 (Mm00435249_m1), NOTCH3 (Hs00166432_m1), Notch3 (Mm00435270_m1), IVL (Hs00846307_s1), CK13 (s00999762_m1), CDH1 (Hs00170423_m1), CDH2 (Hs00983062 _m1), ZEB1 (Hs00232783_m1), SNAI1 (Hs00195591_m1), and PAI1 (Hs01126606_m1), and SYBR ® Green PCR for human ACTB (β-Actin) as well as murine Cdh1 (5′-TCAAGCTCGCGGATAACCAGAACA-3′ and 5′-ATTCCCGCCTTCATGCAGTTGTTG-3′), Cdh2 (5′-ATGGCCTTTCAAACACAGCCACAG-3′ and 5′-ACAATGACGTCCACCCTGTTCTCA-3′), Zeb1 (5′-TGAGCACACAGGTAAGAGGCC-3′ and 5′-GGCTTTTCCCCAGAGTGCA-3′), Zeb2 (5′-TGATAGCCTTGCAAACCCTCTGGA-3′ and 5′-TCCTTCATTTCTTCTGGACCGGCT-3′), Twist (5′-AGCTGAGCAAGATTCAGACCCTCA-3′ and 5′-TGCAGCTTGCCATCTTGGAGT-3′), and Gapdh (5′-GGTGGTCTCCTCTGACTTCAACA-3′ and 5′-GTTGCTGTAGCCAAATTCGTTGT-3′) and as described , , .

Techniques: Blocking Assay, Control, Injection

Journal: Nature Communications

Article Title: Interplay between Notch1 and Notch3 promotes EMT and tumor initiation in squamous cell carcinoma

doi: 10.1038/s41467-017-01500-9

Figure Lengend Snippet: TGFβ-mediated Notch1 activation permits EMT and suppresses differentiation. a Representative flow cytometry scatter and histogram plots for 8×CSL-GFP reporter activity in indicated subpopulations of EPC2T cells treated with TGFβ or CaCl 2 for 72 h. b , c Flow cytometry determined CD44H cells induced in FACS-purified CD44L cells with indicated conditions or genotypes. Cells were treated with TGFβ or DOX for 7 days. * P < 0.0001 vs. TGFβ (−); # P < 0.005 vs. TGFβ (+) and DMSO, GFP , or non-silencing scrambled (NS) shRNA in b ; n = 3. * P < 0.05 vs. TGFβ (−) and DOX (−); ns not significant vs. TGFβ (−) and DOX (−); # P < 0.05 vs. TGFβ (+) and DOX (−) in c ; n = 3. d Representative H&E images of OTC reconstituted with CD44L or CD44H cells. Epi epithelia, Str stroma. Scale bar, 50 μm. e Representative flow cytometry scatter plots of purified CD44H cells treated with SB431542 or vehicle (control) for 14 days. SB431542 increased CD44L cell content (upper left quadrant) to 0.35 ± 0.08% as compared to 1.2 ± 0.1% s.d. in vehicle ( P < 0.0005, n = 3). f qRT-PCR analysis for indicated genes in FACS-purified CD44H cells treated with or without SB431542. mRNA level for each gene in vehicle-treated cells was set as 1. * P < 0.0005 vs. vehicle, n = 3. g Heat map of gene array results from EPC2T derivative (EPC2T- ICN1 TetOn ) treated with DOX and/or TGFβ ( n = 3 per condition). “Differentiation” genes were significantly increased (≥1.5-fold) by DOX-induced ectopic ICN1 alone, but suppressed by TGFβ ( P < 0.05 by two-way ANOVA). “EMT” genes activated by TGFβ (≥1.5-fold) were significantly augmented by ectopic ICN1 ( P < 0.05 by two-way ANOVA). h qRT-PCR analysis validates gene array results in g . mRNA level for DOX (−) and TGFβ (−) was set to 1. * P < 0.001 vs. TGFβ (−) and DOX (−); # P < 0.0001 vs. TGFβ (−) and DOX (+); ns not significant vs. TGFβ (−) and DOX (−); n = 3. All bar diagrams indicate mean ± s.d. Student’s t -test was used for paired data comparisons in e , f . At least two independent replicates were performed for all experiments. ANOVA with Tukey’s post hoc test were used for multiple comparisons in b , c , h

Article Snippet: RNA isolation, cDNA synthesis, and qRT-PCR were done using StepOnePlusTM Real-Time PCR System (Applied Biosystems) by TaqMan ® Gene Expression Assays (Applied Biosystems) for NOTCH1 (Hs01062014_m1), Notch1 (Mm00435249_m1), NOTCH3 (Hs00166432_m1), Notch3 (Mm00435270_m1), IVL (Hs00846307_s1), CK13 (s00999762_m1), CDH1 (Hs00170423_m1), CDH2 (Hs00983062 _m1), ZEB1 (Hs00232783_m1), SNAI1 (Hs00195591_m1), and PAI1 (Hs01126606_m1), and SYBR ® Green PCR for human ACTB (β-Actin) as well as murine Cdh1 (5′-TCAAGCTCGCGGATAACCAGAACA-3′ and 5′-ATTCCCGCCTTCATGCAGTTGTTG-3′), Cdh2 (5′-ATGGCCTTTCAAACACAGCCACAG-3′ and 5′-ACAATGACGTCCACCCTGTTCTCA-3′), Zeb1 (5′-TGAGCACACAGGTAAGAGGCC-3′ and 5′-GGCTTTTCCCCAGAGTGCA-3′), Zeb2 (5′-TGATAGCCTTGCAAACCCTCTGGA-3′ and 5′-TCCTTCATTTCTTCTGGACCGGCT-3′), Twist (5′-AGCTGAGCAAGATTCAGACCCTCA-3′ and 5′-TGCAGCTTGCCATCTTGGAGT-3′), and Gapdh (5′-GGTGGTCTCCTCTGACTTCAACA-3′ and 5′-GTTGCTGTAGCCAAATTCGTTGT-3′) and as described , , .

Techniques: Activation Assay, Flow Cytometry, Activity Assay, Purification, shRNA, Control, Quantitative RT-PCR

Journal: Nature Communications

Article Title: Interplay between Notch1 and Notch3 promotes EMT and tumor initiation in squamous cell carcinoma

doi: 10.1038/s41467-017-01500-9

Figure Lengend Snippet: NOTCH1 activation and ZEB1 expression in invasive ESCC predicts poor prognosis. a , b Representative IHC images for ICN1 and ZEB1 in ESCC cells in a deep invasive tumor nest and ESCC cells invading into lymphatic vessels. a ESCC #55; b ESCC #62 in Supplementary Data . c Representative IF image for ICN1 and CD44 in invasive ESCC cells. Scale bars, 50 μm in a – c . d Survival curve for 185 post-surgical ESCC patients with or without ICN1 expression at the tumor invasive front. e Model of the role of Notch1 in EMT in ESCC. Notch1 activation promotes tumorigenicity and heterogeneity in SCC via EMT. Notch1 drives squamous-cell differentiation by inducing Notch3 in normal squamous epithelia as well as SCC cells. Notch3 limits EMT. In response to TGFβ from the tumor microenvironment (e.g., cancer-associated fibroblasts and inflammatory cells), however, transcriptional repression of Notch3 by ZEB1 permits Notch1-mediated induction of CD44H cells via EMT. Notch activation may result in the generation and maintenance of CD44H cells possessing mesenchymal properties and enhanced malignant potential. EMT allows neoplastic cells to cope with stress during carcinogenesis and disease progression (e.g., genotoxic stress induced by 4NQO). CD44H cells produce pro-tumorigenic cytokines (e.g., IL-6) and tissue remodeling factors (e.g., MMP13, LOX, and POSTN) (Supplementary Fig. )

Article Snippet: RNA isolation, cDNA synthesis, and qRT-PCR were done using StepOnePlusTM Real-Time PCR System (Applied Biosystems) by TaqMan ® Gene Expression Assays (Applied Biosystems) for NOTCH1 (Hs01062014_m1), Notch1 (Mm00435249_m1), NOTCH3 (Hs00166432_m1), Notch3 (Mm00435270_m1), IVL (Hs00846307_s1), CK13 (s00999762_m1), CDH1 (Hs00170423_m1), CDH2 (Hs00983062 _m1), ZEB1 (Hs00232783_m1), SNAI1 (Hs00195591_m1), and PAI1 (Hs01126606_m1), and SYBR ® Green PCR for human ACTB (β-Actin) as well as murine Cdh1 (5′-TCAAGCTCGCGGATAACCAGAACA-3′ and 5′-ATTCCCGCCTTCATGCAGTTGTTG-3′), Cdh2 (5′-ATGGCCTTTCAAACACAGCCACAG-3′ and 5′-ACAATGACGTCCACCCTGTTCTCA-3′), Zeb1 (5′-TGAGCACACAGGTAAGAGGCC-3′ and 5′-GGCTTTTCCCCAGAGTGCA-3′), Zeb2 (5′-TGATAGCCTTGCAAACCCTCTGGA-3′ and 5′-TCCTTCATTTCTTCTGGACCGGCT-3′), Twist (5′-AGCTGAGCAAGATTCAGACCCTCA-3′ and 5′-TGCAGCTTGCCATCTTGGAGT-3′), and Gapdh (5′-GGTGGTCTCCTCTGACTTCAACA-3′ and 5′-GTTGCTGTAGCCAAATTCGTTGT-3′) and as described , , .

Techniques: Activation Assay, Expressing, Cell Differentiation, Biomarker Discovery