Journal: Genome Biology

Article Title: RNA methylomes reveal the m 6 A-mediated regulation of DNA demethylase gene SlDML2 in tomato fruit ripening

doi: 10.1186/s13059-019-1771-7

Figure Lengend Snippet: Dynamics of DNA methylation (5mC) and mRNA m 6 A methylation in tomato fruit ripening. a Images of wild-type (WT) fruit at different ripening stages and Cnr fruit at 42 DPA. DPA, days post-anthesis; scale bar = 1 cm. b Relative 5mC levels of WT and Cnr fruit shown in a . For 5mC assay, 100 ng of genomic DNA was detected in each sample by MethylFlash™ methylated DNA quantification kit. 5mC level in each sample was normalized to that of the positive control according to the manufacturer’s instructions. The plus sign represents the average in each box. c LC-MS/MS assay showing the amount of mRNA m 6 A in WT and Cnr fruit shown in a . Data are presented as mean ± standard deviation ( n = 3). Asterisks indicate significant differences (* P < 0.05, ** P < 0.01; Student’s t test). NS, no significance

Article Snippet: In brief, genomic DNA was extracted from the agroinfiltrated N. benthamiana leaves, and 500 ng of purified DNA was treated with bisulfite to produce mutations from cytosine (C) to thymine (T) using EZ DNA methylation-gold kit (ZYMO Research, D5005).

Techniques: DNA Methylation Assay, Methylation, Positive Control, Liquid Chromatography with Mass Spectroscopy, Standard Deviation

Journal: Genome Biology

Article Title: RNA methylomes reveal the m 6 A-mediated regulation of DNA demethylase gene SlDML2 in tomato fruit ripening

doi: 10.1186/s13059-019-1771-7

Figure Lengend Snippet: SlALKBH2 is transcriptionally regulated by DNA methylation. a The 5mC levels in the differentially methylated region (DMR) of SlALKBH2 promoter in wild-type (WT) and Cnr mutant fruit based on the Tomato Epigenome Database ( http://ted.bti.cornell.edu/epigenome/ ). b The 5mC levels in the DMR of SlALKBH2 promoter in WT and sldml2 mutant fruit based on the DNA methylome database . a , b The numbers indicate the cytosine positions relative to the start codon. Black represents the methylation frequency of cytosines at the indicated positions. DPA, days post-anthesis. c Schematic of the dual-luciferase system used for promoter activity assay. The SlALKBH2 promoter was cloned into the dual-luciferase reporter vector to activate the expression of firefly luciferase (Fluc). The renilla luciferase (Rluc) driven by the CaMV 35S promoter served as an internal control. LB, left border; RB, right border; Ter, terminator. d – f Co-expression of SlDML2 (SlDML2-HA) with the dual-luciferase reporter vector in the Nicotiana benthamiana leaves increased the relative Fluc activity ( d ), facilitated the Fluc gene expression ( e ), and reduced the 5mC level in SlALKBH2 promoter ( f ) compared with the empty plasmid control (HA). d The representative image from a total of six images (left panel). The Fluc activity was normalized against the Rluc activity, followed by normalization against the control (right panel). Data are presented as means ± standard deviation ( n = 6). Asterisks indicate significant differences (*** P < 0.0001; Student’s t test). e Gene expression was determined by quantitative RT-PCR analysis. Error bars represent the standard deviation of three independent experiments. Asterisks indicate significant differences (* P < 0.05; Student’s t test). f The box plot showing 5mC levels of all cytosines ( n = 41) in the DMR analyzed by Sanger bisulfite sequencing. The plus sign represents the average level in each box

Article Snippet: In brief, genomic DNA was extracted from the agroinfiltrated N. benthamiana leaves, and 500 ng of purified DNA was treated with bisulfite to produce mutations from cytosine (C) to thymine (T) using EZ DNA methylation-gold kit (ZYMO Research, D5005).

Techniques: DNA Methylation Assay, Methylation, Mutagenesis, Luciferase, Activity Assay, Clone Assay, Plasmid Preparation, Expressing, Standard Deviation, Quantitative RT-PCR, Methylation Sequencing

Journal: Genome Biology

Article Title: RNA methylomes reveal the m 6 A-mediated regulation of DNA demethylase gene SlDML2 in tomato fruit ripening

doi: 10.1186/s13059-019-1771-7

Figure Lengend Snippet: SlALKBH2 is necessary for normal tomato fruit ripening. a Genotyping of mutations mediated by CRISPR/Cas9 gene-editing system in slalkbh2-23 , slalkbh2-25 , and slalkbh2-28 mutants. Diagram showing the single guide RNAs (sgRNAs) containing different target sequences (T1, T2, and T3), which were designed to specifically target the exons of SlALKBH2 . The red letters indicate the protospacer adjacent motif (PAM). The transgenic plants in the second generation were genotyped by sequencing genomic regions flanking the target sites. Red arrows indicate the editing sites. Two mutants ( slalkbh2-23 and slalkbh2-28 ) have a homozygous 1-bp insertion, and one ( slalkbh2-25 ) has a homozygous 5-bp deletion caused by target T2 in the fourth exon of SlALKBH2 . b Ripening phenotype of slalkbh2 mutants. Fruit from wild-type (WT) and slalkbh2 mutants ( slalkbh2-23 , slalkbh2-25 , and slalkbh2-28 ) at 39, 42, 47, and 52 days post-anthesis (DPA) are shown. c LC-MS/MS assay showing the amount of mRNA m 6 A in WT and slalkbh2 mutant fruit at 39 DPA. Data are presented as mean ± standard deviation ( n = 3). d m 6 A-IP-PCR assay showing the relative m 6 A enrichment in SlDML2 mRNA in WT and slalkbh2 mutant fruit at 39 DPA. e SlDML2 gene expression in WT and slalkbh2 mutant fruit at 39 and 42 DPA. The ACTIN gene was used as an internal control. d , e Error bars represent the standard deviation of three independent experiments. Asterisks indicate significant differences (* P < 0.05, ** P < 0.01; Student’s t test). f Model for the relationship between DNA methylation and m 6 A mRNA methylation in fruit ripening. DNA methylation negatively regulates SlALKBH2 to mediate overall m 6 A mRNA methylation. The m 6 A modification promotes SlDML2 mRNA decay, thereby affecting DNA methylation and fruit ripening

Article Snippet: In brief, genomic DNA was extracted from the agroinfiltrated N. benthamiana leaves, and 500 ng of purified DNA was treated with bisulfite to produce mutations from cytosine (C) to thymine (T) using EZ DNA methylation-gold kit (ZYMO Research, D5005).

Techniques: CRISPR, Transgenic Assay, Sequencing, Liquid Chromatography with Mass Spectroscopy, Mutagenesis, Standard Deviation, Expressing, DNA Methylation Assay, Methylation, Modification

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: Cell Reports Medicine

Article Title: CRISPR screens in the context of immune selection identify CHD1 and MAP3K7 as mediators of cancer immunotherapy resistance

doi: 10.1016/j.xcrm.2025.102565

Figure Lengend Snippet: Autologous tumoroid-T cell co-culture CRISPR screens identify modulators of sensitivity to tumor-reactive T cells (A) Overview of the autologous co-culture CRISPR screens with primary tumoroids (CRC-9) and anti-tumor T cells. Tumoroid screens were performed ± IFN-γ (200 ng/mL) or TNF- α (100 ng/mL), or in the presence of tumor-reactive T cells (1:1 effector:target ratio) for 10 days. Tumoroids underwent one or two rounds of selection with T cells. MSI CRC, microsatellite-unstable colorectal cancer. (B) Genetic modulators of cancer cell sensitivity to autologous human tumor-reactive T cells. Scatterplot comparing CRISPR KO screen log2-fold change (control versus T cells) from the first and second rounds of T cell selection. Data are representative of two independent screens performed on separate days. CHD1 and selected co-culture hits from cytokine tumoroid screens are highlighted ( P adjusted < 0.05). Pearson correlation, r = 0.68. (C) Heatmap displaying clustering of cell models and immunological selection pressures based on CRISPR KO screen log2 fold-changes. Columns represent different CRISPR screens against the control sample (e.g., control versus interferon or WT versus JAK1 KO in the presence of interferon). Genes include published resistance and sensitizing hits and all reach a significance threshold of P < 0.05 in at least one experimental condition shown. See also B. (D) Overview of a competition assay using patient-derived CRC tumoroids co-cultured with autologous tumor-reactive T cells. The ratio of CRISPR-Cas9-edited (BFP + and mCherry + ) and non-targeting gRNA-harboring (GFP + and mCherry + ) tumoroids was monitored over time. (E) CHD1 and MAP3K7 loss additively sensitize cancer cells to killing by autologous T cells. Fluorescence of the different cell populations in the competition assay was measured using an Incucyte. Data represent the mean ± SD of two independent experiments, each performed in technical triplicate. Two-way analysis of variance (ANOVA); ∗∗∗∗ P < 0.0001 (NT versus CHD1 gRNA1 + MAP3K7 gRNA1, NT versus CHD1 gRNA2 + MAP3K7 gRNA2, NT versus CHD1 gRNA1 + MAP3K7 gRNA2, NT versus CHD1 gRNA2 + MAP3K7 gRNA1, CHD1 gRNA1 versus CHD1 gRNA1 + MAP3K7 gRNA1, MAP3K7 gRNA1 versus CHD1 gRNA1 + MAP3K7 gRNA1, CHD1 gRNA2 versus CHD1 gRNA2 + MAP3K7 gRNA1, MAP3K7 gRNA1 versus CHD1 gRNA2 + MAP3K7 gRNA1; ∗∗ P = 0.0012, MAP3K7 gRNA2 versus CHD1 gRNA2 + MAP3K7 gRNA2; ∗ P = 0.04, MAP3K7 gRNA2 versus CHD1 gRNA1 + MAP3K7 gRNA2; n.s. (not significant) CHD1 gRNA2 versus CHD1 gRNA2 + MAP3K7 gRNA2, CHD1 gRNA1 versus CHD1 gRNA1 + MAP3K7 gRNA2.

Article Snippet: Successful clones were expanded for 9 days and assessed for GFP expression (Incucyte S3; Sartorius) to ensure a lack of plasmid integration.

Techniques: Co-Culture Assay, CRISPR, Selection, Control, Competitive Binding Assay, Derivative Assay, Cell Culture, Fluorescence

Journal: Science Advances

Article Title: SirT7 auto-ADP-ribosylation regulates glucose starvation response through mH2A1

doi: 10.1126/sciadv.aaz2590

Figure Lengend Snippet: ( A ) Affinity purification of SirT7-binding factors identified the histone H2A variant mH2A1 by MS analysis (table S2). HA, hemagglutinin. ( B ) Schematic representation of the three mH2A isoforms, of which, only mH2A1.1 binds to ADP-ribose . ( C ) Endogenous SirT7 specifically immunoprecipitates mH2A1.1 in HEK293F cells. IgG, immunoglobulin G. ( D and E ) mH2A1.1 recognizes and binds ADP-ribosylated SirT7. Immunoprecipitation (IP) of bacterially expressed rSirT7 WT or N189A preincubated ± NAD + and added to nuclear extracts of HEK293F SirT7 KO cells (CRISPR-Cas9–mediated KO of SIRT7) expressing mH2A1.1 WT or G224E, a mutant deficient in ADP-ribose binding. Inputs (I) and elutions (E) are shown. A similar experiment with H187Y is shown in fig. S3A. ( F ) Top: Interaction between endogenous SirT7 and mH2A1.1 under high-stringency conditions upon different types of stress in HEK293 cells. C, untreated; IR, 7-gray ionizing irradiation; H 2 O 2 , oxidative stress. Western blot of the input and elution of immunoprecipitation experiments with anti-SirT7 antibody under these conditions. Bottom: Summary of the ADP-ribosylation events detected by MS in SirT7-FLAG expressed in the same cells and purified under the same stress conditions. Further details are shown in table in fig. S3B and in table S1. ( G ) Superose 6 gel filtration chromatography of nuclear endogenous proteins from nuclear extracts of HEK293F cells under normal cell growth (NT) or upon GS. Fraction numbers and approximate molecular weights (MW) are indicated. Western blot of SirT7 and mH2A1.1 are shown. ( H ) High-stringency immunoprecipitation of endogenous mH2A1.1 by WT or N189A SirT7 in HEK293F cells treated under normal conditions or under GS. Inputs (I) and elutions (E) are shown.

Article Snippet: Plasmids encoding mouse shRNAs of mH2A isoforms were obtained from Addgene (Cambridge, MA, USA): pSUPER retro puro mH2A1 shRNA (#30517) and pSUPER retro puro Scr shRNA (#30520).

Techniques: Affinity Purification, Binding Assay, Variant Assay, Immunoprecipitation, CRISPR, Expressing, Mutagenesis, Irradiation, Western Blot, Purification, Filtration, Chromatography

Journal: Science Advances

Article Title: SirT7 auto-ADP-ribosylation regulates glucose starvation response through mH2A1

doi: 10.1126/sciadv.aaz2590

Figure Lengend Snippet: ( A ) Levels of three mH2A isoforms in WCE of NIH3T3 cells transfected with scramble shRNA or mH2A1 shRNA and cultured under normal or GS conditions. ( B ) Levels of endogenous SirT7 and histone H3 in chromatin and nucleoplasm fractions purified from Wt and Sirt7 −/− MEFs cultured under normal or GS conditions during the indicated times. ( C ) Venn diagrams showing the intersection of SIRT7-associated genes with mH2A1-enriched genes in Wt cells under NT (top) or GS (bottom). SirT7-associated and mH2A1-enriched genes were derived from GREAT analysis (fig. S3B and Materials and Methods). ( D ) Average enrichment of mH2A1 at all genes in Wt (left) or SirT7 −/− MEF (right) cells under NT (black) or GS (red) conditions. Data are expressed as the log 2 ratio of reads per kilobase of transcript per million mapped reads–normalized ChIP/input signals. ( E ) Distribution of sites occupied by SirT7 upon GS around the TSS by GREAT analysis. The values for each bin from the TSS are shown above each bar (TSS: −5 kb, 5 to 50 kb, 50 to 500 kb, and >500 kb). ( F ) KEGG cell signaling pathways for SirT7-associated genes mapped by GREAT analysis under GS in MEF cells. The signaling pathways were ranked by their combined score provided by Enrichr analysis. cGMP-PKG, guanosine 3′,5′-monophosphate–protein kinase G; cAMP, cyclic adenosine 3′,5′-monophosphate; TCA, tricarboxylic acid. ( G ) SirT7 ChIP-qPCR (quantitative polymerase chain reaction) analysis of SirT7 binding sites associated with mH2A1 at distal regions upon shRNA-mediated down-regulation of mH2A1 under normal and GS conditions in NIH3T3 cells. The amplified regions (red) and their distance to each gene are indicated in the upper part of each graph. Each SirT7 ChIP was normalized with respect to its own input. SEM from n = 4. Two-tailed t test (* P < 0.05 and *** P < 0.005).

Article Snippet: Plasmids encoding mouse shRNAs of mH2A isoforms were obtained from Addgene (Cambridge, MA, USA): pSUPER retro puro mH2A1 shRNA (#30517) and pSUPER retro puro Scr shRNA (#30520).

Techniques: Transfection, shRNA, Cell Culture, Purification, Derivative Assay, Protein-Protein interactions, ChIP-qPCR, Real-time Polymerase Chain Reaction, Binding Assay, Amplification, Two Tailed Test

Journal: Science Advances

Article Title: SirT7 auto-ADP-ribosylation regulates glucose starvation response through mH2A1

doi: 10.1126/sciadv.aaz2590

Figure Lengend Snippet: ( A ) Heat map showing RNA expression changes relative to NT (as log 2 magnitude of difference between GS and NT) conditions in Wt and SirT7-deficient MEFs. ( B ) Pipeline applied to RNA-seq data to filter genes associated with SirT7/mH2A1 in Wt and Sirt7 −/− MEF cells treated under GS or NT. The analysis was restricted to genes that (i) were associated with SirT7 via GREAT and were mH2A1-enriched, (ii) showed a log 2 fold change (FC) of expression between WT-GS and WT-NT >0.6, and (iii) showed a difference between WT and KO log 2 FC (GS versus NT) of >0.3 (table S3). ( C ) mH2A1 ChIP-seq signals across ctgf , a gene differentially enriched in mH2A1 upon GS in Wt MEFs compared with NT. ( D ) Top: Real-time qPCR (RT-qPCR) analysis of genes regulated by SirT7 upon GS and NT. The expression of SirT7 in SirT7 −/− MEFs was rescued by retroviral-mediated gene transfer of SirT7 WT, H187Y(HY), N189A(NA), and empty vector (−). SEM from n = 4. Two-tailed t tests (* P < 0.05, ** P < 0.01, *** P < 0.005, and **** P < 0.001). Bottom: Relative mH2A1 enrichment (GS versus NT) by ChIP-qPCR analysis at specific regions around the TSS of the indicated genes [ gbp6 , −6 kb; necab1 , −3 kb; lair1 , −27.5 kb; ctgf , +600 base pairs (bp); adra2a , −5 kb; and nrip3 , +7 kb]. SEM from n = 3. One-way analysis of variance (ANOVA) (* P <0.05, ** P < 0.01, and *** P < 0.005). a.u., arbitrary units. ( E ) Chromatin state transitions induced by GS in Wt and SirT7-deficient cells. The colors of the arrows indicate the frequency (%) of the transition as stated in the color scale (right). Bottom right: State map illustrating the specific combination of mH2A1 and/or H3K27me3 in the four chromatin states defined in the analysis. U1, without H3K27me3 or mH2A1; U2, mH2A1; U3, H3K27me3; U4, enriched by H3K27me3 and mH2A1.

Article Snippet: Plasmids encoding mouse shRNAs of mH2A isoforms were obtained from Addgene (Cambridge, MA, USA): pSUPER retro puro mH2A1 shRNA (#30517) and pSUPER retro puro Scr shRNA (#30520).

Techniques: RNA Expression, RNA Sequencing, Expressing, ChIP-sequencing, Quantitative RT-PCR, Retroviral, Plasmid Preparation, Two Tailed Test, ChIP-qPCR

Journal: Science Advances

Article Title: SirT7 auto-ADP-ribosylation regulates glucose starvation response through mH2A1

doi: 10.1126/sciadv.aaz2590

Figure Lengend Snippet: ( A ) Model validation studies in WT and Sirt7 −/− mice fed AL or calorie restricted (CR, 30%) for 8 weeks. ( B ) RT-qPCR analysis of the indicated genes in liver samples from Wt and Sirt7 −/− mice fed AL or CR. Three animals were analyzed for each condition. Each quantification was generated from three replicates. Probabilities are those associated with one-way ANOVA (* P < 0.05, ** P < 0.01, and *** P < 0.005). ( C ) Levels of SirT7 in liver samples from Wt and Sirt7 −/− mice AL and CR after subcellular fractionation. WCE and chromatin fractions are shown. ( D ) SirT7 immunoprecipitation of mH2A1 in the same liver samples. Inputs (I) and elutions (E) are shown. ( E ) Autophagy activity in the Wt and Sirt7 −/− livers under AL or CR monitored by levels of formation of LCIII-2. Left: A representative Western blot of n = 5 replicates used in the quantification shown. Right: Quantification of the relative accumulation of LCIII-2 compared to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) analyzed with a two-tailed t test (* P < 0.05). GAPDH was used as a loading control, as we did not detect a significant alteration of the levels of the protein in our conditions (data not shown). ( F ) Similar analysis ( n = 5) of Beclin-1 as in (E). ( G ) Model proposed for the dual SirT7/mH2A regulatory axis in GS. On the basis of our data, we speculate that this axis is also involved in CR and aging. Nutrient stress induces SirT7 auto-mADPRT, which leads to mH2A-dependent recruitment of SirT7 to distal regulatory regions and subsequent mH2A enrichment around the associated genes. This axis plays a key role in CR in vivo and possibly in aging by modulating key signaling pathways.

Article Snippet: Plasmids encoding mouse shRNAs of mH2A isoforms were obtained from Addgene (Cambridge, MA, USA): pSUPER retro puro mH2A1 shRNA (#30517) and pSUPER retro puro Scr shRNA (#30520).

Techniques: Biomarker Discovery, Quantitative RT-PCR, Generated, Fractionation, Immunoprecipitation, Activity Assay, Western Blot, Two Tailed Test, Control, In Vivo, Protein-Protein interactions

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: PLoS Pathogens

Article Title: Human papillomavirus type 38 alters wild-type p53 activity to promote cell proliferation via the downregulation of integrin alpha 1 expression

doi: 10.1371/journal.ppat.1008792

Figure Lengend Snippet: (A) Primary HKs were transduced with pLXSN HPV38 E6/E7 or pLSXN. mRNA levels were measured by RT-qPCR and normalized to GAPDH . Error bars represent standard deviations from 3 biological replicates of 2 different donors ( n = 6). ***, p <0.001; ****, p <0.0001; ns, not significant. (B) Total RNA levels of HKs expressing or not expressing HPV38 E6 and E7 were analyzed by TaqMan PCR. Commercial probes for ITGA1 and GAPDH were used. Results were normalized to GAPDH . Data shown are the means of 3 independent experiments for 2 different donors ( n = 6). ****, p <0.0001. (C) Proteins extracts from HKs expressing or not expressing HPV38 E6 and E7 were analyzed by immunoblotting (IB) with the indicated antibodies. (D) The TaqMan assay was also performed as previously described in primary HKs previously retrovirally transduced with the hTERT gene and expressing E6 and/or E7 from HPV38 ( n = 3). Results were normalized to GAPDH . ****, p <0.0001. (E) Proteins extracts from hTERT pLXSN or hTERT HPV38 E6/E7 cells were analyzed by IB with the indicated antibodies. Images shown are representative examples of 2 different experiments. (F) hTERT pLXSN or hTERT HPV38 E6/E7 cells were plated on coverslips and after 24 h were probed for ITGA1 using anti-ITGA1 antibody followed by secondary Alexa Fluor 488-conjugated antibody. Nuclei were stained with DAPI (pseudocoloured red), and cells were analyzed under a microscope. Images were merged using ImageJ software.

Article Snippet: A TaqMan assay was performed with the ITGA1 TaqMan gene expression assay probe (Hs00235006_m1; Life Technologies), following the manufacturer’s instructions.

Techniques: Transduction, Quantitative RT-PCR, Expressing, Western Blot, TaqMan Assay, Staining, Microscopy, Software

Journal: PLoS Pathogens

Article Title: Human papillomavirus type 38 alters wild-type p53 activity to promote cell proliferation via the downregulation of integrin alpha 1 expression

doi: 10.1371/journal.ppat.1008792

Figure Lengend Snippet: (A) Electromobility shift assay performed with 38HK nuclear protein extracts and biotinylated probes containing p53RE WT or mutated sequences. Probes were incubated and cross-linked with protein extracts. Unlabeled WT or mutant p53RE2 probes were used as a control. Images shown are representative examples of 2 different experiments. (B) 38HK were cross-linked and chromatin was processed for ChIP using p53 antibody. Results were analyzed by qPCR with primers spanning p53RE1, p53RE2, p53RE3, or the intergenic region of chromosome 22 as a negative control (nc). Error bars represent standard deviations of 3 independent experiments performed in triplicate. **, p <0.01. (C) HKs or 38HK were cross-linked and chromatin was processed for ChIP using p53 or IgG antibodies. Results were analyzed by qPCR using primers spanning for p53 REs of the ITGA1 promoter and normalized to IgG enrichment (negative control). Error bars represent the standard deviation of 2 independent experiments performed in 2 different HKs donors. *, p <0.05, ns, not significant. (D) Cell lysate was incubated with WT biotinylated probe containing p53 REs of the ITGA1 promoter. Incubation without a probe was used as a control. DNA-associated proteins were recovered by precipitation with streptavidin beads and analyzed by IB. Images shown are representative examples of 3 independent experiments. Signals of 3 different IBs were quantified with Image Lab software (right panel). Data shown are the means of 3 independent experiments. *, p <0.05. (E) Chromatin from 38HK was processed for ChIP experiments using p53 or DNMT1 antibodies. Results were obtained by qPCR with primers spanning p53RE2 or the intergenic region of chromosome 22 (nc). Error bars indicate standard deviations from 3 independent experiments performed in duplicate. **, p <0.01; ***, p <0.001. (F) 38HK were cultured in medium containing cyclic pifithrin-α hydrobromide or DMSO as a control. Chromatin was processed for ChIP using p53 or DNMT1 antibodies. Results were obtained by qPCR using primers spanning p53RE2. Data shown are the means of 2 independent experiments performed in triplicate. *, p <0.05, **, p <0.01. (G) Chromatin was processed for a ChIP-reChIP assay in which p53-immunoprecipitated DNA was re-immunoprecipitated by DNMT1. Enrichment of p53RE2 or the intergenic region of chromosome 22 (nc) was obtained by qPCR. Data shown are the means of 3 independent experiments performed in triplicate. **, p <0.01. (H) Nuclear protein extracts from 38HK were processed for IP. Agarose beads were conjugated with IgG or p53 antibodies. Conjugated beads were incubated with protein lysate overnight. IgG was used as a control. Results were obtained by IB using the indicated antibodies. (I) 38HK were transfected with DNMT1 siRNA or control siRNA (Scramble). After 72 h, a ChIP assay was performed with p53 or DNMT1 antibodies. Results were obtained by qPCR using p53RE2 primers. Error bars represent standard deviations from 3 independent experiments. *, p <0.05; **, p <0.01. DNMT1 protein levels in different cells were determined by IB with the indicated antibodies (right panel).

Article Snippet: A TaqMan assay was performed with the ITGA1 TaqMan gene expression assay probe (Hs00235006_m1; Life Technologies), following the manufacturer’s instructions.

Techniques: Electro Mobility Shift Assay, Incubation, Mutagenesis, Control, Negative Control, Standard Deviation, Software, Cell Culture, Immunoprecipitation, Transfection

Journal: PLoS Pathogens

Article Title: Human papillomavirus type 38 alters wild-type p53 activity to promote cell proliferation via the downregulation of integrin alpha 1 expression

doi: 10.1371/journal.ppat.1008792

Figure Lengend Snippet: (A) mRNA levels of 38HK treated with cyclic pifithrin-α hydrobromide or DMSO for 6 h were analyzed by RT-qPCR and normalized to GAPDH . Histograms represent the mean of at least 3 independent experiments. ****, p <0.0001. (B and C) ITGA1 and p53 mRNA and protein levels from 38HK expressing WT p53 (Scramble) or with CRISPR/Cas9-mediated p53 deletion (CRISPR-p53) were measured by RT-qPCR (B) and IB (C). (B) Gene expression was normalized to GAPDH . (C) Protein quantification was normalized to β-actin. Signals of 4 different IBs were quantified with Image Lab software (right panel). Data shown are the means of 4 independent experiments. *, p <0.05. (D and E) 38HK N-HA-p53 or p53-C-HA cells were generated by retroviral transduction with WT p53 tagged at the N- or C-terminus. As a control, 38HK were transduced with the corresponding empty plasmid. Protein extracts and total mRNA levels were processed for IB and RT-qPCR analysis, respectively. (D) IB images shown are representative examples of 3 independent experiments. (E) ITGA1 mRNA levels were normalized to GAPDH . Error bars indicate standard deviations of 4 independent experiments. ***, p <0.001. (F) 38HK were transfected with control siRNA (Scramble) or with DNMT1 siRNA (siDNMT1). After 72 h, cells were collected for RNA extraction and RT-qPCR analysis. Error bars indicate standard deviations of 3 independent experiments. *, p <0.05; ***, p <0.001. (G) ITGA1 expression was evaluated by RT-qPCR after 24 h of treatment with 5-aza-2′- deoxycytidine (Aza) or DMSO at 30 μM final concentration. Error bars represent standard deviations of 3 independent experiments. **, p <0.01. (H) H3K9ac at the ITGA1 promoter was evaluated by ChIP assay after treatment with Aza or DMSO as previously described ( n = 4). Results were obtained by qPCR using primers for p53RE2. *, p <0.05.

Article Snippet: A TaqMan assay was performed with the ITGA1 TaqMan gene expression assay probe (Hs00235006_m1; Life Technologies), following the manufacturer’s instructions.

Techniques: Quantitative RT-PCR, Expressing, CRISPR, Gene Expression, Software, Generated, Retroviral, Transduction, Control, Plasmid Preparation, Transfection, RNA Extraction, Concentration Assay

Journal: PLoS Pathogens

Article Title: Human papillomavirus type 38 alters wild-type p53 activity to promote cell proliferation via the downregulation of integrin alpha 1 expression

doi: 10.1371/journal.ppat.1008792

Figure Lengend Snippet: (A) Protein extracts from 38HK were processed for oligonucleotide pulldown as previously described. Images shown are representative examples of 3 independent experiments. (B) HK and 38HK were processed for protein extraction and IB with the indicated antibodies. After incubation with p446PKR antibody, the membrane was stripped and incubated with total PKR antibody. (C) Proteins extracts from U2OS cells retrovirally transduced with E6 and/or E7 from HPV38 were analyzed by IB with the indicated antibodies. (D) 38HK were treated with PKR inhibitor, 2AP, or PBS:glacial acetic acid (200:1) as a control for 4 h at 10 mM final concentration. p-p53 S392 and p53 band intensities were quantified and normalized to total p53 (central panel) or β-actin (right panel). Membranes were first incubated with p446PKR, then stripped and incubated with total PKR. Data shown are the means of 3 independent experiments. *, p <0.05, **, p <0.01. (E and F) 38HK were treated with 2AP, and ITGA1 mRNA (D) and protein levels (E) were determined by RT-PCR and IB, respectively. (D) Data shown are the means of 3 independent experiments (**, p <0.01). (E) Images shown are representative examples of 3 independent experiments. (G and H) ChIP assay using p53 or DNMT1 antibodies was performed in 38HK treated with or PBS:glacial acetic acid (200:1) as a control (CTR) or 2AP. Data shown are the means of 2 independent experiments performed in duplicate by qPCR using p53RE2 primers. *, p <0.05.

Article Snippet: A TaqMan assay was performed with the ITGA1 TaqMan gene expression assay probe (Hs00235006_m1; Life Technologies), following the manufacturer’s instructions.

Techniques: Protein Extraction, Incubation, Membrane, Transduction, Control, Concentration Assay, Reverse Transcription Polymerase Chain Reaction

Journal: PLoS Pathogens

Article Title: Human papillomavirus type 38 alters wild-type p53 activity to promote cell proliferation via the downregulation of integrin alpha 1 expression

doi: 10.1371/journal.ppat.1008792

Figure Lengend Snippet: (A) 38HK were transfected with ITGA1 cDNA (ITGA1) or empty plasmid control (pcDNA). After zeocin selection, 38HK were fixed with crystal violet (left panel) and total colony number per well was counted (right panel). Data shown are the means of 3 independent experiments. ****, p <0.0001. (B) 38HK transfected with ITGA1 cDNA (ITGA1) or empty plasmid control (pcDNA) were fixed and stained with propidium iodide for flow cytometry analysis. The histograms (right) represent the means of the sub-G0 population of 3 independent experiments. **, p <0.01. (C and D) Total protein and mRNA extracts from transfected 38HK were analyzed by IB and RT-qPCR. (C) Protein band intensity was quantified and normalized to β-actin. (D) ITGA1 , EGFR , and CCND1 mRNA levels were normalized to GAPDH . For (C) and (D), data shown are the means of 4 independent experiments *, p <0.05; **, p <0.01; ***, p <0.001; ns, not significant.

Article Snippet: A TaqMan assay was performed with the ITGA1 TaqMan gene expression assay probe (Hs00235006_m1; Life Technologies), following the manufacturer’s instructions.

Techniques: Transfection, Plasmid Preparation, Control, Selection, Staining, Flow Cytometry, Quantitative RT-PCR

Journal: PLoS Pathogens

Article Title: Human papillomavirus type 38 alters wild-type p53 activity to promote cell proliferation via the downregulation of integrin alpha 1 expression

doi: 10.1371/journal.ppat.1008792

Figure Lengend Snippet: (A) Skin keratinocytes were isolated from WT animals ( n = 4) and K14 HPV38 E6/E7 transgenic mice ( n = 3). After 30 weeks of UV irradiation, cSCC samples (cSCC1–3) were isolated from HPV38 E/E7 transgenic mice. Total RNA extraction was performed and Itga1 mRNA levels were determined by quantitative RT-PCR by normalizing to Gapdh . Whole-exome sequencing of the same mice was also performed. Mutational analysis of the Trp53 , Itga1 , and Egfr genes was performed as described in Materials and Methods. (B) Genomic position of the exonic mutations and the corresponding amino acid change are represented for the Itga1 and Egfr genes. WT exons are represented as blue boxes, and mutated exons are represented in red boxes. Text boxes describe the cSCC sample, the genomic position of the nucleotide change on the GRCm38/mm10 mouse reference genome, the type of mutation, and the corresponding amino acid change.

Article Snippet: A TaqMan assay was performed with the ITGA1 TaqMan gene expression assay probe (Hs00235006_m1; Life Technologies), following the manufacturer’s instructions.

Techniques: Isolation, Transgenic Assay, Irradiation, RNA Extraction, Quantitative RT-PCR, Sequencing, Mutagenesis

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

Journal: Frontiers in Immunology

Article Title: Metallothionein 3-Zinc Axis Suppresses Caspase-11 Inflammasome Activation and Impairs Antibacterial Immunity

doi: 10.3389/fimmu.2021.755961

Figure Lengend Snippet: The MT3-Zn 2+ axis suppresses TRIF signaling resulting in decreased IRF3 phosphorylation. When MT3 is absent, TRIF-IRF3-STAT1 signaling and non-canonical inflammasome activation are exaggerated. A lack of MT3 augments immunity to gram-negative bacteria, an effect, that is further enhanced by the combined absence of MT3 and caspase-11 in vivo . Thus, while MT3 curtails caspase-11 activation, the two molecules act together in compromising antibacterial immunity.

Article Snippet: MT3 , Applied Biosystems , Hs00359394_g1.

Techniques: Phospho-proteomics, Activation Assay, Bacteria, In Vivo

Journal: Frontiers in Immunology

Article Title: Metallothionein 3-Zinc Axis Suppresses Caspase-11 Inflammasome Activation and Impairs Antibacterial Immunity

doi: 10.3389/fimmu.2021.755961

Figure Lengend Snippet: See also Supplementary Table S1 and Files S1 , S2 |Protein interaction network of Mus musculus MT3 to determine functionally enriched GO BP categories using the STRING database.

Article Snippet: MT3 , Applied Biosystems , Hs00359394_g1.

Techniques: Transduction, Cell Differentiation

Journal: Frontiers in Immunology

Article Title: Metallothionein 3-Zinc Axis Suppresses Caspase-11 Inflammasome Activation and Impairs Antibacterial Immunity

doi: 10.3389/fimmu.2021.755961

Figure Lengend Snippet: See also Supplementary Figure S1 MT3 suppresses caspase-11 inflammasome activation in BMDMϕ. qRT-PCR analysis of Mt3 expression in WT BMDMϕ stimulated with (A) iLPS (2 μg/ml) or vehicle control, 3-5 independent experiments and (B) exLPS (10 μg/ml) for 48h, 3 independent experiments, two-tailed t-test. (C) Western Blots of pro- and active-caspase-11, pro-caspase-1, pro-IL1β and β-actin in cell lysates and active-caspase-1 and active-IL-1β in supernatants of WT and Mt3 -/- BMDMϕ stimulated with iLPS (10 μg/ml) or vehicle for 48h. Bar graphs are densitometric analysis of targets normalized to β-actin, 3-4 independent experiments, one-way ANOVA, data are mean ± SEM. (D) Western Blots of pro- and active-caspase-11 and β-actin in lysate + supernatant samples from WT and Mt3 -/- BMDMϕ stimulated with iLPS (2 μg/ml) or vehicle for 48h. Bar graphs are densitometric analysis of targets normalized to β-actin. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: MT3 , Applied Biosystems , Hs00359394_g1.

Techniques: Activation Assay, Quantitative RT-PCR, Expressing, Control, Two Tailed Test, Western Blot

Journal: Frontiers in Immunology

Article Title: Metallothionein 3-Zinc Axis Suppresses Caspase-11 Inflammasome Activation and Impairs Antibacterial Immunity

doi: 10.3389/fimmu.2021.755961

Figure Lengend Snippet: See also Supplementary Figure S2 . MT3 curtails CASPASE-4 and caspase-11 signaling and antibacterial immunity in hMϕ and in vivo . (A) MT3 and MT2A expression analyzed by qRT-PCR in hMϕ transfected with scramble siRNA or MT3 siRNA for 24h, 3 independent experiments, two-tailed t-test. (B) Scramble siRNA or MT3 siRNA treated hMϕ stimulated with iLPS (10 μg/ml) or vehicle for 48h. Immunoblots of pro-CASPASE-4 and active-CASPASE-4 in cell extracts, 3 independent experiments, one-way ANOVA. (C) Active-IL-1β measured by ELISA in supernatants of hMϕ treated as above, 3 independent experiments, one-way ANOVA. (D) E . coli growth inhibition in hMϕ transfected with MT3 siRNA and infected with 25 E . coli (K12): 1 hMϕ for 24h compared to scramble siRNA treated hMϕ, 3 independent experiments, two-tailed t-test. (E) E . coli growth inhibition in WT and Mt3 -/- BMDMϕ infected with 25 E . coli (K12):1 hMϕ for 24h, 4 independent experiments, two-tailed t-test. (F) WT and Mt3 -/- mice infected i.p. with 1X10 9 E . coli for 6h, log CFUs of E . coli in blood, kidney and peritoneal lavage samples, n = 12-15 per group, two-tailed t-test. (G) Western blots of inflammasome mediators in kidney homogenates of WT and Mt3 -/- mice infected as above, n = 6 per group, two-tailed t-test. (H) WT and Mt3 -/- mice infected i.p. with 1 X10 9 E . coli for 1h and IL-1β measured in peritoneal lavage and serum by ELISA. n = 3 per group, two-tailed t-test. (I) WT and Mt3 -/- mice primed i.p. with poly(I:C) (10 mg/kg) for 6h and challenged with LPS (2 mg/kg) i.p. After 18h, IL-1β was measured in peritoneal lavage and serum by ELISA, n = 3/group, two-tailed t-test. (J) Bacterial growth in spleen, lung and kidney of WT and Mt3 -/- mice infected i.n. with K. pneumoniae (4 X10 4 CFUs/mouse) for 48h, n = 8-12 per group, two-tailed t-test, data are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: MT3 , Applied Biosystems , Hs00359394_g1.

Techniques: In Vivo, Expressing, Quantitative RT-PCR, Transfection, Two Tailed Test, Western Blot, Enzyme-linked Immunosorbent Assay, Inhibition, Infection

Journal: Frontiers in Immunology

Article Title: Metallothionein 3-Zinc Axis Suppresses Caspase-11 Inflammasome Activation and Impairs Antibacterial Immunity

doi: 10.3389/fimmu.2021.755961

Figure Lengend Snippet: See also Supplementary Figure S3 Caspase-11 synergizes with MT3 in impairing bacterial clearance. WT, C asp-11 -/- , Mt3 -/- and Casp-11 -/- Mt3 -/- mice were infected i.p. with E . coli (1 X10 9 CFUs/mouse) for 6h. (A) Bacterial CFUs measured in kidney, blood and peritoneal lavage, n = 3-6 per group, one-way ANOVA. (B) Western blots of pro-GSDMD, active-GSDMD (p31), pro-caspase-1, active-caspase-1, pro-IL1β and active-IL-1β in kidney homogenates, n = 3-6 per group, one-way ANOVA, data are mean ± SEM. (C) WT and Mt3 -/- mice treated i.p. with MCC950 (1 mg/mouse) or PBS and infected i.p. with E . coli (1 X10 9 CFUs/mouse) for 6h. IL1β was measured by ELISA in peritoneal lavage, n = 6 per group, one-way ANOVA, data are mean ± SEM. Bacterial CFUs in whole blood and kidney, n = 4 per group, one-way ANOVA, data are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: MT3 , Applied Biosystems , Hs00359394_g1.

Techniques: Infection, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Metallothionein 3-Zinc Axis Suppresses Caspase-11 Inflammasome Activation and Impairs Antibacterial Immunity

doi: 10.3389/fimmu.2021.755961

Figure Lengend Snippet: See also Supplementary Figure S4 Myeloid-MT3 suppresses non-canonical inflammasome activation and blunts gram-negative bacterial clearance in vivo . (A) Generation of Mt3 fl/fl mice by inserting loxp sites flanking exon 3 of the Mt3 gene using the CRISPR-Cas9 gene targeting approach. Mt3 fl/fl mice crossed with Lys2Cre mice to obtain Lys2Cre Mt3 fl/fl mice. (B) Efficacy of myeloid Mt3 deletion assessed by genotyping peritoneal Mϕ (PMϕ) and BMDMϕ from Lys2Cre , Mt3 fl/fl and Lys2Cre Mt3 fl/fl mice. Gel electrophoresis analysis demonstrating efficient deletion of the Mt3 gene from BMDMϕ and PMϕ of Lys2Cre Mt3 fl/fl mice. (C) Western blots of pro-caspase-11, active-caspase-11, pro-GSDMD, active-GSDMD (p31), pro-caspase-1, active-caspase-1, pro-IL1β and active-IL-1β in whole kidney homogenates of mice infected as above, n = 3-5 per group, two-tailed t-test. (D) Bacterial CFUs in kidney and whole blood of Lys2Cre and Lys2Cre Mt3 fl/fl mice infected i.p. with E . coli (1 X10 9 CFUs/mouse) for 6h, n = 3-5 per group, two-tailed t-test, data are mean ± SEM. **p < 0.01, ***p < 0.001.

Article Snippet: MT3 , Applied Biosystems , Hs00359394_g1.

Techniques: Activation Assay, In Vivo, CRISPR, Nucleic Acid Electrophoresis, Western Blot, Infection, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Metallothionein 3-Zinc Axis Suppresses Caspase-11 Inflammasome Activation and Impairs Antibacterial Immunity

doi: 10.3389/fimmu.2021.755961

Figure Lengend Snippet: See also Supplementary Figure S5 MT3 thwarts TRIF-IRF3-STAT1 signaling to suppress non-canonical inflammasome activation. (A) Functional enrichment analysis of differentially expressed genes using RNA-seq data from resting WT and Mt3 -/- BMDMϕ (NCBI SRA: PRJNA533616) FDR, false detection rates. (B, C) Heat map (left) and table (right) show differentially expressed IFN-related genes in resting Mt3 -/- BMDMϕ compared to resting WT BMDMϕ obtained from RNA-seq analysis. (D) Western blots of pIRF3, pSTAT1, STAT1, GBP2 and GBP5 in vehicle or iLPS (10 μg/ml)-treated WT and Mt3 -/- BMDMϕ lysates, 3-4 independent experiments, one-way ANOVA. (E) Western blots of TRIF in lysates from WT and Mt3 -/- BMDMϕ stimulated as above, 3 independent experiments, one-way ANOVA. (F) Scramble and Ticam1 siRNA treated WT and Mt3 -/- BMDMϕ treated with iLPS (10 μg/ml) or vehicle for 48h. Immunoblots of TRIF (2 independent experiments), pro-caspase-11, and active-caspase-11 in lysates and active-IL-1β in supernatants, 3 independent experiments, one-way ANOVA, data are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001; NS, not significant.

Article Snippet: MT3 , Applied Biosystems , Hs00359394_g1.

Techniques: Activation Assay, Functional Assay, RNA Sequencing, Western Blot

Journal: Frontiers in Immunology

Article Title: Metallothionein 3-Zinc Axis Suppresses Caspase-11 Inflammasome Activation and Impairs Antibacterial Immunity

doi: 10.3389/fimmu.2021.755961

Figure Lengend Snippet: See also Supplementary Figures S6 , 7 MT3-Zn 2+ axis drives negative regulation of the non-canonical inflammasome. (A) SEC-ICP-MS of WT and Mt3 -/- BMDMϕ exposed to vehicle or iLPS (10 ug/ml) for the indicated time points, chromatograms depict Zn 2+ distribution in cell lysates across various molecular masses, arrow indicates Zn 2+ associated with the MT-peak (18-21 min.) on the chromatogram, Y axis is off-set to allow easy comparison under the same scale. (B) Bar graphs of total Zn 2+ and MT-Zn 2+ in WT and Mt3 -/- BMDMϕ post iLPS (10 μg/ml) or vehicle exposure. Two-way t-test against respective BMDMϕ controls at each time point, 3 independent experiments, data are mean ± SD. (C) WT BMDMϕ treated with iLPS (10 μg/ml) or vehicle for 24h in Zn 2+ sufficient or Zn 2+ deficient Opti-MEM media, immunoblots of pIRF3, pro-caspase-11, active-caspase-11 and pro-IL-1β in lysates and active-IL-1β in media supernatants, one-way ANOVA, data are mean ± SEM. (D, E) Mt3 -/- BMDMϕ transfected with Pro-Ject™ or Pro-Ject™ complexed with apo-MT3, 4Zn 2+ MT3 or 6Zn 2+ MT3 and treated with iLPS (10 μg/ml) or vehicle for 24h in Zn 2+ deficient Opti-MEM media. (D) Chromatograms depict Zn 2+ distribution in cell lysates across various molecular masses, arrow indicates Zn 2+ signal associated with the MT-peak (18-21 min.) on the chromatogram, Y axis is off-set to allow easy comparison under the same scale. (E) Western blots of pIRF3, pro-caspase-11, active-caspase-11 and pro-IL1β in lysates and active-IL-1β in supernatants, 3 independent experiments, one-way ANOVA, data are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001; NS, not significant.

Article Snippet: MT3 , Applied Biosystems , Hs00359394_g1.

Techniques: Comparison, Western Blot, Transfection

Journal: Frontiers in Immunology

Article Title: Metallothionein 3-Zinc Axis Suppresses Caspase-11 Inflammasome Activation and Impairs Antibacterial Immunity

doi: 10.3389/fimmu.2021.755961

Figure Lengend Snippet: Reagents and resources.

Article Snippet: MT3 , Applied Biosystems , Hs00359394_g1.

Techniques: Expressing, Plasmid Preparation, Control, Extraction, Blocking Assay, Injection, Cell Culture, Enzyme-linked Immunosorbent Assay, Transgenic Assay, Filtration, Transfection, Reverse Transcription, Cytotoxicity Assay, Isolation, Software, Imaging, Real-time Polymerase Chain Reaction