Journal: Neoplasia (New York, N.Y.)

Article Title: SAMSN1 Is a Tumor Suppressor Gene in Multiple Myeloma 1 2

doi: 10.1016/j.neo.2014.07.002

Figure Lengend Snippet: SAMSN1 expression is reduced in CD138 + PCs of patients with MM and HMCL. (A) SAMSN1 expression (as determined by real-time PCR) is significantly reduced in the BMs of patients with MM ( n = 34) compared with patients with MGUS ( n = 9) and healthy age-matched controls ( n = 5; * P < .05, ** P < .001, one-way ANOVA with Tukey’s multiple comparison test). (B) SAMSN1 expression in CD138 + MACS isolated PCs from patients with MM negatively correlates with BM PC burden ( n = 10, r 2 = 0.6147, P = .0043). (C) In silico analysis of published microarray data. CD138 + PCs were isolated by MACS from 414 patients with MM, 44 patients with MGUS, and 22 age-matched controls. RNA was extracted and analyzed using the Affymetrix U133Plus2.0 microarray platform (GEO Accession Nos GSE4581 and GSE5900). Expression of SAMSN1 is significantly reduced in PCs of patients with MM compared to those of patients with MGUS and normal controls. P < 0.0001, one-way ANOVA with Tukey’s multiple comparison test. (D) Total RNA was extracted from six HMCLs and reverse transcribed. The levels of SAMSN1 expression were assessed by real-time PCR.

Article Snippet: PCs were isolated from flushed long bones and identified using rat anti-mouse CD138 (R&D Systems, Minneapolis, MN) followed by PE-conjugated goat anti-rat IgG.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Comparison, Isolation, In Silico, Microarray, Reverse Transcription

Journal: Cancer Science

Article Title: EWS ‐ FLI 1 regulates a transcriptional program in cooperation with Foxq1 in mouse Ewing sarcoma

doi: 10.1111/cas.13710

Figure Lengend Snippet: Global EWS ‐ FLI 1 binding in mouse Ewing sarcoma. A, Histology of mouse Ewing sarcoma, ES 49, used for Ch IP ‐Seq analysis. Hematoxylin and eosin staining (top). FLAG ‐tagged EWS ‐ FLI 1 expression was detected by western blotting. CIC ‐ DUX 4 sarcoma ( CDS 4) was used as a negative control (bottom). B, Global distribution of EWS ‐ FLI 1‐binding sites in relation to known genes. C, Distribution of EWS ‐ FLI 1‐binding peaks and histone H3K27Ac peaks in relation to transcriptional start sites ( TSS ) of known genes. D, Density plots of signal intensities of EWS ‐ FLI 1 and H3K27Ac peaks from TSS . E, Total H3K27Ac Ch IP ‐Seq signal in units of reads per million in enhancer regions for all enhancers in the ES 49 cell line. Enhancers are ranked by increasing H3K27Ac Ch IP ‐Seq signal. F, AME suite motif analysis carried out on EWS ‐ FLI 1 binding sites. Ets binding motifs (Elf3 and ELF 5) and GGAA microsatellites ( EWS ‐ FLI 1) were efficiently detected. G, EWS ‐ FLI 1 binds to GGAA microsatellites at Pvt1 and Sdk1 loci. H3K27Ac signals accompanying these binding sites are highlighted in yellow. Arrows indicate the transcriptional orientation of each gene. H, Venn diagrams for EWS ‐ FLI 1 ( FLAG ) and H3K27Ac Ch IP ‐Seq signals and up‐ or downregulated genes depicted by microarray analysis

Article Snippet: ChIP was carried out with 4 μg anti‐histone H3K27Me3 (Abcam, Cambridge, UK), anti‐histone H3K27Ac (Active Motif, Carlsbad, CA, USA), or anti‐FLAG M2 (Sigma, St Louis, MO, USA) antibodies.

Techniques: Binding Assay, Staining, Expressing, Western Blot, Negative Control, Microarray

Journal: Cancer Science

Article Title: EWS ‐ FLI 1 regulates a transcriptional program in cooperation with Foxq1 in mouse Ewing sarcoma

doi: 10.1111/cas.13710

Figure Lengend Snippet: EWS‐FLI1‐binding peaks in mouse Ewing sarcoma

Article Snippet: ChIP was carried out with 4 μg anti‐histone H3K27Me3 (Abcam, Cambridge, UK), anti‐histone H3K27Ac (Active Motif, Carlsbad, CA, USA), or anti‐FLAG M2 (Sigma, St Louis, MO, USA) antibodies.

Techniques:

Journal: bioRxiv

Article Title: Secreted phospholipase PLA2G12A-driven lysophospholipid signaling via lipolytic modification of extracellular vesicles facilitates pathogenic Th17 differentiation

doi: 10.1101/2024.10.27.620543

Figure Lengend Snippet: (A) qPCR of Pla2g12a in various tissues of 10-week-old C57BL/6 mice. (B) Procedure used for flow cytometry of CD4 + -gated TCRβ + IL-17A + Th17 cells from ex vivo Th17 differentiation culture. (C) qPCR of various PLA 2 enzymes in splenic CD4 + T cells cultured for 3 days under Th0 and Th17 differentiation conditions ( n = 3). (D) Strategy for gene targeting of Pla2g12a . Positions of PCR primers for genotyping are indicated by arrows. (E) PCR genotyping of Pla2g12a +/+ (+/+), Pla2g12a +/– (+/–), and Pla2g12a -/- (–/–) mice. (F) qPCR of Pla2g12a in the skin and spleen of Pla2g12a +/+ and Pla2g12a -/- mice. Values are mean ± s.e.m.. Representative data of two experiments (C) and combined results of two experiments (A, F) are shown. Statistical analysis was performed by Mann-Whitney U test (B, F, G). **, P < 0.01.

Article Snippet: Endogenous peroxidase was inactivated by treatment with 3% ( w / v ) hydrogen peroxide solution (Fujifilm-Wako) for 5 min, and then blocked with Block Ace (DS Pharma Biomedical) for 1 h. The sections were incubated with anti-PLA2G12A antibody (clone #44) biotinylated with a Biotin-Labeling Kit (Dojindo) and rabbit anti-CD4 monoclonal antibody (D7D2Z; Cell Signaling) diluted at 1:1000 at room temperature for 1 h. After washing, the sections were incubated with APC streptavidin (BioLegend) and Alexa Flour 488-conjugated F(ab’) 2 -goat anti-rabbit IgG (H+L) cross-adsorbed secondary antibody (A-11070, Thermo Fisher Scientific) diluted at 1:1000 at room temperature for 1 h. After washing, the sections were sealed with VECTASHIELD mounting medium with 4’,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories).

Techniques: Flow Cytometry, Ex Vivo, Cell Culture, MANN-WHITNEY

Journal: bioRxiv

Article Title: Secreted phospholipase PLA2G12A-driven lysophospholipid signaling via lipolytic modification of extracellular vesicles facilitates pathogenic Th17 differentiation

doi: 10.1101/2024.10.27.620543

Figure Lengend Snippet: (A) Schematic representation of the procedure for ex vivo culture of splenic naïve CD4 + T cells with (Th17) or without (Th0) IL-1β, IL-6, IL-23 and TGF-β on anti-CD3ε/CD28-coated plates. (B) qPCR of Pla2g12a in splenic CD4 + T cells cultured for various periods under Th0 and Th17 differentiation conditions. (C) FACS of Th17 (CD4 + -gated TCRβ + IL-17A + ) cells after culture of naïve T cells prepared from Pla2g12a +/+ (+/+) and Pla2g12a -/- (–/–) mice for 3 days under Th0 and Th17 differentiation conditions. Representative FACS profiles ( left ) and the proportion and number of Th17 cells ( right ) are shown. (D-F) Scatter plots (D) and heatmap (E) of genes expressed in CD4 + T cells from Pla2g12a +/+ and Pla2g12a -/- mice after culture for 3 days under Th0 and Th17 differentiation conditions. Cells from 6-7 mice of each genotype were pooled and subjected to microarray analysis. Colors in (E) show the z-score. (F) Pathway enrichment analysis of genes decreased (>2-fold) in Pla2g12a -/- Th17 cells relative to Pla2g12a +/+ Th17 cells. (G) Heatmap of Th17-related genes in Th17 cells from Pla2g12a -/- mice relative to those from Pla2g12a +/+ mice. Colors indicate fold changes in Pla2g12a -/- cells relative to Pla2g12a +/+ cells. (H) qPCR of Th17 signature genes in Th0 and Th17 cells from Pla2g12a +/+ and Pla2g12a -/- mice. (I) Heatmap of lipid-related genes in Pla2g12a -/- Th17 cells relative to Pla2g12a +/+ Th17 cells. Colors indicate fold changes in Pla2g12a -/- cells relative to Pla2g12a +/+ cells. (J) FACS of non-pathogenic Th17 cells from Pla2g12a +/+ and Pla2g12a -/- mice after culture for 3 days with IL-6 and TGF-β. Values are mean ± s.e.m.. Representative data of three experiments (C) and results of one experiment (B, D–I) are shown. Statistical analysis was performed by one way ANOVA with Tukey’s multiple comparisons test (C, J), Brown-Forsythe and Welch ANOVA with Dunnett’s T3 multiple comparisons test (H), and two-way ANOVA with Sidak’s multiple comparisons test (B). *, P < 0.05; **, P < 0.01.

Article Snippet: Endogenous peroxidase was inactivated by treatment with 3% ( w / v ) hydrogen peroxide solution (Fujifilm-Wako) for 5 min, and then blocked with Block Ace (DS Pharma Biomedical) for 1 h. The sections were incubated with anti-PLA2G12A antibody (clone #44) biotinylated with a Biotin-Labeling Kit (Dojindo) and rabbit anti-CD4 monoclonal antibody (D7D2Z; Cell Signaling) diluted at 1:1000 at room temperature for 1 h. After washing, the sections were incubated with APC streptavidin (BioLegend) and Alexa Flour 488-conjugated F(ab’) 2 -goat anti-rabbit IgG (H+L) cross-adsorbed secondary antibody (A-11070, Thermo Fisher Scientific) diluted at 1:1000 at room temperature for 1 h. After washing, the sections were sealed with VECTASHIELD mounting medium with 4’,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories).

Techniques: Ex Vivo, Cell Culture, Microarray

Journal: bioRxiv

Article Title: Secreted phospholipase PLA2G12A-driven lysophospholipid signaling via lipolytic modification of extracellular vesicles facilitates pathogenic Th17 differentiation

doi: 10.1101/2024.10.27.620543

Figure Lengend Snippet: (A) qPCR of Pla2g12a in the whole spleen and isolated CD4 - and CD4 + cells of Pla2g12a fl/fl (fl/fl) and Pla2g12a fl/fl Cd4 cre (T-KO) mice. (B) FACS of Th17 cells after culture of naïve T cells from fl/fl and T-KO mice for 3 days under Th0 and Th17 differentiation conditions. Representative FACS profiles ( left ) and the proportion of Th17 cells ( right ) are shown. (C) IMQ-induced ear swelling in fl/fl and T-KO mice over 6 days ( n = 20–30). (D) HE staining of ear skin sections from IMQ-treated fl/fl and T-KO mice on day 6. Scale bar, 100 μm. (E, F) FACS of Th17 cells in the spleen (E) and Th17 and γδ T cells in the skin (F) of IMQ-treated fl/fl and T-KO mice on day 6. (G) qPCR of Il17a in the skin of IMQ-treated fl/fl and T-KO mice on day 6. (H) qPCR of Pla2g12a in the whole LNs and isolated CD45 + hematopoietic cells and CD140a + fibroblasts of fl/fl and Pla2g12a fl/fl Col1a2 cre (F-KO) mice. (I) FACS of Th17 cells after culture of naïve T cells from fl/fl and F-KO mice for 3 days under Th0 and Th17 differentiation conditions. Representative FACS profiles ( left ) and the proportion of Th17 cells ( right ) are shown. (J) IMQ-induced ear swelling in fl/fl and F-KO mice over 6 days ( n = 4–6). (K) FACS of splenic Th17 cells in IMQ-treated fl/fl and F-KO mice on day 6. A representative histogram ( left ) and the number of Th17 cells ( right ) are shown. Values are mean ± s.e.m.. Representative data of two (A, F) or three (E) experiments, results of one experiment (B, I–K), and combined results of two (C, G, H) are shown. Statistical analysis was performed using unpaired t test (G, K), Mann-Whitney test (A, E, F, H), one-way ANOVA with Tukey’s multiple comparisons test (B, I), and two-way repeated measures ANOVA with Sidak’s multiple comparisons test (C, J). *, P < 0.05; **, P < 0.01.

Article Snippet: Endogenous peroxidase was inactivated by treatment with 3% ( w / v ) hydrogen peroxide solution (Fujifilm-Wako) for 5 min, and then blocked with Block Ace (DS Pharma Biomedical) for 1 h. The sections were incubated with anti-PLA2G12A antibody (clone #44) biotinylated with a Biotin-Labeling Kit (Dojindo) and rabbit anti-CD4 monoclonal antibody (D7D2Z; Cell Signaling) diluted at 1:1000 at room temperature for 1 h. After washing, the sections were incubated with APC streptavidin (BioLegend) and Alexa Flour 488-conjugated F(ab’) 2 -goat anti-rabbit IgG (H+L) cross-adsorbed secondary antibody (A-11070, Thermo Fisher Scientific) diluted at 1:1000 at room temperature for 1 h. After washing, the sections were sealed with VECTASHIELD mounting medium with 4’,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories).

Techniques: Isolation, Staining, MANN-WHITNEY

Journal: bioRxiv

Article Title: Secreted phospholipase PLA2G12A-driven lysophospholipid signaling via lipolytic modification of extracellular vesicles facilitates pathogenic Th17 differentiation

doi: 10.1101/2024.10.27.620543

Figure Lengend Snippet: (A) FACS of Th17 cells after culture of naïve T cells for 3 days in the presence of the indicated concentrations of the LPA 1/3 antagonist Ki16425. (B) qPCR of Lpar1 in Th0 and Th17 cells after culture for the indicated periods. (C) qPCR of Lpar1 in the LNs of Lpar1 +/+ and Lpar1 -/- mice. (D, E) IMQ-induced ear swelling over 6 days ( n = 8–12) (D) and ear histology on day 6 (E) of Lpar1 +/+ and Lpar1 -/- mice. (F, G) IMQ-induced ear swelling over 6 days ( n = 6) (F) and the number of splenic Th17 cells (G) of Lpar1 +/+ mice that had been transferred with Lpar1 +/+ or Lpar1 -/- BM cells. (H, I) FACS of splenic CDε+ T cells in Lpar1 +/+ mice that had been transferred with Lpar1 +/+ or Lpar1 -/- BM cells (H) or in Pla2g12a +/+ mice that had been transferred with Pla2g12a +/+ or Pla2g12a -/- BM cells (I). Representative FACS profiles ( left ) and the proportion of CD3ε + T cells ( right ) are shown. (J) FACS of CD4 + Th0 cells from Pla2g12a -/- , Lpar1 -/- , and Lpar2 -/- mice in comparison with those from respective control mice. Representative FACS profiles ( upper ) and the proportion of CD4 + T cells ( lower ) are shown. (K) FACS of splenic CD4 + -gated CD44 - CD62L + naïve T cells and CD44 + CD62L - effector memory T cells in Pla2g12a -/- , Lpar1 -/- , and Lpar2 -/- mice in comparison with WT mice. Values are mean ± s.e.m.. Representative data of two experiments (B, J) and results of one experiment (A, C–I, K) are shown. Statistical analysis was performed using unpaired t test (C, I, J), Mann-Whitney U test (G, H), ordinary one-way ANOVA with Dunnett’s multiple comparisons test (A, K), Brown-Forsythe and Welch ANOVA with Dunnett’s T3 multiple comparisons test (B), and two-way ANOVA with Sidak’s multiple comparisons test (D, F). *, P < 0.05; **, P < 0.01.

Article Snippet: Endogenous peroxidase was inactivated by treatment with 3% ( w / v ) hydrogen peroxide solution (Fujifilm-Wako) for 5 min, and then blocked with Block Ace (DS Pharma Biomedical) for 1 h. The sections were incubated with anti-PLA2G12A antibody (clone #44) biotinylated with a Biotin-Labeling Kit (Dojindo) and rabbit anti-CD4 monoclonal antibody (D7D2Z; Cell Signaling) diluted at 1:1000 at room temperature for 1 h. After washing, the sections were incubated with APC streptavidin (BioLegend) and Alexa Flour 488-conjugated F(ab’) 2 -goat anti-rabbit IgG (H+L) cross-adsorbed secondary antibody (A-11070, Thermo Fisher Scientific) diluted at 1:1000 at room temperature for 1 h. After washing, the sections were sealed with VECTASHIELD mounting medium with 4’,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories).

Techniques: Comparison, Control, MANN-WHITNEY

Journal: bioRxiv

Article Title: Secreted phospholipase PLA2G12A-driven lysophospholipid signaling via lipolytic modification of extracellular vesicles facilitates pathogenic Th17 differentiation

doi: 10.1101/2024.10.27.620543

Figure Lengend Snippet: (A) Schematic representation of the procedure used for antibody treatment in IMQ-induced psoriasis. (B) IMQ-induced ear swelling in WT mice treated with anti-PLA2G12A, -IL-17A, or control IgG 1 antibody over 6 days. Antibody treatment started on day 0 (arrow). (C) FACS of splenic Th17 cells in (B) on day 6. (D) IMQ-induced ear swelling in Pla2g12a +/+ (+/+) and Pla2g12a -/- (–/–) mice treated with anti-PLA2G12A or control antibody over 6 days. Antibody treatment started on day 0 (arrow). (E) FACS of splenic Th17 cells in (D) on day 6. (F) IMQ-induced ear swelling in WT mice treated with anti-PLA2G12A or control antibody. Antibody treatment started on day 3 (arrow). (G) FACS of splenic Th17 cells in (F) on day 6. (H) Schematic representation of the procedure used for antibody treatment in CIA. (I, J) Paw thickness (I) and clinical sore (J) in WT mice treated with anti-PLA2G12A, - IL-17A, or control antibody over 42 days. (K) FACS of splenic Th17 cells in (I, J) on day 42. (L) Schematic diagram showing PLA2G12A-driven regulation of Th17 differentiation. PLA2G12A is induced in CD4 + T cells after TCR stimulation and is also supplied from stromal fibroblasts. EV secretion is facilitated by Th17 differentiation driven by Th17-inducing cytokines. PLA2G12A hydrolyzes PE and PC to generate LPE and LPC in EV membranes. The EVs are taken up by Th17-differentiating cells and deliver LPE(1-18:1) intracellularly, which then transactivates RORγt. LPE and LPC are converted to LPA by ATX, which is supplied from Th17 cells, stromal cells ( e.g ., fibroblasts and endothelial cells), and serum. LPA acts mainly on LPA 2 (and to a lesser extent LPA 1 ) on Th17-differentiating cells to augment IL-6/IL23-mediated STAT3 phosphorylation via G 12/13 -Rho-ROCK2 signaling, assisting pathogenic Th17 differentiation in autocrine and paracrine fashions. Gene disruption of PLA2G12A dampens this lipid-orchestrated amplification loop and decreases EV secretion, uptake, and transfer of lipid, miRNA, and protein cargos. Accordingly, antibody-mediated neutralization of PLA2G12A efficiently attenuates psoriasis, arthritis, and probably other Th17-related pathologies such as EAE. Values are mean ± s.e.m.. Results are from one experiment (B–G, I–K). Statistical analysis was performed using unpaired t test (G), ordinary one-way ANOVA with Dunnett’s multiple comparisons test (C, E, K), and two-way repeated measures ANOVA with Sidak’s multiple comparisons test (B, D, F, I, J). *, P < 0.05; **, P < 0.01 versus control IgG 1 .

Article Snippet: Endogenous peroxidase was inactivated by treatment with 3% ( w / v ) hydrogen peroxide solution (Fujifilm-Wako) for 5 min, and then blocked with Block Ace (DS Pharma Biomedical) for 1 h. The sections were incubated with anti-PLA2G12A antibody (clone #44) biotinylated with a Biotin-Labeling Kit (Dojindo) and rabbit anti-CD4 monoclonal antibody (D7D2Z; Cell Signaling) diluted at 1:1000 at room temperature for 1 h. After washing, the sections were incubated with APC streptavidin (BioLegend) and Alexa Flour 488-conjugated F(ab’) 2 -goat anti-rabbit IgG (H+L) cross-adsorbed secondary antibody (A-11070, Thermo Fisher Scientific) diluted at 1:1000 at room temperature for 1 h. After washing, the sections were sealed with VECTASHIELD mounting medium with 4’,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories).

Techniques: Control, Disruption, Amplification, Neutralization

Journal: bioRxiv

Article Title: Secreted phospholipase PLA2G12A-driven lysophospholipid signaling via lipolytic modification of extracellular vesicles facilitates pathogenic Th17 differentiation

doi: 10.1101/2024.10.27.620543

Figure Lengend Snippet: (A) PLA 2 enzyme assay using human and mouse PLA2G12A proteins after incubation with several anti-PLA2G12A antibody clones (100 ng/ml), with the activity in the presence of a control antibody being regarded as 1. *, P < 0.05; **, P < 0.01 versus control. (B) FACS of Th17 cells after culture of WT naïve T cells for 3 days with several anti-PLA2G12A antibody clones (100 ng/ml). *, P < 0.05; **, P < 0.01 versus control. (C) Immunoblotting of human and mouse PLA2G12As and human PLA2G3 (50 ng/lane for each) with an anti-PLA2G12A antibody (clone #44). (D) ELISA of various sPLA 2 s with an anti-PLA2G12A antibody (clone #44). hIIA, human PLA2G2A; hIID, human PLA2G2D; hIIE, human PLA2G2E; hIIF, human PLA2G2F; hIII, human PLA2G3; hV, human PLA2G5; hX, human PLA2G10; hXIIA, human PLA2G12A; mXIIA, mouse PLA2G12A. *, P < 0.05; **, P < 0.01. (E) Immunohistochemistry of mouse skin sections with or without IMQ treatment with an anti-PLA2G12A antibody (clone #44). Red, PLA2G12A; green, CD4 + T cells. Scale bar, 100 µm. Values are mean ± s.e.m.. Results of one experiment is shown (A–D). Statistical analysis was performed using Brown-Forsythe and Welch ANOVA with Dunnett’s T3 multiple comparisons test (A) and ordinary one-way ANOVA with Dunnett’s multiple comparisons test (B, D).

Article Snippet: Endogenous peroxidase was inactivated by treatment with 3% ( w / v ) hydrogen peroxide solution (Fujifilm-Wako) for 5 min, and then blocked with Block Ace (DS Pharma Biomedical) for 1 h. The sections were incubated with anti-PLA2G12A antibody (clone #44) biotinylated with a Biotin-Labeling Kit (Dojindo) and rabbit anti-CD4 monoclonal antibody (D7D2Z; Cell Signaling) diluted at 1:1000 at room temperature for 1 h. After washing, the sections were incubated with APC streptavidin (BioLegend) and Alexa Flour 488-conjugated F(ab’) 2 -goat anti-rabbit IgG (H+L) cross-adsorbed secondary antibody (A-11070, Thermo Fisher Scientific) diluted at 1:1000 at room temperature for 1 h. After washing, the sections were sealed with VECTASHIELD mounting medium with 4’,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories).

Techniques: Enzymatic Assay, Incubation, Clone Assay, Activity Assay, Control, Western Blot, Enzyme-linked Immunosorbent Assay, Immunohistochemistry

Journal: bioRxiv

Article Title: Histone H3K27 methyltransferase EZH2 interacts with MEG3-lncRNA to directly regulate integrin signaling and endothelial cell function

doi: 10.1101/2022.05.20.492787

Figure Lengend Snippet: a) Distribution of annotated single hits over MEG3 gene, with statistically filtered EZH2-FLASH reads from two biological replicates in HUVECs. b) The occupancy of EZH2 hits over MEG3 features. Total reads per feature are given with exons being mostly occupies vs introns. c) Proportion of overlapping features over MEG3. The occupancy of EZH2 over each MEG3 exon is shown for two constitutively expressed transcripts. For both given transcripts there is high occupancy of exon 3. d) RNA immunoprecipitation (RIP) for EZH2 and H3K27me3 (repressive chromatin) followed by qPCR analysis. RIP-purified RNA from UV crosslinked HUVECs was used to prepare cDNA for qPCR analysis with primers against MEG3 (exon 3 region). Primers against U1snRNA gene serves as a negative control. Side diagram of EHZ2-MEG3 interacting region is charted as per FLASH hits and sequence. e) Distribution of EZH2 hybrids hits over MEG3 gene. Intermolecular MEG3-RNA interactions found in chimeras are captured by EZH2-FLASH-seq. Hits represent MEG3:MEG3 hybrids (black). IgG hybrids are plotted but are <1. f) Total MEG3:MEG3 hybrid count against predicted free energy of hybridization (dG) for MEG3 interactions ( red lncRNA:MEG3, blue mRNA:MEG3, green MEG3:antisense, purple snoRNA:MEG3) with free hybridization energy cutoff at dG<-10 kcal mol -1 , as captured by EZH2-FLASH-seq ( i ) vs. IgG control ( ii ) .

Article Snippet: Following sonication as described, samples were immunoprecipitated using EZH2 (D2C9) XP(R) Rabbit mAb, (5246S Cell signalling technology), Tri-Methyl-Histone H3 (H3K27me3) (C36B11) Rabbit mAb (9733S, CST) antibodies or IgG control (Normal Rabbit IgG, 2729S, CST) and captured on beads using Protein G Dyneabeads (10003D, Life Technologies).

Techniques: RNA Immunoprecipitation, Purification, Negative Control, Sequencing, Hybridization, Control

Journal: bioRxiv

Article Title: Histone H3K27 methyltransferase EZH2 interacts with MEG3-lncRNA to directly regulate integrin signaling and endothelial cell function

doi: 10.1101/2022.05.20.492787

Figure Lengend Snippet: a. Overview of the critical steps to obtain MEG3-bound genomic loci and intersections with EZH2 and H3K27me3 signals (obtained from GEO databases for HUVECs). In addition, enhancer regions were mapped within the genomic tracks. The intersection between GEO EZH2 ChIP data, GEO H3K27me3 ChIP data and statistically filtered MEG3-ChIRP data from two biological replicates was performed. The number of genes and degree of overlap is obtained between MEG3 and PRC2-dependent genes. The p-values are a result of hypergeometric test. b. Distribution of MEG3 peaks overlapping EZH2-ChIP peaks or H3K27me3-peaks with intersecting reads in relation to (i) gene regions and (ii) gene-type. c. Maximum peak score of ChIP signal for EZH2 and H3K27me3 intersecting the top enriched MEG3 peaks associated with nearest genes. Highest EZH2 peak score is over ITGA4, whereas H3K27me3 was detected in ITGA4, ITGA7, ITGA8 and ITGA9, members of ITGA family. d. Normalized reads from RNA-seq de novo analysis of GEO: GSE71164 dataset on Hg38, and expression of ITGA4 gene between Scr and siEZH2 depleted HUVECs, showing that ITGA4 is targeted by EZH2. Dataset in d and e is compared using Student’s t-test. e. ITGA4 expression from microarray analysis in C2C12 cells depleted of MEG3 (10nM, LNA GapMer) as per GEO dataset: GSE73524. The data shows that ITGA4 is a direct target of MEG3. f. (i) Total number of representable peaks (mRNA, antisense and lncRNA genes) from ChIP-seq analysis of Scr vs. MEG3 KD HUVECs. (ii ) Depletion of MEG3 gene in HUVECs (10nM LNA gapmers) was achieved with relative expression showing ∼70% reduction compared with Scr control. g. (i) Heat map showing distribution of reads and EZH2 densities at all unique RefSeq genes within TSSs ± 3 kb, sorted by EZH2 occupancy, in Control vs. MEG3 deficient (10nM) HUVECs. (ii) Overlap of ChIP-results between MEG3 and EZH2-dependent genes, with overlapped genes belonging to the biological pathway regulating cell adhesion. The common targets had lost or reduced EZH2 ChIP-signal.

Article Snippet: Following sonication as described, samples were immunoprecipitated using EZH2 (D2C9) XP(R) Rabbit mAb, (5246S Cell signalling technology), Tri-Methyl-Histone H3 (H3K27me3) (C36B11) Rabbit mAb (9733S, CST) antibodies or IgG control (Normal Rabbit IgG, 2729S, CST) and captured on beads using Protein G Dyneabeads (10003D, Life Technologies).

Techniques: RNA Sequencing, Expressing, Microarray, ChIP-sequencing, Control

Journal: bioRxiv

Article Title: Histone H3K27 methyltransferase EZH2 interacts with MEG3-lncRNA to directly regulate integrin signaling and endothelial cell function

doi: 10.1101/2022.05.20.492787

Figure Lengend Snippet: a) Computational analysis pipeline used to obtain orthologous peaks in human and intersect regions and genes enriched in repressive chromatin (H3K27me3) from ChIP-seq public dataset GSE114283. Up- and down-regulated genes were obtained associated with the peak region within 2000bp, and relevant function and biological pathway were associated using GREAT and DAVID analysis b) Overlap of the GEO datasets from a (Microarray GSE73524 ) and b (RNA-seq GSE71164 ) and the GSE114283 ChIP-seq reads of H3K27me 3 distribution in mouse MN cells depleted of MEG3 vs. control. ChIP extracted peaks unique to Ctrl vs. MEG3 KD were obtained, and associated mouse gene list composed based on reduction in H3K27me 3 signal. Using gene orthologous analysis in gProfiler we obtained human orthologous targets that was used for data intersection. c) Maximum peak scores of the overlapping signal over ITGA4 promoter, obtained by intersection of EZH2 ChIP signal with MEG3-ChIRP signal at this region. Upon depletion of MEG3 the EZH2 signal is significantly reduced whereby no overlap with MEG3 ChIRP signal is seen. d) Relative expression of ITGA4 in HUVEC measuring the levels of ITGA4 following addition of siRNA (50nM).

Article Snippet: Following sonication as described, samples were immunoprecipitated using EZH2 (D2C9) XP(R) Rabbit mAb, (5246S Cell signalling technology), Tri-Methyl-Histone H3 (H3K27me3) (C36B11) Rabbit mAb (9733S, CST) antibodies or IgG control (Normal Rabbit IgG, 2729S, CST) and captured on beads using Protein G Dyneabeads (10003D, Life Technologies).

Techniques: ChIP-sequencing, Microarray, RNA Sequencing, Control, Expressing

Journal: bioRxiv

Article Title: Histone H3K27 methyltransferase EZH2 interacts with MEG3-lncRNA to directly regulate integrin signaling and endothelial cell function

doi: 10.1101/2022.05.20.492787

Figure Lengend Snippet: a. Venn diagram showing the intersection between statistically filtered FLASH data from two biological replicates of our MEG3-ChIRP-seq-data (green), de novo hg38 analysed GEO RNA-seq data from siEZH2 deficient HUVECs (GSE71164, blue), and EZH2 ChIP-seq following MEG3 KD (yellow) and FLASH-seq transcriptome following EZH2 IP (pink). b. Correlation between gene expression levels and FLASH signal. Gray, expressed RefSeq genes with reproducible FLASH signal consistently detected in RNA-seq. Blue, genes with the highest RNA-seq signals and no reproducible FLASH signal belonging to integrin cell surface interaction pathway. Red , expressed ITGA4 gene, and green, ITGB1 gene, without reproducible FLASH signals. Data are from two biological replicates of each EZH2 FLASH sample and three biological replicates of EZH2 RNA-seq samples (Scr vs. siEZH2, GSE71164). c. Genomic tracks showing ChIRP-seq signal (MEG3 Odd, Even and LacZ) in HUVECs over ITGA4 gene only. The MEG3 binding site is located upstream of the ITGA4 gene in the promoter region, and it overlaps with the H3K27me3 signal and EZH2; as well as downstream within the ITGA4 gene body, where it overlaps with within the EZH2 signal in the intronic region of the gene. d. MEG3-ChIRP followed by qPCR, analysis of MEG3 binding region on ITGA4 in HUVECs. The crosslinked cell lysates were incubated with combined biotinylated probes against MEG3 lncRNA and the binding complexes recovered by magnetic streptavidin-conjugated beads. The qPCR was performed to detect the enrichment of specific region that associated with MEG3, peaks were related to input control and compared vs. the non-biotynilated control. e. ChIP-QPCR enrichment for EZH2 and H3K27me3 over ITGA4 promoter region in HUVECs depleted of MEG3 vs. Control.

Article Snippet: Following sonication as described, samples were immunoprecipitated using EZH2 (D2C9) XP(R) Rabbit mAb, (5246S Cell signalling technology), Tri-Methyl-Histone H3 (H3K27me3) (C36B11) Rabbit mAb (9733S, CST) antibodies or IgG control (Normal Rabbit IgG, 2729S, CST) and captured on beads using Protein G Dyneabeads (10003D, Life Technologies).

Techniques: RNA Sequencing, ChIP-sequencing, Gene Expression, Binding Assay, Incubation, Control, ChIP-qPCR

Journal: bioRxiv

Article Title: Histone H3K27 methyltransferase EZH2 interacts with MEG3-lncRNA to directly regulate integrin signaling and endothelial cell function

doi: 10.1101/2022.05.20.492787

Figure Lengend Snippet: a. ChIP signal enrichment vs . 1% input for EZH2 and H3K27me3 mark over ITGA4 promoter regions in HUVECs treated with A-395 (5µM, 24h) inhibitor of PRC2 vs. Control (DMSO). The expression was measured using two sets of primers against the same promoter region of ITGA4. Representative graphs are average of three qPCR datasets ± SEM. b. ITGA4 expression in the presence of A-395 vs . DMSO control, N=6 independent experiments compared using t -test. c. Measuring the expression levels of ITGA4 upon depletion of MEG3 using LNA GapmeRs (10nM, 48h), data is mean of N=5 independent experiments (biological replicates). d. Representative image of immunofluorescence staining for ITGA4 protein levels in ECs treated with A-395 vs . DMSO, or upon MEG3 depletion like in b . e. Intra-cellular localisation of MEG3 (chromatin associated lncRNA) between different cellular compartments in HUVECs treated with A-395 vs. DMSO, whereby the distribution of MEG3 has shifted upon PRC2 inhibition with A-395; from the nucleus (where it was highly chromatin bound) into the cytoplasm. Representative bars were compared by t-test and on-way Anova. f. MEG3-ChIRP followed by qPCR, N =3, analysis of MEG3 binding over ITGA4 promoter region in HUVECs treated with A-395 (5µM, 24h) vs. DMSO. MEG3-ChIRP HUVEC lysates treated with A-395 resulted in reduced engagement of MEG3 with ITGA4 site compared with either DMSO control or ChIRP with non-biotinylated probes. The non-biotin probes served as a negative control, and we detected the background level <1.

Article Snippet: Following sonication as described, samples were immunoprecipitated using EZH2 (D2C9) XP(R) Rabbit mAb, (5246S Cell signalling technology), Tri-Methyl-Histone H3 (H3K27me3) (C36B11) Rabbit mAb (9733S, CST) antibodies or IgG control (Normal Rabbit IgG, 2729S, CST) and captured on beads using Protein G Dyneabeads (10003D, Life Technologies).

Techniques: Control, Expressing, Immunofluorescence, Staining, Inhibition, Binding Assay, Negative Control

Journal: bioRxiv

Article Title: Histone H3K27 methyltransferase EZH2 interacts with MEG3-lncRNA to directly regulate integrin signaling and endothelial cell function

doi: 10.1101/2022.05.20.492787

Figure Lengend Snippet: a. Measure of cell migratory capacity using ECIS functional analysis in ECs treated with control or A-395 (5µM, 24h) inhibitor. Experiments were performed in duplicates (technical replicates) and four experiments were run for migration assay and six for adhesion (biological replicates). The data showing ECIS trace (left hand side) is mean ±SD as calculated by the ECIS. The graph on the right is mean±SEM with N =6, data was compared using ordinary one-way ANOVA with Dunnett’s multiple comparisons tests. b. Adhesion to Fibronectin, FN (20µg/ml) was used to coat the culture plates and assess adhesion of endothelial cells within 3h of ECIS assay, following cell pre-treatment with A-395, 24h. The difference in resistance change was calculated over 3h. c. Subcutaneous Matrigel plug injection (200µl) into mice ( N =5) treated with DMSO (control, left flange) and A-395 (1mg/ml, right flange) was done for 2 weeks. Matrigel plugs were collected and processed for histology. Staining for H3K27me3 was done, displaying nuclear positivity with strong intensity in control (<0.02% DMSO in water) and the A-395 treatment decreased total H3K27me3 staining, as compared by t-test. d. Staining for arterioles was performed to assess vessel growth as angiogenesis and data was compared using Student’s t-test. The data shows increased area of staining for Isolectin B4 (Iso-B4) dye in A-395 vs. DMSO treated Matrigel plugs with increased neovascularization, P<0.05. e. A-395 has increased the percentage of vessels positive for ITGA4 (red) within the Isolectin B4 positive cells, compared with the DMSO using t -test. f. Graphical abstract. 1 Maternally Expressed Gene–MEG3 is highly expressed with hypoxia and bound to EZH2 in endothelial cells (EC) affected by ischaemic insult. 2 Such MEG3:EZH2 complex assembles onto the target genes to 3 direct the EZH2 activity to “write” H3K27me3 trimethylation repressive mark and block expression of target gene i.e. integrin alpha 4 (ITGA4) and its ability to dimerise with integrin beta 1 (ITGB1), leading to 4 reduced EC function as measured by adhesion and migration. Hence 5 targeted disruptions of MEG3:EZH2 interaction, or inhibition of EZH2 activity could increase EC function under ischaemia.

Article Snippet: Following sonication as described, samples were immunoprecipitated using EZH2 (D2C9) XP(R) Rabbit mAb, (5246S Cell signalling technology), Tri-Methyl-Histone H3 (H3K27me3) (C36B11) Rabbit mAb (9733S, CST) antibodies or IgG control (Normal Rabbit IgG, 2729S, CST) and captured on beads using Protein G Dyneabeads (10003D, Life Technologies).

Techniques: Functional Assay, Control, Migration, Injection, Staining, Activity Assay, Blocking Assay, Expressing, Inhibition

Journal: Cell Reports

Article Title: Mutant TP53 switches therapeutic vulnerability during gastric cancer progression within interleukin-6 family cytokines

doi: 10.1016/j.celrep.2024.114616

Figure Lengend Snippet:

Article Snippet: Ani-STAT3 antibody, rabbit monoclonal, clone 79D7 , Cell Signaling Technology , Cat# 4904; RRID:AB_331269.

Techniques: Plasmid Preparation, Microarray, Recombinant, Protease Inhibitor, Membrane, Gentle, Blocking Assay, Transfection, Reverse Transcription, Bicinchoninic Acid Protein Assay, Viability Assay, Mutagenesis, CRISPR, Software, Activation Assay

Journal: Cancer cell

Article Title: Oncogenic KRAS regulates amino acid homeostasis and asparagine biosynthesis via ATF4 and alters sensitivity to L-asparaginase

doi: 10.1016/j.ccell.2017.12.003

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit monoclonal anti-ERK , Cell Signaling Technologies , Cat# 4695, RRID:AB_390779.

Techniques: Virus, Plasmid Preparation, Recombinant, Labeling, cDNA Synthesis, SYBR Green Assay, Microarray, Expressing, Gene Expression, Mutagenesis, Software

Journal: Current Biology

Article Title: Long-Fiber Carbon Nanotubes Replicate Asbestos-Induced Mesothelioma with Disruption of the Tumor Suppressor Gene Cdkn2a ( Ink4a/Arf )

doi: 10.1016/j.cub.2017.09.007

Figure Lengend Snippet:

Article Snippet: Rabbit monoclonal anti-phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (clone 197G2) , Cell Signaling Technology , Cat# 4377; RRID: AB_331775.

Techniques: Recombinant, Produced, Reverse Transcription, Microarray, Labeling, DNA Purification, Methylation, TA Cloning, Sequencing, SYBR Green Assay, Software

Journal: Journal for Immunotherapy of Cancer

Article Title: Potent STING activation stimulates immunogenic cell death to enhance antitumor immunity in neuroblastoma

doi: 10.1136/jitc-2019-000282

Figure Lengend Snippet: Nanoparticle-enabled cytosolic delivery of 2’3’-cGAMP activates the STING pathway in neuroblastoma cells. (A) Integrated molecular analysis of mRNA expression of genes from the pediatric neuroblastoma TARGET dataset that have been distinguished by functional significance and clustered into evenly split tertiles based on high (upper tertile, n=47), intermediate (median tertile, n=47), and low (bottom tertile, n=47) TMEM173 mRNA expression. (B) TMEM173 mRNA expression in MYCN non-amplified and amplified samples profiled by microarray in the TARGET pediatric neuroblastoma (n=55) datasets. Data were accessed through the cBioPortal. Mann-Whitney U test (two-tailed) was used for statistical comparison.(C) Schematic representation of STING-NPs designed to enhance cytosolic delivery of cGAMP via endosomal escape, resulting in potent activation of STING signaling. (D) Neuroblastoma cell lines (Neuro-2a, 9464D, SK-N-SH, and LAN-1) were treated with vehicle (PBS), empty nanoparticles (NP), 200 nM (or 400 nM for 9464D) cGAMP, or STING-NPs for 48 hours; cells were collected for western blot analysis using anti-IRF3 and anti-phospho-IRF3 (p-IRF3) antibodies. Gel loading was normalized for equal actin; representative blots from one of two independent experiments. The relative density of bands is shown under each immunoblot, after normalization to the levels of actin. (E) qRT-PCR gene expression of IFNB1, TNF, CXCL10, and IL12 in neuroblastoma cell lines at 6, 24 and 48 hours after treatment with cGAMP or STING-NPs. (n=2, data shown as mean±SD; data was analyzed by two-way ANOVA followed by Dunnett’s posthoc test, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 indicate a statistically significant difference relative to vehicle (PBS). ANOVA, analysis of variance; cGAMP, cyclic guanosine monophosphate–adenosine monophosphate; qRT-PCR, quantitative real-time PCR; STING, stimulator of interferon genes; STING-NPs, STING-activating nanoparticles.

Article Snippet: Antibodies against STING (13 647S), p-IRF3 (4997S), IRF3 (4302S), Cleaved-Caspase 3 (9664T), and p-STAT1 (9167S) were purchased from Cell Signaling Technology (Beverly, Massachusetts).

Techniques: Expressing, Functional Assay, Amplification, Microarray, MANN-WHITNEY, Two Tailed Test, Comparison, Activation Assay, Western Blot, Quantitative RT-PCR, Gene Expression, Real-time Polymerase Chain Reaction

Journal: Journal for Immunotherapy of Cancer

Article Title: Potent STING activation stimulates immunogenic cell death to enhance antitumor immunity in neuroblastoma

doi: 10.1136/jitc-2019-000282

Figure Lengend Snippet: STING activation in neuroblastoma generates an immunogenic, tumoricidal, and T-cell inflamed tumor microenvironment. Mice with 200 mm 3 Neuro-2a tumors were administered PBS or STING-NPs (10 µg cGAMP) intratumorally three times, spaced 3 days apart, and tumors were harvested 48 hours following the last treatment. (A) qRT-PCR analysis of IFNB1, TNF, CXCL10, and IL12 gene expression in injected Neuro-2a tumors. n=5 to 7 mice per group represented as mean±SEM, *p<0.05, **p<0.01 indicate a statistically significant difference using a two-tailed Mann-Whitney U test. (B) Neuro-2a tumor lysates (n=4–5) were analyzed using western blot for phospho-STAT1 (p-STAT1), IRF3, and phospho-IRF3 (p-IRF3), caspase 3, and cleaved caspase 3. Gel loading was normalized for equal actin. The density of bands is shown under each immunoblot, after normalization to the levels of actin. (C/D) Immunohistochemical staining of tumor sections for cleaved caspase-3 (apoptosis), Ki-67 (proliferation), CD8, and FoxP3 and corresponding quantification of staining intensity using ImageJ software. Data shown as mean±SD for n=3 Neuro-2a tumors, ***p<0.001, ****p<0.0001 indicate a statistically significant difference using a Student’s t-test. cGAMP, cyclic guanosine monophosphate–adenosine monophosphate; qRT-PCR, quantitative real-time PCR; STING, stimulator of interferon genes; STING-NPs, STING-activating nanoparticles.

Article Snippet: Antibodies against STING (13 647S), p-IRF3 (4997S), IRF3 (4302S), Cleaved-Caspase 3 (9664T), and p-STAT1 (9167S) were purchased from Cell Signaling Technology (Beverly, Massachusetts).

Techniques: Activation Assay, Quantitative RT-PCR, Gene Expression, Injection, Two Tailed Test, MANN-WHITNEY, Western Blot, Immunohistochemical staining, Staining, Software, Real-time Polymerase Chain Reaction

Journal: The Journal of Pathology

Article Title: Constitutive ERK1/2 activation contributes to production of double minute chromosomes in tumour cells

doi: 10.1002/path.4439

Figure Lengend Snippet: Double minute chromosome (DM)-containing tumour cells often show constitutive activation of the ERK1/2–MAPK pathway. (A, F) The number of DMs/cell in various human malignant tumour cells (bars represent mean ± SD). (B–D, G) Phosphorylation status of ERK1/2, p38 and JNK1/2/3 in DM-containing tumour cells; phosphorylation of ERK1/2, p38 and JNK1/2/3 in MEKK3- or p38-over-expressing or TNF α -treated HEK293T cells (10 ng/ml for 30 min) are shown as positive controls. (E) Microarray analysis of UACC-1598DM compared to UACC-1598HSR cells

Article Snippet: The anti-phospho-ERK1/2 (Thr202/Tyr204), anti-ERK1/2, anti-phospho-SAPK/JNK (Thr183/Tyr185), anti-SAPK/JNK, anti-phospho-p38 MAP kinase (Thr180/Tyr182) and anti-p38 MAP kinase antibodies were obtained from Cell Signalling Technology (Danvers, MA, USA); anti-MCL1 and anti-MYC from Santa Cruz Biotechnology (Dallas, TX, USA); anti-EIF5A2 from Sigma-Aldrich (St. Louis, MO, USA); anti-GAPDH from KangChen Bio-tech Inc. (Shanghai, China); and anti-phospho-histone H2A.X (Ser139) from Merck Millipore (Bedford, MA, USA).

Techniques: Activation Assay, Phospho-proteomics, Expressing, Microarray

Journal: The Journal of Pathology

Article Title: Constitutive ERK1/2 activation contributes to production of double minute chromosomes in tumour cells

doi: 10.1002/path.4439

Figure Lengend Snippet: The number of double minute chromosomes (DMs) decreases after inhibition of ERK1/2 phosphorylation. (A, B) ERK1/2 inhibitors decrease the number of DMs in UACC-1598 and Colo320DM cells (mean number of DMs/cell ± SD); * p < 0.05, ** p < 0.01, *** p < 0.001, by ANOVA and Dunnett's multiple comparison post-test. (C, D) The numbers of DMs in UACC-1598 or Colo320DM cells are not affected by JNK1/2/3 or p38 inhibitor treatment (SP600125 or SB203580). (E, F) The numbers of DMs in NCI-H716 and NCI-H508 cells are not affected by ERK1/2 inhibitor treatment

Article Snippet: The anti-phospho-ERK1/2 (Thr202/Tyr204), anti-ERK1/2, anti-phospho-SAPK/JNK (Thr183/Tyr185), anti-SAPK/JNK, anti-phospho-p38 MAP kinase (Thr180/Tyr182) and anti-p38 MAP kinase antibodies were obtained from Cell Signalling Technology (Danvers, MA, USA); anti-MCL1 and anti-MYC from Santa Cruz Biotechnology (Dallas, TX, USA); anti-EIF5A2 from Sigma-Aldrich (St. Louis, MO, USA); anti-GAPDH from KangChen Bio-tech Inc. (Shanghai, China); and anti-phospho-histone H2A.X (Ser139) from Merck Millipore (Bedford, MA, USA).

Techniques: Inhibition, Phospho-proteomics, Comparison

Journal: Signal Transduction and Targeted Therapy

Article Title: USP7 deubiquitinates and stabilizes NOTCH1 in T-cell acute lymphoblastic leukemia

doi: 10.1038/s41392-018-0028-3

Figure Lengend Snippet: USP7 interacts with ICN1. a HEK293T cells were transfected with plasmids encoding FLAG-tagged USP7 and/or Myc-tagged ICN1. Cell extracts were prepared and immunoprecipitated with anti-FLAG or anti-Myc antibodies. The protein interactions were analyzed by western blotting. b Whole-cell lysates from JURKAT and MOLT-4 cells were subjected to immunoprecipitation with a control IgG or an anti-ICN1 antibody. The immunoprecipitates were detected by western blotting. The input represented ~5% of the total protein extract used for immunoprecipitation. c The direct interaction between USP7 and ICN1 was detected using a GST pull-down assay, and the indicated proteins were examined by western blotting. d USP7 was co-localized with NOTCH1. CUTLL1 cells were fixed and immunostained with anti-USP7 (green) and anti-NOTCH1 (red) antibodies. The cell nuclei were counterstained with DAPI (blue). e Mapping of the ICN1-interacting domain in the USP7 protein. Top panel, a schematic representation of various USP7 truncated mutants. Bottom panel, HEK293T cells were co-transfected with constructs encoding FLAG-tagged ICN1 and GFP-tagged USP7 or truncated mutants. FLAG-tagged ICN1 proteins were immunoprecipitated with an anti-FLAG antibody, and the presence of USP7 protein and truncated mutants was examined by western blotting using an anti-GFP antibody

Article Snippet: The following antibodies were used in this study: anti-cleaved NOTCH1 (Val1744) and anti-β-Actin (CST, Danves, MA, USA); anti-USP7 (Bethyl Laboratories, Montgomery, TX, USA); anti-NOTCH1 and anti-GFP (Santa Cruz, Dallas, TX, USA); anti-HA and anti-Myc epitope tag (MBL, Nagoya, Japan); anti-FLAG (M2) (Sigma-Aldrich, Louis, MO, USA); and anti-HRP-conjugated secondary antibody (Millipore, Bedford, MA, USA).

Techniques: Transfection, Immunoprecipitation, Western Blot, Pull Down Assay, Construct

Journal: Signal Transduction and Targeted Therapy

Article Title: USP7 deubiquitinates and stabilizes NOTCH1 in T-cell acute lymphoblastic leukemia

doi: 10.1038/s41392-018-0028-3

Figure Lengend Snippet: USP7 is overexpressed in T-ALL. a USP7 microarray gene expression data were obtained from the Cancer Cell Line Encyclopedia (CCLE). The data are presented with box plots. The sample number ( n ) are indicated in parentheses. RMA represents Robust Multi-array Average. b Analysis of TCGA leukemia dataset from the Oncomine database to assess the expression of USP7 in normal bone marrow or peripheral blood cells and in T-ALL patient samples. The data are presented with box plots. Fold change, p- value (determined by Student’s t -test), and sample size are shown. c Comparison of USP7 gene expression levels between NOTCH1 WT ( n = 31) and NOTCH1 mutated ( n = 87) T-ALL cases. The read counts mapped to the USP7 transcript were normalized and applied with variance-stabilizing transformation. The p- value was determined using Student’s t -test. Dots represent the value of the USP7 expression level in each of the T-ALL cases. The mean and 25th and 75th percentiles are represented by the midline and line edges in the plots, respectively. d western blotting analysis of the USP7 protein levels in normal PBMCs and T-ALL patient samples (top panel) along with various T-ALL cell lines (bottom panel)

Article Snippet: The following antibodies were used in this study: anti-cleaved NOTCH1 (Val1744) and anti-β-Actin (CST, Danves, MA, USA); anti-USP7 (Bethyl Laboratories, Montgomery, TX, USA); anti-NOTCH1 and anti-GFP (Santa Cruz, Dallas, TX, USA); anti-HA and anti-Myc epitope tag (MBL, Nagoya, Japan); anti-FLAG (M2) (Sigma-Aldrich, Louis, MO, USA); and anti-HRP-conjugated secondary antibody (Millipore, Bedford, MA, USA).

Techniques: Microarray, Expressing, Transformation Assay, Western Blot

Journal: iScience

Article Title: Type 2 diabetes sex-specific effects associated with E167K coding variant in TM6SF2

doi: 10.1016/j.isci.2021.103196

Figure Lengend Snippet:

Article Snippet: Phospho-GSK-3β (Ser9) (D85E12) Rabbit mAb , Cell Signaling , #5558; RRID: AB_10013750.

Techniques: Virus, Recombinant, Protease Inhibitor, SYBR Green Assay, Enzyme-linked Immunosorbent Assay, Microarray, Software