Journal: Diagnostic Pathology

Article Title: Establishment of an antibody specific for AMIGO2 improves immunohistochemical evaluation of liver metastases and clinical outcomes in patients with colorectal cancer

doi: 10.1186/s13000-021-01176-2

Figure Lengend Snippet: Specific detection of AMIGO2 by the monoclonal antibody rTNK1A0012. The large membrane was reacted with anti-AMIGO2 antibody, while the small membrane was reacted with an anti-β-actin antibody. A Cell lysates were prepared from HepG2 cells transfected with human AMIGO1 (A1), AMIGO2 (A2), AMIGO3 (A3), and an empty vector (E). Immunoblotting with rTNK1A0012 (rTNK mAb). B Immunoblotting with sc-373699 (sc mAb). C Cell lysates were treated with or without PNGase F and immunoblotted with rTNK mAb. D Cell lysates were treated with or without alkaline β-elimination and immunoblotted with rTNK mAb

Article Snippet: Expression plasmids for human AMIGO1 (pEZ-M02/AMIGO1), AMIGO2 (pEZ-M02/AMIGO2), and AMIGO3 (pEZ-M02/AMIGO3) were purchased from GeneCopoeia (EX-E1371-M02, EX-E1271-M02, EX-E1133-M02, Rockville, MD).

Techniques: Membrane, Transfection, Plasmid Preparation, Western Blot

Journal: Diagnostic Pathology

Article Title: Establishment of an antibody specific for AMIGO2 improves immunohistochemical evaluation of liver metastases and clinical outcomes in patients with colorectal cancer

doi: 10.1186/s13000-021-01176-2

Figure Lengend Snippet: Detection of three types of AMIGO family molecules by commercially available antibodies. A The same cell lysates as shown in Fig. was used. Lysates were prepared from HepG2 cells transfected with human AMIGO1 (A1), AMIGO2 (A2), AMIGO3 (A3), and an empty vector (E). Immunoblotting with LS-C404504 polyclonal antibody. B Immunoblotting with #36094 polyclonal antibody. C Immunoblotting with HPA054004 polyclonal antibody

Article Snippet: Expression plasmids for human AMIGO1 (pEZ-M02/AMIGO1), AMIGO2 (pEZ-M02/AMIGO2), and AMIGO3 (pEZ-M02/AMIGO3) were purchased from GeneCopoeia (EX-E1371-M02, EX-E1271-M02, EX-E1133-M02, Rockville, MD).

Techniques: Transfection, Plasmid Preparation, Western Blot

Journal: Diagnostic Pathology

Article Title: Establishment of an antibody specific for AMIGO2 improves immunohistochemical evaluation of liver metastases and clinical outcomes in patients with colorectal cancer

doi: 10.1186/s13000-021-01176-2

Figure Lengend Snippet: Immunohistochemical staining for AMIGO2 expression by the monoclonal antibody rTNK1A0012 in CRC tissues. AMIGO2 negative expression ( A and C ) and AMIGO2 high expression ( B and D ) are shown. The lower row are shown at higher magnification. Scale bars = 400 μm ( A and B ) and 100 μm ( C and D )

Article Snippet: Expression plasmids for human AMIGO1 (pEZ-M02/AMIGO1), AMIGO2 (pEZ-M02/AMIGO2), and AMIGO3 (pEZ-M02/AMIGO3) were purchased from GeneCopoeia (EX-E1371-M02, EX-E1271-M02, EX-E1133-M02, Rockville, MD).

Techniques: Immunohistochemical staining, Staining, Expressing

Journal: Diagnostic Pathology

Article Title: Establishment of an antibody specific for AMIGO2 improves immunohistochemical evaluation of liver metastases and clinical outcomes in patients with colorectal cancer

doi: 10.1186/s13000-021-01176-2

Figure Lengend Snippet: AMIGO2 expression and clinicopathological factors affecting overall survival rate in 173 CRC patients

Article Snippet: Expression plasmids for human AMIGO1 (pEZ-M02/AMIGO1), AMIGO2 (pEZ-M02/AMIGO2), and AMIGO3 (pEZ-M02/AMIGO3) were purchased from GeneCopoeia (EX-E1371-M02, EX-E1271-M02, EX-E1133-M02, Rockville, MD).

Techniques: Expressing

Journal: Diagnostic Pathology

Article Title: Establishment of an antibody specific for AMIGO2 improves immunohistochemical evaluation of liver metastases and clinical outcomes in patients with colorectal cancer

doi: 10.1186/s13000-021-01176-2

Figure Lengend Snippet: Relationship between AMIGO2 expression in primary colorectal cancer and metastatic site. A high expression of AMIGO2 was significantly associated with liver metastases ( A ) and lung metastases ( B ), but not with peritoneal dissemination ( C ), as calculated using the X 2 test

Article Snippet: Expression plasmids for human AMIGO1 (pEZ-M02/AMIGO1), AMIGO2 (pEZ-M02/AMIGO2), and AMIGO3 (pEZ-M02/AMIGO3) were purchased from GeneCopoeia (EX-E1371-M02, EX-E1271-M02, EX-E1133-M02, Rockville, MD).

Techniques: Expressing

Journal: Diagnostic Pathology

Article Title: Establishment of an antibody specific for AMIGO2 improves immunohistochemical evaluation of liver metastases and clinical outcomes in patients with colorectal cancer

doi: 10.1186/s13000-021-01176-2

Figure Lengend Snippet: Univariate and multivariate analysis for overall survival in CRC patients (Cox proportional hazard regression model)

Article Snippet: Expression plasmids for human AMIGO1 (pEZ-M02/AMIGO1), AMIGO2 (pEZ-M02/AMIGO2), and AMIGO3 (pEZ-M02/AMIGO3) were purchased from GeneCopoeia (EX-E1371-M02, EX-E1271-M02, EX-E1133-M02, Rockville, MD).

Techniques: Expressing

Journal: Diagnostic Pathology

Article Title: Establishment of an antibody specific for AMIGO2 improves immunohistochemical evaluation of liver metastases and clinical outcomes in patients with colorectal cancer

doi: 10.1186/s13000-021-01176-2

Figure Lengend Snippet: Univariate and multivariate analysis of factors affecting liver metastases in CRC patients (Logistic regression model)

Article Snippet: Expression plasmids for human AMIGO1 (pEZ-M02/AMIGO1), AMIGO2 (pEZ-M02/AMIGO2), and AMIGO3 (pEZ-M02/AMIGO3) were purchased from GeneCopoeia (EX-E1371-M02, EX-E1271-M02, EX-E1133-M02, Rockville, MD).

Techniques: Expressing

Journal: Diagnostic Pathology

Article Title: Establishment of an antibody specific for AMIGO2 improves immunohistochemical evaluation of liver metastases and clinical outcomes in patients with colorectal cancer

doi: 10.1186/s13000-021-01176-2

Figure Lengend Snippet: Comparison of two AMIGO2 antibodies in AMIGO2 expression and survival in CRC patients. Cumulative survival rates were assessed using the Kaplan-Meier plot method. Differences were analyzed using the log-rank test. Overall survival ( A ) and disease-specific survival ( B ) are shown

Article Snippet: Expression plasmids for human AMIGO1 (pEZ-M02/AMIGO1), AMIGO2 (pEZ-M02/AMIGO2), and AMIGO3 (pEZ-M02/AMIGO3) were purchased from GeneCopoeia (EX-E1371-M02, EX-E1271-M02, EX-E1133-M02, Rockville, MD).

Techniques: Comparison, Expressing

Journal: The Journal of Cell Biology

Article Title: AMIGO2, a novel membrane anchor of PDK1, controls cell survival and angiogenesis via Akt activation

doi: 10.1083/jcb.201503113

Figure Lengend Snippet: AMIGO2 regulates EC viability and angiogenesis. (A–J) HUVECs were pretransfected with scrambled siRNA and AMIGO2-specific siRNA and harvested for analysis 48 h after transfection. (A) FAK immunostaining was performed in parallel. Images on the right are enlargements of the boxed regions on the left. Bars, 20 µm. (B) Cell viability of AMIGO2 siRNA–transfected HUVECs in complete media and under conditions of serum-free starvation with or without VEGF ( n = 6). Data were collected from independent experiments and analyzed using a two-tailed unpaired t test. Data are means ± SD. (C) Expression of active caspase-3 and FAK fragments in AMIGO2 siRNA–transfected ECs. (D) Different stages of apoptosis were detected by phycoerythrin-conjugated annexin V flow cytometry and PerCP–7-AAD staining. Quadrant gates are based on unstained, annexin V, or 7-AAD alone. Quadrant regions show the percentage of living cells (7-AAD − /annexin V − ), early apoptotic cells (7-AAD − /annexin V + ), late apoptotic cells (7-AAD + /annexin V + ), and necrotic cells (7-AAD + /annexin V − ). The data are representative of three experiments conducted by using different samples. (E and F) Wound healing migration and gelatin-coated Transwell migrations with or without VEGF were performed. Two types of siA2 were evaluated for wound healing migration. n = 3 and n = 6. (G) Matrigel-induced tube formation by AMIGO2 siRNA–transfected HUVECs was assessed. (H and J) Mean numbers of tubule branch points and of tubes per field in randomly selected images were quantified. (I) Tube length is presented as the percentage of total tube length per field versus that of untreated control cells. (H–J) Data were collected from independent experiments and analyzed using a two-tailed unpaired t test. Data are means ± SD. n = 4. *, P < 0.05; **, P < 0.005; ***, P < 0.0001. sc, scrambled siRNA; siA2, AMIGO2 siRNA.

Article Snippet: Mouse and goat anti-AMIGO2 antibodies for immunoprecipitation and Western blotting were purchased from R&D Systems and Santa Cruz Biotechnology, Inc., respectively.

Techniques: Transfection, Immunostaining, Two Tailed Test, Expressing, Flow Cytometry, Staining, Migration, Control

Journal: The Journal of Cell Biology

Article Title: AMIGO2, a novel membrane anchor of PDK1, controls cell survival and angiogenesis via Akt activation

doi: 10.1083/jcb.201503113

Figure Lengend Snippet: AMIGO2 is required for angiogenesis in vivo. P3.5 mice were injected with Amigo2 -specific or control siRNA and shRNA, and experiments were performed at P5.5, P8.5, and P15.5. (A) Impairment of hyaloid vessel structures in Amigo2 siRNA–injected mice. Bars, 200 µm. (B) Capillary quantification. n = 5. (C) Hyaloid vessel staining with CD31, AMIGO2, and TUNEL was performed in Amigo2 shRNA–injected P5.5 mice. (D and E) Visualization of blood vessels by iB4 staining with AMIGO2 and TUNEL staining of Amigo2 -depleted retinas from P5.5 mice. Bars: (D) 50 µm; (E, left to right) 500 µm, 200 µm, and 50 µm. (E) Middle and right images are enlargements of the left image. (F–H) Radial expansions, retinal vascular branch points, and sprouts per field were quantified. n = 5. (I–L) Immunostaining of blood vessels by iB4-staining of Amigo2 shRNA–injected P8.5 retinas (I and K) and P15.5 retinas (J and L). Superficial (S), intermediate (I), and deep (D) layers of retinal vessels were taken and quantified. Bars, 50 µm. n = 5. Data were collected from mice ( n = 5–6) and analyzed using a two-tailed unpaired t test. *, P < 0.05; **, P < 0.005; ***, P < 0.0001. Data are means ± SD. shCon, control shRNA; shA2, Amigo2 shRNA.

Article Snippet: Mouse and goat anti-AMIGO2 antibodies for immunoprecipitation and Western blotting were purchased from R&D Systems and Santa Cruz Biotechnology, Inc., respectively.

Techniques: In Vivo, Injection, Control, shRNA, Staining, TUNEL Assay, Immunostaining, Two Tailed Test

Journal: The Journal of Cell Biology

Article Title: AMIGO2, a novel membrane anchor of PDK1, controls cell survival and angiogenesis via Akt activation

doi: 10.1083/jcb.201503113

Figure Lengend Snippet: AMIGO2 modulates the PDK1–Akt pathway. (A) The endogenous effect of AMIGO2 knockdown on Akt signaling in HUVECs. Two types of siA2 were evaluated. (B) Endogenous effects of AMIGO overexpression on Akt signaling activation in HUVECs. (C and D) Akt phosphorylation in hyaloid vessels (C) and retinas (D) from Amigo2 -depleted mice was evaluated. The data are representative of six mice. (E) Temporal effects of AMIGO2 knockdown on VEGF-induced signaling pathways (20 ng/ml) in HUVECs. (F) VEGF-induced PI3K–PDK1–Akt signaling was evaluated in AMIGO2 siRNA–transfected HUVECs.

Article Snippet: Mouse and goat anti-AMIGO2 antibodies for immunoprecipitation and Western blotting were purchased from R&D Systems and Santa Cruz Biotechnology, Inc., respectively.

Techniques: Knockdown, Over Expression, Activation Assay, Phospho-proteomics, Protein-Protein interactions, Transfection

Journal: The Journal of Cell Biology

Article Title: AMIGO2, a novel membrane anchor of PDK1, controls cell survival and angiogenesis via Akt activation

doi: 10.1083/jcb.201503113

Figure Lengend Snippet: AMIGO2 regulates subcellular localization and interacts with PDK1. (A) PDK1 immunostaining was performed in AMIGO2-inhibited HUVECs in the presence or absence of VEGF. Cells were starved for 6 h and treated with VEGF. White arrowheads indicate membrane localization of PDK1. Bars, 20 µm. (B) Subcellular localization analysis was performed by cell fractionation in VEGF-treated, AMIGO2-deficient HUVECs. (C) Quantification of Western blots. Subcellular localization analysis was performed by cell fractionation with or without VEGF in control siRNA and AMIGO2-deficient HUVECs. ***, P < 0.0001. Data are means ± SD. (D) Endogenous AMIGO2 interacts with PDK1 in HUVECs. AMIGO2 was immunoprecipitated and blotted with anti-PDK1 and -Akt antibodies. (E) Colocalization of PDK1 and AMIGO2 in the plasma membrane and cytosol of HUVECs in the presence of VEGF. HUVECs were starved for 6 h and stimulated with or without VEGF. White arrowheads indicate membrane edge areas of HUVECs. Bars, 20 µm. (F) HUVECs were starved for 6 h, stimulated with VEGF for 4 h, and immunoprecipitated with an AMIGO2 antibody and blotted with endogenous PDK1. (G) AMIGO2-deficient HUVECs were immunoprecipitated with an AMIGO2 antibody and blotted with an anti-PDK1 antibody. (H) Flag-PDK1 was immunoprecipitated and blotted with an anti-GFP antibody tagged to AMIGO2. A2, AMIGO2; HC, heavy chain; IB, immunoblotting; IgG, normal IgG; IP, immunoprecipitation; ns, not significant; WCL, whole cell lysate.

Article Snippet: Mouse and goat anti-AMIGO2 antibodies for immunoprecipitation and Western blotting were purchased from R&D Systems and Santa Cruz Biotechnology, Inc., respectively.

Techniques: Immunostaining, Membrane, Cell Fractionation, Western Blot, Control, Immunoprecipitation, Clinical Proteomics

Journal: The Journal of Cell Biology

Article Title: AMIGO2, a novel membrane anchor of PDK1, controls cell survival and angiogenesis via Akt activation

doi: 10.1083/jcb.201503113

Figure Lengend Snippet: The PH domain of PDK1 binds to the CD of AMIGO2. (A and B) The CD of AMIGO2 interacts with PDK1. HEK293 cells were transfected with Flag-PDK1 and GFP-tagged AMIGO2 or domain deletion mutants. Lysates were immunoprecipitated with GFP (A) or Flag (B) antibodies and blotted with the GFP or Flag antibody. PDK1 was immunoprecipitated with the Flag antibody and detected with the anti-GFP antibody. A2, AMIGO2 WT ; ΔLRR, AMIGO2 ΔLRR ; ΔIgG, AMIGO2 ΔIgG ; ΔCD, AMIGO2 ΔCD ; CD, CD domain of AMIGO2. (C) Schematic of 491 aa and 457 aa of the AMIGO2 constructs. (D) PDK1 associates with the region between 491 aa and 457 aa of AMIGO2. HEK293 cells were transfected with Flag-PDK1– and GFP-tagged AMIGO2 or domain deletion mutants. Lysates were immunoprecipitated with GFP antibody and blotted with the Flag and GFP antibodies. (E) Each His-tagged PDK1 kinase and His-tagged PDK1 PH was incubated with purified GST-AMIGO2 CD . Mixtures of His-tagged PDK1 kinase or PDK1 PH proteins and GST-AMIGO2 CD proteins were pulled down with GST resin and analyzed by Western blotting using anti-His and -GST antibodies. His-kinase, His-PDK1 kinase ; His-PH, His-PDK1 PH ; GST-CD, GST-AMIGO2 CD . (F) Mixtures of His-PDK1 PH and GST-CD proteins were incubated with PIP3-coated or control beads and treated with DM-PIT-1. The PIP3-bound proteins were analyzed with His and GST antibodies. (G) Multiple sequence alignments of the AMIGO family CD domains were performed. An asterisk indicates that the alignment contains identical amino acid residues in all sequences (or identical bases if DNA sequences are aligned); a colon indicates that the alignment contains different but highly conserved (very similar) amino acids; a period indicates that the alignment contains different amino acids that are somewhat similar; and a blank space indicates that the alignment contains dissimilar amino acids or gaps (or different bases if DNA sequences are aligned). (H) Diagram showing the protein sequences of cytosolic-truncated mutants. The total length of the cytosolic tail is 103 aa. (I) Far-western analysis was performed for each truncated mutant. Coomassie blue staining revealed purified GST-tagged truncated mutants that were previously loaded for far-western analysis. (J) Mixtures of purified GST-tagged truncated mutants and His-tagged PH domain proteins were pulled down with GST beads and eluted, and the Western blot was performed using His and GST antibodies. HC, heavy chain; IP, immunoprecipitation.

Article Snippet: Mouse and goat anti-AMIGO2 antibodies for immunoprecipitation and Western blotting were purchased from R&D Systems and Santa Cruz Biotechnology, Inc., respectively.

Techniques: Transfection, Immunoprecipitation, Construct, Incubation, Purification, Western Blot, Control, Sequencing, Mutagenesis, Staining

Journal: The Journal of Cell Biology

Article Title: AMIGO2, a novel membrane anchor of PDK1, controls cell survival and angiogenesis via Akt activation

doi: 10.1083/jcb.201503113

Figure Lengend Snippet: PTD-A2, an AMIGO2 competitive peptide, blocks PDK1 translocation and Akt activation. (A) The TAT-A2 binding assay was performed. PH domain proteins (0–60 µg) were incubated with 1-µM FITC–TAT-A2 and detected by fluorescence. Data were collected from independent experiments and analyzed using a two-tailed unpaired t test. n = 4. (B) Peptide competition assay with Con or TAT-A2 and CD to PH domains. His-tagged PDK1 PH was incubated with purified GST-AMIGO2 CD and Con or TAT-A2 in a dose-dependent manner. His-tagged PH domain proteins were pulled down with GST resins, eluted, and analyzed by Western blotting. (C) Con- and TAT-A2– treated HUVECs were immunoprecipitated with an AMIGO2 antibody and blotted with anti-PDK1 and AMIGO2 antibodies. (D) Localization of PDK1 in the plasma membrane and cytosol of HUVECs in the presence of VEGF after Con or TAT-A2 treatment. Bars, 20 µm. (E) Subcellular localization analysis was performed by cell fractionation in VEGF-treated, TAT-A2–treated HUVECs. (F) Quantification of Western blots. Subcellular localization analysis was performed by cell fractionation with or without VEGF in control and TAT-A2 HUVECs. HUVECs were affected by both PTD-A2 peptides. (G) Effects of TAT-A2 on VEGF-induced Akt signaling in HUVECs. Con, control peptide; TAT-A2, TAT-RVVFLEPLKD peptide. PTD-A2 means both TAT-A2 and NGR-A2. Data are means ± SD. *, P < 0.05; **, P < 0.005; ***, P < 0.0001. HC, heavy chain; IB, immunoblotting; IP, immunoprecipitation; WCL, whole cell lysate.

Article Snippet: Mouse and goat anti-AMIGO2 antibodies for immunoprecipitation and Western blotting were purchased from R&D Systems and Santa Cruz Biotechnology, Inc., respectively.

Techniques: Translocation Assay, Activation Assay, Binding Assay, Incubation, Fluorescence, Two Tailed Test, Competitive Binding Assay, Purification, Western Blot, Immunoprecipitation, Clinical Proteomics, Membrane, Cell Fractionation, Control

Journal: The Journal of Cell Biology

Article Title: AMIGO2, a novel membrane anchor of PDK1, controls cell survival and angiogenesis via Akt activation

doi: 10.1083/jcb.201503113

Figure Lengend Snippet: Treatment with PTD-A2 inhibits tumor growth and tumor angiogenesis. (A–E) Treatment with FITC–NGR-A2 inhibits tumor growth, neovessel formation, and tumor cell death in B16F10 tumors. (A) Photograph of B16F10 melanoma tumors resected after 13 d. Bar, 5 mm. (B) Comparison of tumor volume ( n = 7 mice per group; 4 mg/kg NGR-Con or NGR-A2 was administered every other day). (C–E) Images (C) and quantification (D) of CD31-positive blood vessels (red) and TUNEL-positive cells (pink) over CD31-positive blood vessels (E) in B16F10 melanoma tumors. FITC, green; DAPI, blue. Bars, 50 µm. (F–I) Amigo2 shRNA–transfected B16F10 tumors with NGR-A2 display dramatically inhibited tumor growth and tumor vessel formation. (F) Image of Amigo2 shRNA–transfected B16F10 melanoma tumors resected after 14 d. Bar, 5 mm. (G) Comparison of tumor volumes ( n = 7–8 mice per group; 4 mg/kg NGR-Con or NGR-A2 was administered every other day). (H and I) Images (H) and quantification (I) of CD31-positive blood vessels (red). (B and G) Data were analyzed using repeated measures of two-way analysis of variance. *, P < 0.05; **, P < 0.005; ***, P < 0.0001. ns, not significant. Data are means ± SD.

Article Snippet: Mouse and goat anti-AMIGO2 antibodies for immunoprecipitation and Western blotting were purchased from R&D Systems and Santa Cruz Biotechnology, Inc., respectively.

Techniques: Comparison, TUNEL Assay, shRNA, Transfection

Journal: The Journal of Cell Biology

Article Title: AMIGO2, a novel membrane anchor of PDK1, controls cell survival and angiogenesis via Akt activation

doi: 10.1083/jcb.201503113

Figure Lengend Snippet: The AMIGO2–PDK1–Akt signaling pathway. (A) This model proposes that the direct binding of AMIGO2 to the PDK1 PH domain enhanced the activation of PDK1 and Akt in the plasma membrane, which resulted in increased EC viability, adhesion, migration, and angiogenesis. (B) Loss of AMIGO2 attenuated the phosphorylation of PDK1 and Akt and EC survival and adhesion. (C) PTD-A2, a specific peptide in the C-terminal region of AMIGO2, effectively abrogated the phosphorylation of PDK1 and Akt, cell survival, OIR-induced angiogenesis, tumor angiogenesis, and tumor growth. P, phosphorylation.

Article Snippet: Mouse and goat anti-AMIGO2 antibodies for immunoprecipitation and Western blotting were purchased from R&D Systems and Santa Cruz Biotechnology, Inc., respectively.

Techniques: Binding Assay, Activation Assay, Clinical Proteomics, Membrane, Migration, Phospho-proteomics

Journal: International Journal of Medical Sciences

Article Title: AMIGO2 as a Novel Biomarker Predicting Poor Prognosis and Associated with Adhesion-Driven Metastasis in Pancreatic Adenocarcinoma

doi: 10.7150/ijms.121794

Figure Lengend Snippet: Transcriptional expression and pathological stage analysis of AMIGO family members in PAAD using GEPIA2 database. (A-B) Comparison of AMIGO1, AMIGO2 and AMIGO3 mRNA expression levels between PAAD and normal pancreatic tissues. (C) Expression levels of AMIGOs across different tumor stages in PAAD patients.

Article Snippet: Primary antibodies used in this study included: AMIGO2 (E-AB-10790, Elabscience), E-cadherin (R55-180, BD Biosciences), Vimentin (E-AB-22085, Elabscience), and α-Tubulin (T6199, Sigma-Aldrich).

Techniques: Expressing, Comparison

Journal: International Journal of Medical Sciences

Article Title: AMIGO2 as a Novel Biomarker Predicting Poor Prognosis and Associated with Adhesion-Driven Metastasis in Pancreatic Adenocarcinoma

doi: 10.7150/ijms.121794

Figure Lengend Snippet: Prognostic value of AMIGO family in pancreatic adenocarcinoma (PAAD) patients. (A) Relapse-free survival (RFS) curves and (B) Overall survival (OS) of AMIGO1, AMIGO2 and AMIGO3 in PAAD.

Article Snippet: Primary antibodies used in this study included: AMIGO2 (E-AB-10790, Elabscience), E-cadherin (R55-180, BD Biosciences), Vimentin (E-AB-22085, Elabscience), and α-Tubulin (T6199, Sigma-Aldrich).

Techniques:

Journal: International Journal of Medical Sciences

Article Title: AMIGO2 as a Novel Biomarker Predicting Poor Prognosis and Associated with Adhesion-Driven Metastasis in Pancreatic Adenocarcinoma

doi: 10.7150/ijms.121794

Figure Lengend Snippet: Genetic alterations, gene and protein interaction networks and co-expression analysis of AMIGO family members in pancreatic adenocarcinoma (PAAD). (A-B) Genetic alteration analysis of AMIGO1, AMIGO2 and AMIGO3 in PAAD patients using cBioPortal. (C) Gene-gene interaction (GGI) network of AMIGOs constructed by GeneMANIA. (D) Protein-protein interaction (PPI) network of AMIGOs generated using the STRING database. (E) Co-expression gene analysis of AMIGOs based on the intersection of top 1000 correlated genes, visualized using VENNY.

Article Snippet: Primary antibodies used in this study included: AMIGO2 (E-AB-10790, Elabscience), E-cadherin (R55-180, BD Biosciences), Vimentin (E-AB-22085, Elabscience), and α-Tubulin (T6199, Sigma-Aldrich).

Techniques: Expressing, Construct, Generated

Journal: International Journal of Medical Sciences

Article Title: AMIGO2 as a Novel Biomarker Predicting Poor Prognosis and Associated with Adhesion-Driven Metastasis in Pancreatic Adenocarcinoma

doi: 10.7150/ijms.121794

Figure Lengend Snippet: Functional enrichment analysis of AMIGO2 in pancreatic adenocarcinoma (PAAD) by using the DAVID platform. (A-C) Gene Ontology (GO) enrichment analysis of AMIGO2-related genes, categorized into biological processes (BP), cellular components (CC), and molecular functions (MF), respectively. (D) Top 20 GO terms enriched in AMIGO2 co-expressed genes. (E) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of AMIGO2-associated genes.

Article Snippet: Primary antibodies used in this study included: AMIGO2 (E-AB-10790, Elabscience), E-cadherin (R55-180, BD Biosciences), Vimentin (E-AB-22085, Elabscience), and α-Tubulin (T6199, Sigma-Aldrich).

Techniques: Functional Assay

Journal: International Journal of Medical Sciences

Article Title: AMIGO2 as a Novel Biomarker Predicting Poor Prognosis and Associated with Adhesion-Driven Metastasis in Pancreatic Adenocarcinoma

doi: 10.7150/ijms.121794

Figure Lengend Snippet: Correlation between AMIGO2 and EMT-related transcription factors in PAAD. (A) Protein expression of AMIGO2 in normal pancreatic tissues and PAAD tissues based on immunohistochemistry (IHC) images from the Human Protein Atlas (HPA). Representative IHC staining shows weak or absent AMIGO2 expression in normal pancreatic tissues (left) and stronger staining intensity in PAAD tissues (right). (B-G) Correlation analyses between AMIGO2 and key EMT-related transcription factors in TCGA-PAAD. Scatter plots show the Spearman correlation between AMIGO2 expression and ZEB1 (ρ = 0.27, p = 2.7 × 10⁻⁴), SNAI1 (ρ = 0.23, p = 1.8 × 10⁻³), SP1 (ρ = 0.40, p = 3.6 × 10⁻⁸), NFKB1 (ρ = 0.22, p = 3.8 × 10⁻³), RELA (ρ = 0.41, p = 1.8 × 10⁻⁸) and TWIST1 (ρ = 0.37, p = 4.1 × 10⁻⁷). (H) Correlation between AMIGO2 expression and immune cell infiltration across TCGA cancer types, including pancreatic adenocarcinoma (PAAD), pheochromocytoma and paraganglioma (PCPG), and prostate adenocarcinoma (PRAD), analyzed using TIMER2.0 with tumor purity adjustment. The heatmap displays partial Spearman correlation coefficients across multiple immune deconvolution algorithms. (I) Correlation between AMIGO2 and CD274 (PD-L1) expression in TCGA-PAAD, analyzed using GEPIA2 (Spearman correlation). Scatter plot shows a significant positive correlation (R = 0.34, p = 3.9 × 10⁻⁶), indicating that AMIGO2-high tumors exhibit elevated PD-L1 expression.

Article Snippet: Primary antibodies used in this study included: AMIGO2 (E-AB-10790, Elabscience), E-cadherin (R55-180, BD Biosciences), Vimentin (E-AB-22085, Elabscience), and α-Tubulin (T6199, Sigma-Aldrich).

Techniques: Expressing, Immunohistochemistry, Staining

Journal: International Journal of Medical Sciences

Article Title: AMIGO2 as a Novel Biomarker Predicting Poor Prognosis and Associated with Adhesion-Driven Metastasis in Pancreatic Adenocarcinoma

doi: 10.7150/ijms.121794

Figure Lengend Snippet: AMIGO2 promotes migration and invasion by inducing EMT in pancreatic cancer cells. (A) Western blot analysis of AMIGO2 protein levels in MiaPaCa-2 cells transfected with two independent AMIGO2 shRNAs (kd-AMIGO2 A1 and kd-AMIGO2 B2) compared with control cells. α-Tubulin served as a loading control. (B) Western blot analysis of EMT markers showing that AMIGO2 knockdown upregulated E-cadherin and downregulated Vimentin expression. α-Tubulin was used as an internal control. (C) Representative images of wound-healing assays performed in MiaPaCa-2 cells after AMIGO2 knockdown, captured at 0, 24, and 48 h. (D) Quantitative analysis of wound closure rates (%) in MiaPaCa-2 control and AMIGO2-silenced cells. (E) Representative images of Transwell invasion assays showing a reduced number of invading cells following AMIGO2 knockdown. (F) Statistical quantification of invaded cell area (%) based on Transwell assays. ***, p<0.01.

Article Snippet: Primary antibodies used in this study included: AMIGO2 (E-AB-10790, Elabscience), E-cadherin (R55-180, BD Biosciences), Vimentin (E-AB-22085, Elabscience), and α-Tubulin (T6199, Sigma-Aldrich).

Techniques: Migration, Western Blot, Transfection, Control, Knockdown, Expressing

Journal: NPJ Precision Oncology

Article Title: Metabolomic and transcriptomic profiling of HNSCC identifies AMIGO2 as a therapeutic target modulating tumor microenvironment

doi: 10.1038/s41698-025-01132-z

Figure Lengend Snippet: a Expression of AMIGO2, STC2, INHBA, DKK1, VEGFC, SPOCK1, MT1E, and FOXD1 in scRNA-seq UMAP plots. b Bar plot showing cell type proportions under LRS subtypes from scRNA-seq data. c AMIGO2, ranked highly for prognostic importance, was selected for further analysis. d AMIGO2 expression is upregulated in most tumor tissues across various cancers in TCGA pan-cancer dataset. e Evaluation of AMIGO2’s prognostic value (OS) in TCGA pan-cancer dataset. f Correlation analysis between AMIGO2 expression and representative signatures in TCGA pan-cancer dataset. Ns: no significant difference, * P < 0.05, ** P ≤ 0.01, *** P ≤ 0.001.

Article Snippet: The precleared lysates were then incubated overnight at 4°C with either anti-AMIGO2 antibody (Santa Cruz Biotechnology, Cat# sc-373699, RRID: AB_10920216) or anti-PDK1 antibody (Thermo Fisher Scientific Cat# A302-130A, RRID:AB_1720395).

Techniques: Expressing

Journal: NPJ Precision Oncology

Article Title: Metabolomic and transcriptomic profiling of HNSCC identifies AMIGO2 as a therapeutic target modulating tumor microenvironment

doi: 10.1038/s41698-025-01132-z

Figure Lengend Snippet: a IHC images and quantitative statistics of AMIGO2 expression in adjacent normal tissue, precancerous lesions, and tumor tissues. b Representative images and quantification of CD8, CD56, FAP, and PanCK staining in tumor tissues from high-AMIGO2 and low-AMIGO2 patient groups. c Graphical abstract for this study. Ns: no significant difference. * P < 0.05, ** P ≤ 0.01, *** P ≤ 0.001.

Article Snippet: The precleared lysates were then incubated overnight at 4°C with either anti-AMIGO2 antibody (Santa Cruz Biotechnology, Cat# sc-373699, RRID: AB_10920216) or anti-PDK1 antibody (Thermo Fisher Scientific Cat# A302-130A, RRID:AB_1720395).

Techniques: Expressing, Staining

Journal: Molecular cell

Article Title: Harnessing BET Inhibitor Sensitivity Reveals AMIGO2 as a Melanoma Survival Gene

doi: 10.1016/j.molcel.2017.11.004

Figure Lengend Snippet: (A) AMIGO2 expression levels detected by qRT-PCR in NHM and a panel of metastatic melanoma cell lines harboring distinct melanoma mutations. Data are normalized to GAPDH.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies BRD2 Bethyl Laboratories A302-583A BRD4 Bethyl Laboratories A301-985A H3K4me1 Abcam ab8895 H3K4me3 Abcam ab1012 H3K27ac Abcam ab177178 H3K27me3 EMD 07-449 MED1 Bethyl Laboratories A300-793A macroH2A2 Bernstein Laboratory, ISMMS in house macroH2A1 Abcam ab37264 CTCF EMD 07-729 FOSL2 Santa Cruz SC-604 TEAD4 Santa Cruz SC-101184 HSP90 Santa Cruz SC-13119 GAPDH Santa Cruz SC-32233 GFP Roche 11814460001 LAMIN B1 Santa Cruz SC-6217 ALCAM Abcam ab109215 L1CAM Abcam ab20148 NRP2 Santa Cruz SC-13117 NRP1 Abcam ab81321 TUBULIN Sigma T9026 FOXM1 Cell Signaling Technology 5436 Casp3 Cell Signaling Technology 9662 PARP Cell Signaling Technology 9542 AMIGO2 (for IHC) Abnova H00347902-B01P Lot# F8271 RRID:AB_1672842 AMIGO2 (for IF and WB) R&D systems AF2080 PTK7 (N-terminal) (for WB) R&D systems AF4499 PTK7 (C-terminal) (for IF and WB) Cell Signaling 11926 Secondary rabbit anti goat (for AMIGO2 detection on WB) Sigma A5420 HRP-conjugated ACTIN Sigma A3854 Biological Samples Melanoma and normal skin tissue microarray Biomax ME208 Nevus tissue microarray IMCG, NYU MT032808 Patient derived short term cultures Osman Laboratory, NYU de Miera et al., 2012 NHM (this paper) Bernstein Laboratory, ISMMS NHM1-5 Chemicals, Peptides, and Recombinant Proteins GFP Trap-A beads Chromotek gta-10 BET inhibitor JQ1 Bradner Laboratory; Agreement # {"type":"entrez-nucleotide","attrs":{"text":"A09608","term_id":"490581","term_text":"A09608"}} A09608 N/A BET inhibitor I-BET762 Apex Bio 501014747 IGEPAL Sigma I-8896 Micrococcal Nuclease Affymetrix 70196Y Dynabeads Protein A Immunoprécipitation kit Invitrogen 10006D Magna ChIP Protein A+G Magnetic beads Millipore 16-663 DSG Pierce (ThermoFisher) 20593 Critical Commercial Assays Click- Click-IT EdU Pacific Blue Flow Cytometry Assay Kit ThermoFisher {"type":"entrez-nucleotide","attrs":{"text":"C10636","term_id":"1535707","term_text":"C10636"}} C10636 Caspase Activity assay MBL Int.

Techniques: Expressing, Quantitative RT-PCR

Journal: Molecular cell

Article Title: Harnessing BET Inhibitor Sensitivity Reveals AMIGO2 as a Melanoma Survival Gene

doi: 10.1016/j.molcel.2017.11.004

Figure Lengend Snippet: (A) Relative growth curves of 501MEL and SKmel147 cells stably transduced with non-targeting scrambled control shRNA (shSCR) and two AMIGO2 shRNAs (shA2 #1 and #2). Values are normalized to seeding control (n = 3).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies BRD2 Bethyl Laboratories A302-583A BRD4 Bethyl Laboratories A301-985A H3K4me1 Abcam ab8895 H3K4me3 Abcam ab1012 H3K27ac Abcam ab177178 H3K27me3 EMD 07-449 MED1 Bethyl Laboratories A300-793A macroH2A2 Bernstein Laboratory, ISMMS in house macroH2A1 Abcam ab37264 CTCF EMD 07-729 FOSL2 Santa Cruz SC-604 TEAD4 Santa Cruz SC-101184 HSP90 Santa Cruz SC-13119 GAPDH Santa Cruz SC-32233 GFP Roche 11814460001 LAMIN B1 Santa Cruz SC-6217 ALCAM Abcam ab109215 L1CAM Abcam ab20148 NRP2 Santa Cruz SC-13117 NRP1 Abcam ab81321 TUBULIN Sigma T9026 FOXM1 Cell Signaling Technology 5436 Casp3 Cell Signaling Technology 9662 PARP Cell Signaling Technology 9542 AMIGO2 (for IHC) Abnova H00347902-B01P Lot# F8271 RRID:AB_1672842 AMIGO2 (for IF and WB) R&D systems AF2080 PTK7 (N-terminal) (for WB) R&D systems AF4499 PTK7 (C-terminal) (for IF and WB) Cell Signaling 11926 Secondary rabbit anti goat (for AMIGO2 detection on WB) Sigma A5420 HRP-conjugated ACTIN Sigma A3854 Biological Samples Melanoma and normal skin tissue microarray Biomax ME208 Nevus tissue microarray IMCG, NYU MT032808 Patient derived short term cultures Osman Laboratory, NYU de Miera et al., 2012 NHM (this paper) Bernstein Laboratory, ISMMS NHM1-5 Chemicals, Peptides, and Recombinant Proteins GFP Trap-A beads Chromotek gta-10 BET inhibitor JQ1 Bradner Laboratory; Agreement # {"type":"entrez-nucleotide","attrs":{"text":"A09608","term_id":"490581","term_text":"A09608"}} A09608 N/A BET inhibitor I-BET762 Apex Bio 501014747 IGEPAL Sigma I-8896 Micrococcal Nuclease Affymetrix 70196Y Dynabeads Protein A Immunoprécipitation kit Invitrogen 10006D Magna ChIP Protein A+G Magnetic beads Millipore 16-663 DSG Pierce (ThermoFisher) 20593 Critical Commercial Assays Click- Click-IT EdU Pacific Blue Flow Cytometry Assay Kit ThermoFisher {"type":"entrez-nucleotide","attrs":{"text":"C10636","term_id":"1535707","term_text":"C10636"}} C10636 Caspase Activity assay MBL Int.

Techniques: Stable Transfection, Transduction, shRNA

Journal: Molecular cell

Article Title: Harnessing BET Inhibitor Sensitivity Reveals AMIGO2 as a Melanoma Survival Gene

doi: 10.1016/j.molcel.2017.11.004

Figure Lengend Snippet: (A) Immunofluorescence of SKmel147 cells stably expressing AMIGO2-GFP (green), stained with AMIGO2 antibody (red) and Hoechst 33342 (blue). Scale bar, 20 μm.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies BRD2 Bethyl Laboratories A302-583A BRD4 Bethyl Laboratories A301-985A H3K4me1 Abcam ab8895 H3K4me3 Abcam ab1012 H3K27ac Abcam ab177178 H3K27me3 EMD 07-449 MED1 Bethyl Laboratories A300-793A macroH2A2 Bernstein Laboratory, ISMMS in house macroH2A1 Abcam ab37264 CTCF EMD 07-729 FOSL2 Santa Cruz SC-604 TEAD4 Santa Cruz SC-101184 HSP90 Santa Cruz SC-13119 GAPDH Santa Cruz SC-32233 GFP Roche 11814460001 LAMIN B1 Santa Cruz SC-6217 ALCAM Abcam ab109215 L1CAM Abcam ab20148 NRP2 Santa Cruz SC-13117 NRP1 Abcam ab81321 TUBULIN Sigma T9026 FOXM1 Cell Signaling Technology 5436 Casp3 Cell Signaling Technology 9662 PARP Cell Signaling Technology 9542 AMIGO2 (for IHC) Abnova H00347902-B01P Lot# F8271 RRID:AB_1672842 AMIGO2 (for IF and WB) R&D systems AF2080 PTK7 (N-terminal) (for WB) R&D systems AF4499 PTK7 (C-terminal) (for IF and WB) Cell Signaling 11926 Secondary rabbit anti goat (for AMIGO2 detection on WB) Sigma A5420 HRP-conjugated ACTIN Sigma A3854 Biological Samples Melanoma and normal skin tissue microarray Biomax ME208 Nevus tissue microarray IMCG, NYU MT032808 Patient derived short term cultures Osman Laboratory, NYU de Miera et al., 2012 NHM (this paper) Bernstein Laboratory, ISMMS NHM1-5 Chemicals, Peptides, and Recombinant Proteins GFP Trap-A beads Chromotek gta-10 BET inhibitor JQ1 Bradner Laboratory; Agreement # {"type":"entrez-nucleotide","attrs":{"text":"A09608","term_id":"490581","term_text":"A09608"}} A09608 N/A BET inhibitor I-BET762 Apex Bio 501014747 IGEPAL Sigma I-8896 Micrococcal Nuclease Affymetrix 70196Y Dynabeads Protein A Immunoprécipitation kit Invitrogen 10006D Magna ChIP Protein A+G Magnetic beads Millipore 16-663 DSG Pierce (ThermoFisher) 20593 Critical Commercial Assays Click- Click-IT EdU Pacific Blue Flow Cytometry Assay Kit ThermoFisher {"type":"entrez-nucleotide","attrs":{"text":"C10636","term_id":"1535707","term_text":"C10636"}} C10636 Caspase Activity assay MBL Int.

Techniques: Immunofluorescence, Stable Transfection, Expressing, Staining

Journal: Molecular cell

Article Title: Harnessing BET Inhibitor Sensitivity Reveals AMIGO2 as a Melanoma Survival Gene

doi: 10.1016/j.molcel.2017.11.004

Figure Lengend Snippet: (A) Capture of the UCSC genome browser (GRCh37/hg19) showing 800 kb region on human chromosome 12 (y axis = reads per kilobase per million reads). Ensembl genes (blue) and AMIGO2 SEs (red) are shown at the top. Putative TAD/LAD domain is shown at the bottom.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies BRD2 Bethyl Laboratories A302-583A BRD4 Bethyl Laboratories A301-985A H3K4me1 Abcam ab8895 H3K4me3 Abcam ab1012 H3K27ac Abcam ab177178 H3K27me3 EMD 07-449 MED1 Bethyl Laboratories A300-793A macroH2A2 Bernstein Laboratory, ISMMS in house macroH2A1 Abcam ab37264 CTCF EMD 07-729 FOSL2 Santa Cruz SC-604 TEAD4 Santa Cruz SC-101184 HSP90 Santa Cruz SC-13119 GAPDH Santa Cruz SC-32233 GFP Roche 11814460001 LAMIN B1 Santa Cruz SC-6217 ALCAM Abcam ab109215 L1CAM Abcam ab20148 NRP2 Santa Cruz SC-13117 NRP1 Abcam ab81321 TUBULIN Sigma T9026 FOXM1 Cell Signaling Technology 5436 Casp3 Cell Signaling Technology 9662 PARP Cell Signaling Technology 9542 AMIGO2 (for IHC) Abnova H00347902-B01P Lot# F8271 RRID:AB_1672842 AMIGO2 (for IF and WB) R&D systems AF2080 PTK7 (N-terminal) (for WB) R&D systems AF4499 PTK7 (C-terminal) (for IF and WB) Cell Signaling 11926 Secondary rabbit anti goat (for AMIGO2 detection on WB) Sigma A5420 HRP-conjugated ACTIN Sigma A3854 Biological Samples Melanoma and normal skin tissue microarray Biomax ME208 Nevus tissue microarray IMCG, NYU MT032808 Patient derived short term cultures Osman Laboratory, NYU de Miera et al., 2012 NHM (this paper) Bernstein Laboratory, ISMMS NHM1-5 Chemicals, Peptides, and Recombinant Proteins GFP Trap-A beads Chromotek gta-10 BET inhibitor JQ1 Bradner Laboratory; Agreement # {"type":"entrez-nucleotide","attrs":{"text":"A09608","term_id":"490581","term_text":"A09608"}} A09608 N/A BET inhibitor I-BET762 Apex Bio 501014747 IGEPAL Sigma I-8896 Micrococcal Nuclease Affymetrix 70196Y Dynabeads Protein A Immunoprécipitation kit Invitrogen 10006D Magna ChIP Protein A+G Magnetic beads Millipore 16-663 DSG Pierce (ThermoFisher) 20593 Critical Commercial Assays Click- Click-IT EdU Pacific Blue Flow Cytometry Assay Kit ThermoFisher {"type":"entrez-nucleotide","attrs":{"text":"C10636","term_id":"1535707","term_text":"C10636"}} C10636 Caspase Activity assay MBL Int.

Techniques:

Journal: Molecular cell

Article Title: Harnessing BET Inhibitor Sensitivity Reveals AMIGO2 as a Melanoma Survival Gene

doi: 10.1016/j.molcel.2017.11.004

Figure Lengend Snippet: (A) Schematic of AMIGO2 SE showing enhancers E1–E5 (B), deleted sequences for CRISPR-Cas9 editing (C), and DNA segments used for E3 lucif-erase assay (D).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies BRD2 Bethyl Laboratories A302-583A BRD4 Bethyl Laboratories A301-985A H3K4me1 Abcam ab8895 H3K4me3 Abcam ab1012 H3K27ac Abcam ab177178 H3K27me3 EMD 07-449 MED1 Bethyl Laboratories A300-793A macroH2A2 Bernstein Laboratory, ISMMS in house macroH2A1 Abcam ab37264 CTCF EMD 07-729 FOSL2 Santa Cruz SC-604 TEAD4 Santa Cruz SC-101184 HSP90 Santa Cruz SC-13119 GAPDH Santa Cruz SC-32233 GFP Roche 11814460001 LAMIN B1 Santa Cruz SC-6217 ALCAM Abcam ab109215 L1CAM Abcam ab20148 NRP2 Santa Cruz SC-13117 NRP1 Abcam ab81321 TUBULIN Sigma T9026 FOXM1 Cell Signaling Technology 5436 Casp3 Cell Signaling Technology 9662 PARP Cell Signaling Technology 9542 AMIGO2 (for IHC) Abnova H00347902-B01P Lot# F8271 RRID:AB_1672842 AMIGO2 (for IF and WB) R&D systems AF2080 PTK7 (N-terminal) (for WB) R&D systems AF4499 PTK7 (C-terminal) (for IF and WB) Cell Signaling 11926 Secondary rabbit anti goat (for AMIGO2 detection on WB) Sigma A5420 HRP-conjugated ACTIN Sigma A3854 Biological Samples Melanoma and normal skin tissue microarray Biomax ME208 Nevus tissue microarray IMCG, NYU MT032808 Patient derived short term cultures Osman Laboratory, NYU de Miera et al., 2012 NHM (this paper) Bernstein Laboratory, ISMMS NHM1-5 Chemicals, Peptides, and Recombinant Proteins GFP Trap-A beads Chromotek gta-10 BET inhibitor JQ1 Bradner Laboratory; Agreement # {"type":"entrez-nucleotide","attrs":{"text":"A09608","term_id":"490581","term_text":"A09608"}} A09608 N/A BET inhibitor I-BET762 Apex Bio 501014747 IGEPAL Sigma I-8896 Micrococcal Nuclease Affymetrix 70196Y Dynabeads Protein A Immunoprécipitation kit Invitrogen 10006D Magna ChIP Protein A+G Magnetic beads Millipore 16-663 DSG Pierce (ThermoFisher) 20593 Critical Commercial Assays Click- Click-IT EdU Pacific Blue Flow Cytometry Assay Kit ThermoFisher {"type":"entrez-nucleotide","attrs":{"text":"C10636","term_id":"1535707","term_text":"C10636"}} C10636 Caspase Activity assay MBL Int.

Techniques: CRISPR

Journal: Molecular cell

Article Title: Harnessing BET Inhibitor Sensitivity Reveals AMIGO2 as a Melanoma Survival Gene

doi: 10.1016/j.molcel.2017.11.004

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies BRD2 Bethyl Laboratories A302-583A BRD4 Bethyl Laboratories A301-985A H3K4me1 Abcam ab8895 H3K4me3 Abcam ab1012 H3K27ac Abcam ab177178 H3K27me3 EMD 07-449 MED1 Bethyl Laboratories A300-793A macroH2A2 Bernstein Laboratory, ISMMS in house macroH2A1 Abcam ab37264 CTCF EMD 07-729 FOSL2 Santa Cruz SC-604 TEAD4 Santa Cruz SC-101184 HSP90 Santa Cruz SC-13119 GAPDH Santa Cruz SC-32233 GFP Roche 11814460001 LAMIN B1 Santa Cruz SC-6217 ALCAM Abcam ab109215 L1CAM Abcam ab20148 NRP2 Santa Cruz SC-13117 NRP1 Abcam ab81321 TUBULIN Sigma T9026 FOXM1 Cell Signaling Technology 5436 Casp3 Cell Signaling Technology 9662 PARP Cell Signaling Technology 9542 AMIGO2 (for IHC) Abnova H00347902-B01P Lot# F8271 RRID:AB_1672842 AMIGO2 (for IF and WB) R&D systems AF2080 PTK7 (N-terminal) (for WB) R&D systems AF4499 PTK7 (C-terminal) (for IF and WB) Cell Signaling 11926 Secondary rabbit anti goat (for AMIGO2 detection on WB) Sigma A5420 HRP-conjugated ACTIN Sigma A3854 Biological Samples Melanoma and normal skin tissue microarray Biomax ME208 Nevus tissue microarray IMCG, NYU MT032808 Patient derived short term cultures Osman Laboratory, NYU de Miera et al., 2012 NHM (this paper) Bernstein Laboratory, ISMMS NHM1-5 Chemicals, Peptides, and Recombinant Proteins GFP Trap-A beads Chromotek gta-10 BET inhibitor JQ1 Bradner Laboratory; Agreement # {"type":"entrez-nucleotide","attrs":{"text":"A09608","term_id":"490581","term_text":"A09608"}} A09608 N/A BET inhibitor I-BET762 Apex Bio 501014747 IGEPAL Sigma I-8896 Micrococcal Nuclease Affymetrix 70196Y Dynabeads Protein A Immunoprécipitation kit Invitrogen 10006D Magna ChIP Protein A+G Magnetic beads Millipore 16-663 DSG Pierce (ThermoFisher) 20593 Critical Commercial Assays Click- Click-IT EdU Pacific Blue Flow Cytometry Assay Kit ThermoFisher {"type":"entrez-nucleotide","attrs":{"text":"C10636","term_id":"1535707","term_text":"C10636"}} C10636 Caspase Activity assay MBL Int.

Techniques: Microarray, Derivative Assay, Recombinant, Magnetic Beads, Flow Cytometry, Caspase Activity Assay, Blocking Assay, TA Cloning, Sequencing, RNA Sequencing Assay, Western Blot, Mass Spectrometry, Expressing, Software

Journal: NPJ Precision Oncology

Article Title: Metabolomic and transcriptomic profiling of HNSCC identifies AMIGO2 as a therapeutic target modulating tumor microenvironment

doi: 10.1038/s41698-025-01132-z

Figure Lengend Snippet: a Expression of AMIGO2, STC2, INHBA, DKK1, VEGFC, SPOCK1, MT1E, and FOXD1 in scRNA-seq UMAP plots. b Bar plot showing cell type proportions under LRS subtypes from scRNA-seq data. c AMIGO2, ranked highly for prognostic importance, was selected for further analysis. d AMIGO2 expression is upregulated in most tumor tissues across various cancers in TCGA pan-cancer dataset. e Evaluation of AMIGO2’s prognostic value (OS) in TCGA pan-cancer dataset. f Correlation analysis between AMIGO2 expression and representative signatures in TCGA pan-cancer dataset. Ns: no significant difference, * P < 0.05, ** P ≤ 0.01, *** P ≤ 0.001.

Article Snippet: The following primary antibodies were used for Western blotting: AMIGO2 (1:500, HUAAN, catalog No: ER1903-66), GAPDH (1:1000, CST, catalog No: 5174S, RRID: AB_10622025), E-cadherin (1:1000, CST, catalog No: 3195S, RRID: AB_2291471), N-cadherin (1:1000, CST, catalog No: 13116S, RRID: AB_2687616), HPRT1 (1:1000, Proteintech Cat# 15059-1-AP, RRID: AB_10638622), PAICS (1:1000, Proteintech Cat# 12967-1-AP, RRID: AB_10638449), Vimentin (1:1000, CST, catalog No: 5741S, RRID: AB_10695459), MMP9 (1:1000, CST, catalog No: 13667S, RRID: AB_2798289), FAP (Thermo Fisher Scientific Cat# PA5-99313, RRID: AB_2818246), α-SMA (Cell Signaling Technology Cat# 19245, RRID: AB_2734735), PI3K (Cell Signaling Technology Cat# 4292, RRID:AB_329869), Phospho-PI3K (Cell Signaling Technology Cat# 17366, RRID:AB_2895293), AKT (Cell Signaling Technology Cat# 9272, RRID: AB_329827), Phospho-Akt (Thr308) (Cell Signaling Technology Cat# 9275, RRID: AB_329828), Phospho-Akt (Ser473) (Cell Signaling Technology Cat# 9271, RRID: AB_329825), PDK1 (Thermo Fisher Scientific Cat# A302-130A, RRID:AB_1720395), Phospho-PDK1 (Ser241) (Cell Signaling Technology Cat# 3061, RRID: AB_2161919), mTOR (Abcam Cat# ab134903, RRID: AB_2800465) and mTOR (phospho S2481) (Abcam Cat# ab137133, RRID:AB_2800466).

Techniques: Expressing

Journal: NPJ Precision Oncology

Article Title: Metabolomic and transcriptomic profiling of HNSCC identifies AMIGO2 as a therapeutic target modulating tumor microenvironment

doi: 10.1038/s41698-025-01132-z

Figure Lengend Snippet: a IHC images and quantitative statistics of AMIGO2 expression in adjacent normal tissue, precancerous lesions, and tumor tissues. b Representative images and quantification of CD8, CD56, FAP, and PanCK staining in tumor tissues from high-AMIGO2 and low-AMIGO2 patient groups. c Graphical abstract for this study. Ns: no significant difference. * P < 0.05, ** P ≤ 0.01, *** P ≤ 0.001.

Article Snippet: The following primary antibodies were used for Western blotting: AMIGO2 (1:500, HUAAN, catalog No: ER1903-66), GAPDH (1:1000, CST, catalog No: 5174S, RRID: AB_10622025), E-cadherin (1:1000, CST, catalog No: 3195S, RRID: AB_2291471), N-cadherin (1:1000, CST, catalog No: 13116S, RRID: AB_2687616), HPRT1 (1:1000, Proteintech Cat# 15059-1-AP, RRID: AB_10638622), PAICS (1:1000, Proteintech Cat# 12967-1-AP, RRID: AB_10638449), Vimentin (1:1000, CST, catalog No: 5741S, RRID: AB_10695459), MMP9 (1:1000, CST, catalog No: 13667S, RRID: AB_2798289), FAP (Thermo Fisher Scientific Cat# PA5-99313, RRID: AB_2818246), α-SMA (Cell Signaling Technology Cat# 19245, RRID: AB_2734735), PI3K (Cell Signaling Technology Cat# 4292, RRID:AB_329869), Phospho-PI3K (Cell Signaling Technology Cat# 17366, RRID:AB_2895293), AKT (Cell Signaling Technology Cat# 9272, RRID: AB_329827), Phospho-Akt (Thr308) (Cell Signaling Technology Cat# 9275, RRID: AB_329828), Phospho-Akt (Ser473) (Cell Signaling Technology Cat# 9271, RRID: AB_329825), PDK1 (Thermo Fisher Scientific Cat# A302-130A, RRID:AB_1720395), Phospho-PDK1 (Ser241) (Cell Signaling Technology Cat# 3061, RRID: AB_2161919), mTOR (Abcam Cat# ab134903, RRID: AB_2800465) and mTOR (phospho S2481) (Abcam Cat# ab137133, RRID:AB_2800466).

Techniques: Expressing, Staining

Journal: bioRxiv

Article Title: The AMIGO1 adhesion protein activates Kv2.1 voltage sensors

doi: 10.1101/2021.06.20.448455

Figure Lengend Snippet: ( A ) Experimental set up: Whole-cell K + currents (arrow) from Kv2.1–CHO transfected with GFP (red), rAMIGO2–YFP (dark blue), or rAMIGO3–YFP (light blue). Same voltage protocols and representation as . ( B , C, D ) Representative Kv2.1–control (5.1 pF), Kv2.1 + AMIGO2 (6.6 pF) or Kv2.1 + AMIGO3 (2.4 pF) cells. ( E , F, G ) Normalized G–V relationships. 5 of the Kv2.1–control cells were recorded from side by side with the Kv2.1 + AMIGO2 cells and Kv2.1 + AMIGO3 cells (light red). There was no statistical difference between these 5 cells and the data previously acquired during Kv2.1 + AMIGO1 recordings for (assessed by t-test), and data was pooled. Solid lines a 4 th order Boltzmann fits . ( H ) Reconstructed 4 th order Boltzmann fits from average V i,Mid and z i . Shading V i,Mid ± SEM. ( I ) Steepness and ( J ) midpoint of fits. Statistics in . ***: p = ≤0.001, **: p = ≤0.01, *: p = ≤0.05. Bars are mean ± SEM.

Article Snippet: Cells were incubated in the transfection cocktail and 2 mL of unsupplemented media for 6-8 hours before being returned to regular growth media, and used for experiments 40-48 hours after transfection. pEGFP, mAMIGO1–pEYFP–N1, and pCAG–ChroME-mRuby2-ST ( ) plasmids were gifts from James Trimmer. mAMIGO1–pEYFP–N1 uses a VPRARDPPVAT linker to tag the internal C–terminus of wild–type mouse AMIGO1 (NM_001004293.2 or NM_146137.3) with eYFP. pCAG– ChroME–mRuby2–ST encodes an mRuby2–tagged channelrhodopsin with a Kv2.1 PRC trafficking sequence ( , ). mKv2.1 (NM_008420) was purchased from OriGene (MG210968). hSCN1β–pIRES2–GFP was a gift from Vladimir Yarov-Yarovoy. mAMIGO1 was subcloned into pIRES2–GFP between NheI and BamHI restriction sites. rAMIGO2–pEYFP–N1 and rAMIGO3–pEYFP–N1 were generated by subcloning rat AMIGO2 (NM_182816.2) or rat AMIGO3 (NM_178144.1) in place of mAMIGO1 in the mAMIGO1–pEYFP–N1 vector.

Techniques: Transfection

Journal: bioRxiv

Article Title: The AMIGO1 adhesion protein activates Kv2.1 voltage sensors

doi: 10.1101/2021.06.20.448455

Figure Lengend Snippet: ( A ) Coefficient of variation of fluorescence from AMIGO2–YFP (dark blue circles), AMIGO3–YFP (light blue circles), or GxTX–594 (red circles). COV from confocal images of glass-adhered membranes (exemplar images in B-G ). AMIGO2-YFP fluorescence from cells ( B ) not induced for Kv2.1 expression (COVΔ2,oh = 0.2090 ± 0.0062, n = 144), ( C ) induced 48 h for Kv2.1 expression (COV A2,48h = 0.342 ± 0.022, n = 65). ( D ) GxTX–594 labeling of the cells in C (COV A2,48h(GxTX–594) = 0.631 ± 0.013, n = 65 cells). AMIGO3–YFP fluorescence from cells ( E ) not induced for Kv2.1 expression (COV A3,0h = 0.2186 ± 0.0052, n = 160), ( F ) induced 48 h for Kv2.1 expression (COV A3,48h = 0.503 ± 0.014, n = 109). ( G ) GxTX–594 labeling of the cells in panel F (COV A3,48h(GxTX–594) = 0.650 ± 0.013, n = 109 cells). ( H ) Costes thresholded, Pearson’s colocalization coefficients from cells induced for Kv2.1 expression 48 h prior to imaging.. From left to right: PCC A2,GxTX–594 = 0.342 ± 0.022, ≥ 0 (p < 0.0001, one–tailed, t-test), n = 65; PCC 594 = 0.597 ± 0.020, ≥ 0 (p < 0.0001, one–tailed, t-test), n = 108. ( I, J, K) Exemplar images where merge overlay (white) shows colocalization between GxTX–594 (red) and AMIGO2-YFP (cyan) or (L, M, N ) AMIGO2-YFP (cyan) Arithmetic means and standard errors are plotted. ( Statistics ) Outliers were removed using ROUT, Q = 1%. An ordinary one-way ANOVA with multiple comparisons was used to evaluate the differences between groups in COV analysis, while a t-test was used to evaluate the PCC data. ****: p = ≤0.0001. Bars are mean ± SEM. All scale bars are 10 μm.

Article Snippet: Cells were incubated in the transfection cocktail and 2 mL of unsupplemented media for 6-8 hours before being returned to regular growth media, and used for experiments 40-48 hours after transfection. pEGFP, mAMIGO1–pEYFP–N1, and pCAG–ChroME-mRuby2-ST ( ) plasmids were gifts from James Trimmer. mAMIGO1–pEYFP–N1 uses a VPRARDPPVAT linker to tag the internal C–terminus of wild–type mouse AMIGO1 (NM_001004293.2 or NM_146137.3) with eYFP. pCAG– ChroME–mRuby2–ST encodes an mRuby2–tagged channelrhodopsin with a Kv2.1 PRC trafficking sequence ( , ). mKv2.1 (NM_008420) was purchased from OriGene (MG210968). hSCN1β–pIRES2–GFP was a gift from Vladimir Yarov-Yarovoy. mAMIGO1 was subcloned into pIRES2–GFP between NheI and BamHI restriction sites. rAMIGO2–pEYFP–N1 and rAMIGO3–pEYFP–N1 were generated by subcloning rat AMIGO2 (NM_182816.2) or rat AMIGO3 (NM_178144.1) in place of mAMIGO1 in the mAMIGO1–pEYFP–N1 vector.

Techniques: Fluorescence, Expressing, Labeling, Imaging, One-tailed Test