rabbit anti en2 antibody  (Novus Biologicals)

Journal: Cell Death & Disease

Article Title: A novel super-enhancer-driven lncRNA LINC00973 governs head and neck squamous cell carcinoma progression through EN2

doi: 10.1038/s41419-025-08380-8

Figure Lengend Snippet: Heatmap ( a ) and volcano plot ( b ) displayed the expression profile of DEGs following LINC00973 knockdown or control cells. The mRNA ( c ) and protein ( d ) expression of EN2 were measured in Cal27 and HN6 cells with/without EN2 knockdown. e , f The correlations between EN2 and LINC00973 RNA expression were assessed in the public HNSCC datasets ( GSE41613 and GSE42743 ) and our in-house samples. Spearman’s correlation. The mRNA ( g ) and protein ( h ) abundance of EN2 were measured in Cal27 and HN6 cells under four conditions. Cell proliferation, migration and invasion were significantly reduced following LINC00973 knockdown but restored by ectopic EN2 overexpression as gauged by CCK-8 ( i ), wound healing ( j ) and Transwell invasion assays ( k ). Scale bar: 50 μm. l The migration/invasion-related markers were determined by western blot in Cal27 and HN6 cells with/without LINC00973 knockdown coupled with/without EN2 overexpression. m 1 × 10 5 HN6 cells with LINC00973 or/and EN2 manipulations were injected submucosally into the floor of the mouth. Fourteen days post-injection, tumor masses and cervical lymph nodes were harvested. Representative tumor masses images and estimated tumor volumes were showed. Scale bar: 1 cm. Representative IHC staining of Ki67 and EN2 in xenograft tumors ( n ) and their quantification data were shown ( o ). Scale bar: 50 μm. p Representative IHC staining of CK5/6 in lymph nodes (left panel) from orthotopic tumor-bearing mice among four indicated groups were displayed. The percentage of lymph nodes metastasis were shown and compared (right panel). Fisher’s exact test. Data were presented as mean ± SD, ** P < 0.01, Student’s t test.

Article Snippet: Next, membranes were blocked with 5% non-fat dry milk and incubated overnight at 4 °C with the indicated primary antibodies: EN2 (1:500, CSB-PA007660LA01HU, Cusabio, China), N-cadherin (1:2000, #22018-1-AP, Proteintech, China), E-cadherin (1:20000, #20874-1-AP, Proteintech, China), Vimentin (1:20000, #10366-1-AP, Proteintech, China), GAPDH (1:50000, #60004-1-Ig, Proteintech, China), FOSL1 (1:1000, #5281, Cell signaling, USA), NOTCH1 (1:1000, #20687-1-AP, Proteintech, China), BRD4 (1:1000, #13440, Cell signaling, USA), HEY1 (1:1000, #19929-1-AP, Proteintech, China), N1ICD (1:1000, #4147, Cell signaling, USA) or Rpb1 (1:1000, #14958, Cell signaling, USA).

Techniques: Expressing, Knockdown, Control, RNA Expression, Migration, Over Expression, CCK-8 Assay, Western Blot, Injection, Immunohistochemistry

Journal: Cell Death & Disease

Article Title: A novel super-enhancer-driven lncRNA LINC00973 governs head and neck squamous cell carcinoma progression through EN2

doi: 10.1038/s41419-025-08380-8

Figure Lengend Snippet: a , b The half-life of EN2 mRNA were determined by qRT-PCR in LINC00973 knockdown or overexpressed Cal27 and HN6 cells with time-course Actinomycin D exposure. c Experimental design of AGO2-RIP was depicted by schematic diagram (left panel). Endogenous LINC00973 was assessed by qRT-PCR in cell lyses immunoprecipitated with either AGO2 antibody or mouse IgG (right panel). d , e The putative miRNA candidates with high EN2 mRNA binding affinity were computed by TargetScan webtool ( https://www.targetscan.org/vert_80/ ), while LINC00973 sponged miRNAs (top10) were predicted by DIANA tool kits ( http://diana.imis.athena-innovation.gr/DianaTools/index.php ). Six overlapped miRNA candidates were identified ( d ). Only miR-6756-3p expression were shown with significant upregulation followed by LINC00973 silencing in both Cal27 and HN6 cells ( e ). f The expression of miR-6756-3p in 50 HNSCC clinical samples and paired normal counterparts were determined by qRT-PCR. Paired Wilcoxon rank-sum test. g Kaplan-Meier analyses of OS and DSS stratified by median miR-6756-3p expression in 50 in-house samples Log-rank test. h , i Schematic diagram depicted the experimental design of MS2-RIP assays to detect the direct binding between LINC00973 and miR-6756-3p in HNSCC ( h ). The LINC00973-MS2-GFP complex was pulled down by anti-GFP antibody and the interacted miR-6756-3p were assessed by qRT-PCR in Cal27, HN6, and HEK293T cells ( i ). j The sequence alignment displayed the binding sequence between miR-6756-3p and LINC00973 [LINC00973 (WT)] or 3’UTR of EN2 [3’UTR EN2 (WT)]. The miR-6756-3p binding site mutated LINC00973 and EN2 transcripts [LINC00973 (MT) or 3’UTR EN2 (MT)] were showed below. k , l Cal27 and HN6 cells transfected with LINC00973/3’UTR EN2 wild type or miR-6756-3p binding site mutated pmirGLO luciferase reporter vectors and treated with miR-6756-3p or control mimics. The luciferase activated was measured. m The expression levels of EN2 were measured by qRT-PCR in Cal27 and HN6 treated with sh-LINC00973-1 lentivirus or in combination with miR-6756-3p inhibitor (inhibitor). n The correlations between EN2, LINC00973, and miR-6756-3p expression in HNSCC clinical samples were estimated by using Spearman’s correlation. o Schematic illustration of the regulatory model of LINC00973 decoys miR-6756-3p to stabilize EN2 mRNA. Data were presented as mean ± SD, # P ≥ 0.05, * P < 0.05, ** P < 0.01, Student’s t test.

Article Snippet: Next, membranes were blocked with 5% non-fat dry milk and incubated overnight at 4 °C with the indicated primary antibodies: EN2 (1:500, CSB-PA007660LA01HU, Cusabio, China), N-cadherin (1:2000, #22018-1-AP, Proteintech, China), E-cadherin (1:20000, #20874-1-AP, Proteintech, China), Vimentin (1:20000, #10366-1-AP, Proteintech, China), GAPDH (1:50000, #60004-1-Ig, Proteintech, China), FOSL1 (1:1000, #5281, Cell signaling, USA), NOTCH1 (1:1000, #20687-1-AP, Proteintech, China), BRD4 (1:1000, #13440, Cell signaling, USA), HEY1 (1:1000, #19929-1-AP, Proteintech, China), N1ICD (1:1000, #4147, Cell signaling, USA) or Rpb1 (1:1000, #14958, Cell signaling, USA).

Techniques: Quantitative RT-PCR, Knockdown, Immunoprecipitation, Binding Assay, Expressing, Sequencing, Transfection, Luciferase, Control

Journal: Cell Death & Disease

Article Title: A novel super-enhancer-driven lncRNA LINC00973 governs head and neck squamous cell carcinoma progression through EN2

doi: 10.1038/s41419-025-08380-8

Figure Lengend Snippet: a SEs were calculated and ranked by H3K27ac signal using ROSE algorithm. And a proximal SE within LINC00973 gene locus were identified in 3 HNSCC cell lines (Cal27, HN12, and HSC3). b Genomic tracks plot displayed the H3K27ac enrichment (including 6 cell lines, 2 primary HNSCC and 1 human normal oral mucosa (NOM) tissues), BRD4, and EP300 binding and chromatin accessibility at the LINC00973-SE region. Four individual enhancer fragments within LINC00973-SE were observed and referred as E1-E4. c The H3K27ac, BRD4 and EP300 enrichment on E1-E4 or E-NC were measured by ChIP-qPCR in Cal27 and HN6 cells. d , e The expression changes of LINC00973 were measured by qRT-PCR and the binding changes of H3K27ac, BRD4, and EP300 on E1-E4 were determined by ChIP-qPCR in Cal27 and HN6 cells treated with JQ1 (1 μM, 12 h) or NEO2734 (1 μM, 12 h). f Representative FISH staining of LINC00973 and IHC staining of EN2 in PDX samples treated with vehicle, JQ1 or NEO2734. g Schematic diagram showing the procedure of E1-E4 repression by dCas9-KRAB-MeCP2 system. h LINC00973 expression in Cal27-dCas9 and HN6-dCas9 cells with repressed LINC00973 enhancer activity were assessed by qRT-PCR. Cell proliferation, migration and invasion were significantly suppressed following sgE2 treatment as gauged by CCK-8 ( i ), wound healing ( j ) and Transwell invasion assays ( k ). Scale bar: 50 μm. l De novo motif discovery at LINC00973-SE regions via HOMER algorithm. m LINC00973 expression was detected by qRT-PCR in Cal27 and HN6 cell with/without FOSL1 silencing. n , o co-IP assays revealed the endogenous FOSL1 protein interacted with Rpb1 (RNA pol II subunit) and BRD4 in both Cal27 and HN6 cells ( i ), while FOSL1-depletion disrupted these interactions ( j ). p The FOSL1 binding enrichments at E2 region in SCC1 with/without FOSL1 knockdown were displayed by track plot. q FOSL1 binding on E1-E4 or E-NC regions in Cal27 and HN6 transfected with si-FOSL1 were measured by ChIP-qPCR. r The core DNA sequences of E2 or E-NC were subcloned into pGL3 vectors and the luciferase activities were detected in Cal27 and HN6 cells transfected with si-FOSL1 or control siRNAs. s Schematic model of LINC00973 transcription regulation: cis -activation by LINC00973-SE and trans -regulation by AP-1/FOSL1. Data were presented as mean ± SD, # P ≥ 0.05,* P < 0.05, ** P < 0.01, Student’s t test.

Article Snippet: Next, membranes were blocked with 5% non-fat dry milk and incubated overnight at 4 °C with the indicated primary antibodies: EN2 (1:500, CSB-PA007660LA01HU, Cusabio, China), N-cadherin (1:2000, #22018-1-AP, Proteintech, China), E-cadherin (1:20000, #20874-1-AP, Proteintech, China), Vimentin (1:20000, #10366-1-AP, Proteintech, China), GAPDH (1:50000, #60004-1-Ig, Proteintech, China), FOSL1 (1:1000, #5281, Cell signaling, USA), NOTCH1 (1:1000, #20687-1-AP, Proteintech, China), BRD4 (1:1000, #13440, Cell signaling, USA), HEY1 (1:1000, #19929-1-AP, Proteintech, China), N1ICD (1:1000, #4147, Cell signaling, USA) or Rpb1 (1:1000, #14958, Cell signaling, USA).

Techniques: Binding Assay, ChIP-qPCR, Expressing, Quantitative RT-PCR, Staining, Immunohistochemistry, Activity Assay, Migration, CCK-8 Assay, Co-Immunoprecipitation Assay, Knockdown, Transfection, Luciferase, Control, Activation Assay

Journal: Cell Death & Disease

Article Title: A novel super-enhancer-driven lncRNA LINC00973 governs head and neck squamous cell carcinoma progression through EN2

doi: 10.1038/s41419-025-08380-8

Figure Lengend Snippet: a Schematic diagram outlining the experimental design to identify and characterize downstream signaling pathways modulated by EN2 in HNSCC. b Differential analyses of KEGG pathways were performed in Cal27 and HN6 cells with/without EN2 silencing and the results were represented as scatter plot (left panel) and heatmap (right panel). KEGG pathway scores were calculated by GSVA method. c Correlation analyses between LINC00973 expression and KEGG_NOTCH_SIGNALING_PATHWAY scores (GSVA method) in TCGA-HNSC, GSE186775 and GSE65858 datasets. d The transcription activity of NOTCH1, the core member of NOTCH pathway, was measured by NOTCH1 luciferase reporter assays in Cal27 and HN6 with EN2 manipulations. e The protein abundance of NOTCH1, N1ICD and HEY1 were determined by western blot in Cal27 and HN6 cells with EN2 knockdown. f The endogenous NOTCH1, N1ICD and HEY1 protein expression was detected in Cal27 and HN6 cells followed by LINC00973 siRNAs or HN30 cells with LINC00973 overexpression, respectively. g NOTCH1, N1ICD and HEY1 were determined by western blot in Cal27 and HN6 cells with/without LINC00973 knockdown coupled with/withoutEN2 overexpression. Cell proliferation, migration and invasion were significantly increased following LINC00973 overexpression but reduced by DAPT treatment as gauged by CCK-8 ( h ), wound healing ( i ) and Transwell invasion assays ( j ).Scale bar: 50 μm. k Representative H&E, IHC staining of FOSL1, EN2, and NOTCH1 as well as FISH staining of LINC00973, in serial sections of the same HNSCC clinical samples were shown. Scale bar: 50 μm. l The correlation among FOSL1, EN2 and NOTCH1 protein expression (IHC quantifications data) and LINC00973 RNA abundance (FISH signal intensity) were analyzed by using Spearman’s correlation. Data were presented as mean ± SD, ** P < 0.01, Student’s t test.

Article Snippet: Next, membranes were blocked with 5% non-fat dry milk and incubated overnight at 4 °C with the indicated primary antibodies: EN2 (1:500, CSB-PA007660LA01HU, Cusabio, China), N-cadherin (1:2000, #22018-1-AP, Proteintech, China), E-cadherin (1:20000, #20874-1-AP, Proteintech, China), Vimentin (1:20000, #10366-1-AP, Proteintech, China), GAPDH (1:50000, #60004-1-Ig, Proteintech, China), FOSL1 (1:1000, #5281, Cell signaling, USA), NOTCH1 (1:1000, #20687-1-AP, Proteintech, China), BRD4 (1:1000, #13440, Cell signaling, USA), HEY1 (1:1000, #19929-1-AP, Proteintech, China), N1ICD (1:1000, #4147, Cell signaling, USA) or Rpb1 (1:1000, #14958, Cell signaling, USA).

Techniques: Protein-Protein interactions, Expressing, Activity Assay, Luciferase, Quantitative Proteomics, Western Blot, Knockdown, Over Expression, Migration, CCK-8 Assay, Immunohistochemistry, Staining

Journal: Cell Death & Disease

Article Title: A novel super-enhancer-driven lncRNA LINC00973 governs head and neck squamous cell carcinoma progression through EN2

doi: 10.1038/s41419-025-08380-8

Figure Lengend Snippet: The proximal SE in LINC00973 locus cis -activates LINC00973 transcription via recruiting AP-1/FOSL1, BRD4 and EP300; the cytoplasmically localized LINC00973 sponges miR-6756-3p to increase the mRNA half-life of EN2; EN2 activates downstream NOTCH signaling to modulate the aggressiveness of HNSCC.

Article Snippet: Next, membranes were blocked with 5% non-fat dry milk and incubated overnight at 4 °C with the indicated primary antibodies: EN2 (1:500, CSB-PA007660LA01HU, Cusabio, China), N-cadherin (1:2000, #22018-1-AP, Proteintech, China), E-cadherin (1:20000, #20874-1-AP, Proteintech, China), Vimentin (1:20000, #10366-1-AP, Proteintech, China), GAPDH (1:50000, #60004-1-Ig, Proteintech, China), FOSL1 (1:1000, #5281, Cell signaling, USA), NOTCH1 (1:1000, #20687-1-AP, Proteintech, China), BRD4 (1:1000, #13440, Cell signaling, USA), HEY1 (1:1000, #19929-1-AP, Proteintech, China), N1ICD (1:1000, #4147, Cell signaling, USA) or Rpb1 (1:1000, #14958, Cell signaling, USA).

Techniques:

Journal: Clinical & translational oncology : official publication of the Federation of Spanish Oncology Societies and of the National Cancer Institute of Mexico

Article Title: Engrailed-2 (EN2) protein in cervical mucus: a novel biomarker for endometrial carcinoma.

doi: 10.1007/s12094-024-03799-5

Figure Lengend Snippet: Fig. 1 The comparison of mean EN2 concentrations in cervical mucus. (A:Normal:Benign P = 0.477;Normal:EINI-II P = 0.885;N ormal:EINIII:P < 0.001;Normal:Stage 1a P < 0.001;Normal:Stage 1b P < 0.001;Benign:EINI-II P = 0.880;Benign:EINIII P < 0.001;Benign:Stage 1a:P < 0.001;Benign:Stage 1b:P < 0.001;EINI-II:EINIII P < 0.001;EINI-II:Stage 1a P = 0.002;EINI-II:Stage 1b P < 0.001; Stage 1a:1B P = 0.305)

Article Snippet: They were then blocked with goat serum and incubated with 1:250 rabbit anti-EN2 antibody (Novus, Florida, USA).

Techniques: Comparison

Journal: Clinical & translational oncology : official publication of the Federation of Spanish Oncology Societies and of the National Cancer Institute of Mexico

Article Title: Engrailed-2 (EN2) protein in cervical mucus: a novel biomarker for endometrial carcinoma.

doi: 10.1007/s12094-024-03799-5

Figure Lengend Snippet: Fig. 2 Receiver operating characteristic (ROC) curves of the prediction performance of EN2 protein

Article Snippet: They were then blocked with goat serum and incubated with 1:250 rabbit anti-EN2 antibody (Novus, Florida, USA).

Techniques:

Journal: Clinical & translational oncology : official publication of the Federation of Spanish Oncology Societies and of the National Cancer Institute of Mexico

Article Title: Engrailed-2 (EN2) protein in cervical mucus: a novel biomarker for endometrial carcinoma.

doi: 10.1007/s12094-024-03799-5

Figure Lengend Snippet: Fig. 4 EN2 protein was expressed in endometrial cancer cell lines

Article Snippet: They were then blocked with goat serum and incubated with 1:250 rabbit anti-EN2 antibody (Novus, Florida, USA).

Techniques:

Journal: Bioengineering & translational medicine

Article Title: A simple urine test by 3D-plus-3D immunoassay guides precise in vitro cancer diagnosis.

doi: 10.1002/btm2.10489

Figure Lengend Snippet: FIGURE 1 Expression of EN2 and PSA in prostate cancer- and normal cells. (a) Confocal microscopy images of prostate normal (RWPE-1 [1 105 cells/dish]) and cancer cells (DU-145 [1 105

Article Snippet: 64 Rabbit anti-EN2 polyclonal IgG antibodies (Cat. No. TA324936; Origene) (200 μl, 10 mg/L in PBS buffer [2.7 mM KCl, 137 mM NaCl, 2 mM KH2PO4, and 10 mM Na2HPO4 at pH 7.4]) were added to the H6-SPAB-capsid (800 μl, 12.5 mg/L in Tris buffer [50 mM Tris–HCl and 500 mM NaCl at pH 7.0]), followed by incubation with mild stirring at 4 C for 12–16 h to make the anti-EN2 3D-IgG probes.

Techniques: Expressing, Confocal Microscopy

Journal: Bioengineering & translational medicine

Article Title: A simple urine test by 3D-plus-3D immunoassay guides precise in vitro cancer diagnosis.

doi: 10.1002/btm2.10489

Figure Lengend Snippet: FIGURE 2 Schematic illustration of 3p3 immunoassay. (a) Interaction between antibody probe (red) and protein disease markers (gray) in serum and urine, showing denatured conformation of antibodies and protein markers and altered hydrogen and ionic bonds by urea (green) and uric acid (yellow) in urine. (b) Comparison of important characteristics between 2D and 3p3 immunoassay. (c) 3p3 immunoassay-based PCa diagnosis through detecting PCa- specific urinary EN2. 3p3, 3D-plus-3D; PCa, prostate cancer

Article Snippet: 64 Rabbit anti-EN2 polyclonal IgG antibodies (Cat. No. TA324936; Origene) (200 μl, 10 mg/L in PBS buffer [2.7 mM KCl, 137 mM NaCl, 2 mM KH2PO4, and 10 mM Na2HPO4 at pH 7.4]) were added to the H6-SPAB-capsid (800 μl, 12.5 mg/L in Tris buffer [50 mM Tris–HCl and 500 mM NaCl at pH 7.0]), followed by incubation with mild stirring at 4 C for 12–16 h to make the anti-EN2 3D-IgG probes.

Techniques: Comparison, Biomarker Discovery

Journal: Bioengineering & translational medicine

Article Title: A simple urine test by 3D-plus-3D immunoassay guides precise in vitro cancer diagnosis.

doi: 10.1002/btm2.10489

Figure Lengend Snippet: FIGURE 3 3p3 immunoassay of standard EN2 spiked in healthy urine. (a) Photographic images of 384-well plates, showing time-course change of optical assay signals in the 3p3 immunoassay performed with urine samples of standard EN2 at different concentrations (C: 0 ng/ml, 1: 0.01 ng/ml, 2: 0.1 ng/ml, 3: 1.0 ng/ml, 4: 5.0 ng/ml, 5: 10 ng/ml, 6: 20 ng/ml). (b) Time-course change of light absorption spectra of Sample 6 in (a), showing that maximum light absorption happens always at 620 nm. (c) Time-course change of OD620 measured for the optical assay signals of (a). (d) OD620 measured at 80 min after the EN2 detection by 3p3 immunoassay of (a) begins. 3p3, 3D-plus-3D; EN2, engrailed-2 protein

Article Snippet: 64 Rabbit anti-EN2 polyclonal IgG antibodies (Cat. No. TA324936; Origene) (200 μl, 10 mg/L in PBS buffer [2.7 mM KCl, 137 mM NaCl, 2 mM KH2PO4, and 10 mM Na2HPO4 at pH 7.4]) were added to the H6-SPAB-capsid (800 μl, 12.5 mg/L in Tris buffer [50 mM Tris–HCl and 500 mM NaCl at pH 7.0]), followed by incubation with mild stirring at 4 C for 12–16 h to make the anti-EN2 3D-IgG probes.

Techniques:

Journal: Bioengineering & translational medicine

Article Title: A simple urine test by 3D-plus-3D immunoassay guides precise in vitro cancer diagnosis.

doi: 10.1002/btm2.10489

Figure Lengend Snippet: FIGURE 4 3p3 and 2D immunoassay performance in urine and serum. (a) Results of 3p3 immunoassay of standard EN2 in urine and serum. (b) Time-course change of enzyme (HRP) and fluorescent protein (EGFP, mOrange, and DsRed) activities in urine and serum, which was estimated by measuring OD450 of enzymatic reaction solution and fluorescence intensity, respectively. Fluorescence intensity was measured at the following excitation (Ex) and emission (Em) wavelengths: EGFP (Ex/Em: 475/509 nm), mOrange (Ex/Em: 520/562 nm), and DsRed (Ex/Em: 541/596 nm). Relative activity/intensity was calculated by considering the activity/intensity value at 0 min as 1.0. (c) Dissociation constants (Kd) estimated in the binding between EN2 and anti-EN2 IgG probes (3D-IgG and 2D-IgG probe) under serum and urine environment. 2D-IgG probe means the IgG probe immobilized on a 2D surface. (d) Results of ELISA of standard EN2 in urine and serum. 3p3, 3D-plus-3D; EN2, engrailed-2 protein; HRP, horse radish peroxidase; IgG, immunoglobulin G; PSA, prostate-specific antigen

Article Snippet: 64 Rabbit anti-EN2 polyclonal IgG antibodies (Cat. No. TA324936; Origene) (200 μl, 10 mg/L in PBS buffer [2.7 mM KCl, 137 mM NaCl, 2 mM KH2PO4, and 10 mM Na2HPO4 at pH 7.4]) were added to the H6-SPAB-capsid (800 μl, 12.5 mg/L in Tris buffer [50 mM Tris–HCl and 500 mM NaCl at pH 7.0]), followed by incubation with mild stirring at 4 C for 12–16 h to make the anti-EN2 3D-IgG probes.

Techniques: Fluorescence, Activity Assay, Binding Assay, Enzyme-linked Immunosorbent Assay

Journal: Bioengineering & translational medicine

Article Title: A simple urine test by 3D-plus-3D immunoassay guides precise in vitro cancer diagnosis.

doi: 10.1002/btm2.10489

Figure Lengend Snippet: FIGURE 5 PCa diagnosis through detecting EN2 and PSA in PCa patient urine. (a) Results of 3p3 immunoassay-based PCa diagnosis through EN2 detection using the urine of 30 PCa patients and 30 healthy individuals, presented in box and dot plots. (b) Results of ELISA-based PCa diagnosis through EN2 detection using the urine of 30 PCa patients and 30 healthy individuals. COV represent cut-off-value, calculated as per instructions from ELISA kit supplier. False-positive and -negative signals are marked by red and green bars, respectively. (c) Results of 3p3 immunoassay-based PCa diagnosis through PSA detection using the urine of 30 PCa patients and 30 healthy individuals, presented in box and dot plots. (d) Correlation between urinary EN2 and PSA levels, indicating a very low Spearman's rank correlation coefficient (ρ = 0.176). 3p3, 3D-plus- 3D; EN2, engrailed-2 protein; PCa, prostate cancer; PSA, prostate-specific antigen

Article Snippet: 64 Rabbit anti-EN2 polyclonal IgG antibodies (Cat. No. TA324936; Origene) (200 μl, 10 mg/L in PBS buffer [2.7 mM KCl, 137 mM NaCl, 2 mM KH2PO4, and 10 mM Na2HPO4 at pH 7.4]) were added to the H6-SPAB-capsid (800 μl, 12.5 mg/L in Tris buffer [50 mM Tris–HCl and 500 mM NaCl at pH 7.0]), followed by incubation with mild stirring at 4 C for 12–16 h to make the anti-EN2 3D-IgG probes.

Techniques: Biomarker Discovery, Enzyme-linked Immunosorbent Assay

Journal: Bioengineering & translational medicine

Article Title: A simple urine test by 3D-plus-3D immunoassay guides precise in vitro cancer diagnosis.

doi: 10.1002/btm2.10489

Figure Lengend Snippet: FIGURE 6 Diagnostic selectivity of 3p3 immunoassay in urinary EN2 detection. (a) Result of immunoblotting analysis to detect EN2 in the urine of PCa, BPH, and BCa patient and healthy individual. (b) Comparison of diagnostic results by 3p3 immunoassays using the urine of 30 PCa, 15 BPH, and 15 BCa patients and 30 healthy individuals: EN2 versus PSA detection, presented in box plot, showing excellent selectivity of PCa diagnosis performed through urinary EN2 detection-based 3p3 immunoassay. 3p3, 3D-plus-3D; BPH, benign prostatic hyperplasia; EN2, engrailed-2 protein; PCa, prostate cancer; PSA, prostate-specific antigen

Article Snippet: 64 Rabbit anti-EN2 polyclonal IgG antibodies (Cat. No. TA324936; Origene) (200 μl, 10 mg/L in PBS buffer [2.7 mM KCl, 137 mM NaCl, 2 mM KH2PO4, and 10 mM Na2HPO4 at pH 7.4]) were added to the H6-SPAB-capsid (800 μl, 12.5 mg/L in Tris buffer [50 mM Tris–HCl and 500 mM NaCl at pH 7.0]), followed by incubation with mild stirring at 4 C for 12–16 h to make the anti-EN2 3D-IgG probes.

Techniques: Diagnostic Assay, Western Blot, Comparison, Biomarker Discovery

Journal: Bioengineering & translational medicine

Article Title: A simple urine test by 3D-plus-3D immunoassay guides precise in vitro cancer diagnosis.

doi: 10.1002/btm2.10489

Figure Lengend Snippet: FIGURE 1 Expression of EN2 and PSA in prostate cancer- and normal cells. (a) Confocal microscopy images of prostate normal (RWPE-1 [1 105 cells/dish]) and cancer cells (DU-145 [1 105

Article Snippet: RWPE-1, DU-145, and PC-3 cells were incubated with phycoerythrin (PE)-labeled anti-PSA antibody (Cat. No. 70-XG69; Fitzgerald, Acton, MA, USA) and also with fluorescein isothiocyanate (FITC)-labeled anti-EN2 antibody (Cat. No. TA32493; Origene, Rockville, MD, USA), and the fluorescence from the cell cultures was analyzed using confocal fluorescence microscopy.

Techniques: Expressing, Confocal Microscopy

Journal: Bioengineering & translational medicine

Article Title: A simple urine test by 3D-plus-3D immunoassay guides precise in vitro cancer diagnosis.

doi: 10.1002/btm2.10489

Figure Lengend Snippet: FIGURE 2 Schematic illustration of 3p3 immunoassay. (a) Interaction between antibody probe (red) and protein disease markers (gray) in serum and urine, showing denatured conformation of antibodies and protein markers and altered hydrogen and ionic bonds by urea (green) and uric acid (yellow) in urine. (b) Comparison of important characteristics between 2D and 3p3 immunoassay. (c) 3p3 immunoassay-based PCa diagnosis through detecting PCa- specific urinary EN2. 3p3, 3D-plus-3D; PCa, prostate cancer

Article Snippet: RWPE-1, DU-145, and PC-3 cells were incubated with phycoerythrin (PE)-labeled anti-PSA antibody (Cat. No. 70-XG69; Fitzgerald, Acton, MA, USA) and also with fluorescein isothiocyanate (FITC)-labeled anti-EN2 antibody (Cat. No. TA32493; Origene, Rockville, MD, USA), and the fluorescence from the cell cultures was analyzed using confocal fluorescence microscopy.

Techniques: Comparison, Biomarker Discovery

Journal: Bioengineering & translational medicine

Article Title: A simple urine test by 3D-plus-3D immunoassay guides precise in vitro cancer diagnosis.

doi: 10.1002/btm2.10489

Figure Lengend Snippet: FIGURE 3 3p3 immunoassay of standard EN2 spiked in healthy urine. (a) Photographic images of 384-well plates, showing time-course change of optical assay signals in the 3p3 immunoassay performed with urine samples of standard EN2 at different concentrations (C: 0 ng/ml, 1: 0.01 ng/ml, 2: 0.1 ng/ml, 3: 1.0 ng/ml, 4: 5.0 ng/ml, 5: 10 ng/ml, 6: 20 ng/ml). (b) Time-course change of light absorption spectra of Sample 6 in (a), showing that maximum light absorption happens always at 620 nm. (c) Time-course change of OD620 measured for the optical assay signals of (a). (d) OD620 measured at 80 min after the EN2 detection by 3p3 immunoassay of (a) begins. 3p3, 3D-plus-3D; EN2, engrailed-2 protein

Article Snippet: RWPE-1, DU-145, and PC-3 cells were incubated with phycoerythrin (PE)-labeled anti-PSA antibody (Cat. No. 70-XG69; Fitzgerald, Acton, MA, USA) and also with fluorescein isothiocyanate (FITC)-labeled anti-EN2 antibody (Cat. No. TA32493; Origene, Rockville, MD, USA), and the fluorescence from the cell cultures was analyzed using confocal fluorescence microscopy.

Techniques:

Journal: Bioengineering & translational medicine

Article Title: A simple urine test by 3D-plus-3D immunoassay guides precise in vitro cancer diagnosis.

doi: 10.1002/btm2.10489

Figure Lengend Snippet: FIGURE 4 3p3 and 2D immunoassay performance in urine and serum. (a) Results of 3p3 immunoassay of standard EN2 in urine and serum. (b) Time-course change of enzyme (HRP) and fluorescent protein (EGFP, mOrange, and DsRed) activities in urine and serum, which was estimated by measuring OD450 of enzymatic reaction solution and fluorescence intensity, respectively. Fluorescence intensity was measured at the following excitation (Ex) and emission (Em) wavelengths: EGFP (Ex/Em: 475/509 nm), mOrange (Ex/Em: 520/562 nm), and DsRed (Ex/Em: 541/596 nm). Relative activity/intensity was calculated by considering the activity/intensity value at 0 min as 1.0. (c) Dissociation constants (Kd) estimated in the binding between EN2 and anti-EN2 IgG probes (3D-IgG and 2D-IgG probe) under serum and urine environment. 2D-IgG probe means the IgG probe immobilized on a 2D surface. (d) Results of ELISA of standard EN2 in urine and serum. 3p3, 3D-plus-3D; EN2, engrailed-2 protein; HRP, horse radish peroxidase; IgG, immunoglobulin G; PSA, prostate-specific antigen

Article Snippet: RWPE-1, DU-145, and PC-3 cells were incubated with phycoerythrin (PE)-labeled anti-PSA antibody (Cat. No. 70-XG69; Fitzgerald, Acton, MA, USA) and also with fluorescein isothiocyanate (FITC)-labeled anti-EN2 antibody (Cat. No. TA32493; Origene, Rockville, MD, USA), and the fluorescence from the cell cultures was analyzed using confocal fluorescence microscopy.

Techniques: Fluorescence, Activity Assay, Binding Assay, Enzyme-linked Immunosorbent Assay

Journal: Bioengineering & translational medicine

Article Title: A simple urine test by 3D-plus-3D immunoassay guides precise in vitro cancer diagnosis.

doi: 10.1002/btm2.10489

Figure Lengend Snippet: FIGURE 5 PCa diagnosis through detecting EN2 and PSA in PCa patient urine. (a) Results of 3p3 immunoassay-based PCa diagnosis through EN2 detection using the urine of 30 PCa patients and 30 healthy individuals, presented in box and dot plots. (b) Results of ELISA-based PCa diagnosis through EN2 detection using the urine of 30 PCa patients and 30 healthy individuals. COV represent cut-off-value, calculated as per instructions from ELISA kit supplier. False-positive and -negative signals are marked by red and green bars, respectively. (c) Results of 3p3 immunoassay-based PCa diagnosis through PSA detection using the urine of 30 PCa patients and 30 healthy individuals, presented in box and dot plots. (d) Correlation between urinary EN2 and PSA levels, indicating a very low Spearman's rank correlation coefficient (ρ = 0.176). 3p3, 3D-plus- 3D; EN2, engrailed-2 protein; PCa, prostate cancer; PSA, prostate-specific antigen

Article Snippet: RWPE-1, DU-145, and PC-3 cells were incubated with phycoerythrin (PE)-labeled anti-PSA antibody (Cat. No. 70-XG69; Fitzgerald, Acton, MA, USA) and also with fluorescein isothiocyanate (FITC)-labeled anti-EN2 antibody (Cat. No. TA32493; Origene, Rockville, MD, USA), and the fluorescence from the cell cultures was analyzed using confocal fluorescence microscopy.

Techniques: Biomarker Discovery, Enzyme-linked Immunosorbent Assay

Journal: Bioengineering & translational medicine

Article Title: A simple urine test by 3D-plus-3D immunoassay guides precise in vitro cancer diagnosis.

doi: 10.1002/btm2.10489

Figure Lengend Snippet: FIGURE 6 Diagnostic selectivity of 3p3 immunoassay in urinary EN2 detection. (a) Result of immunoblotting analysis to detect EN2 in the urine of PCa, BPH, and BCa patient and healthy individual. (b) Comparison of diagnostic results by 3p3 immunoassays using the urine of 30 PCa, 15 BPH, and 15 BCa patients and 30 healthy individuals: EN2 versus PSA detection, presented in box plot, showing excellent selectivity of PCa diagnosis performed through urinary EN2 detection-based 3p3 immunoassay. 3p3, 3D-plus-3D; BPH, benign prostatic hyperplasia; EN2, engrailed-2 protein; PCa, prostate cancer; PSA, prostate-specific antigen

Article Snippet: RWPE-1, DU-145, and PC-3 cells were incubated with phycoerythrin (PE)-labeled anti-PSA antibody (Cat. No. 70-XG69; Fitzgerald, Acton, MA, USA) and also with fluorescein isothiocyanate (FITC)-labeled anti-EN2 antibody (Cat. No. TA32493; Origene, Rockville, MD, USA), and the fluorescence from the cell cultures was analyzed using confocal fluorescence microscopy.

Techniques: Diagnostic Assay, Western Blot, Comparison, Biomarker Discovery