Journal: The Journal of Immunology Author Choice

Article Title: An Isolated TCR αβ Restricted by HLA-A*02:01/CT37 Peptide Redirecting CD8 + T Cells To Kill and Secrete IFN-γ in Response to Lung Adenocarcinoma Cell Lines

doi: 10.4049/jimmunol.1701054

Figure Lengend Snippet: CT37 Ag peptides selected according to in silico–determined HLA specificity and affinity

Article Snippet: Weak IHC staining in healthy kidney and urinary bladder tissues is mentioned in the Human Protein Atlas, using one of two Abs, in this case using the rabbit anti-human CT37 polyclonal IgG purified Ab HPA011284 ( http://www.proteinatlas.org ).

Techniques: Binding Assay, Selection

Journal: The Journal of Immunology Author Choice

Article Title: An Isolated TCR αβ Restricted by HLA-A*02:01/CT37 Peptide Redirecting CD8 + T Cells To Kill and Secrete IFN-γ in Response to Lung Adenocarcinoma Cell Lines

doi: 10.4049/jimmunol.1701054

Figure Lengend Snippet: Patients included: characteristics

Article Snippet: Weak IHC staining in healthy kidney and urinary bladder tissues is mentioned in the Human Protein Atlas, using one of two Abs, in this case using the rabbit anti-human CT37 polyclonal IgG purified Ab HPA011284 ( http://www.proteinatlas.org ).

Techniques: Tomography

Journal: The Journal of Immunology Author Choice

Article Title: An Isolated TCR αβ Restricted by HLA-A*02:01/CT37 Peptide Redirecting CD8 + T Cells To Kill and Secrete IFN-γ in Response to Lung Adenocarcinoma Cell Lines

doi: 10.4049/jimmunol.1701054

Figure Lengend Snippet: Clinical laboratory features considered for selection of patient NSCLC_7. IHC of NSCLC_7 patient’s tumor sample showing CT37 Ag staining (A); FACS analysis of PBMCs obtained from patient NSCLC_7; upper section, HLA-A*02:01/CT37 YLCSGSSYFV peptide pentamer; lower section, the control HLA-A*02:01/NY-ESO-1 SLLMWITQV peptide pentamer (B). Procedures and reagents are explained in the Materials and Methods.

Article Snippet: Weak IHC staining in healthy kidney and urinary bladder tissues is mentioned in the Human Protein Atlas, using one of two Abs, in this case using the rabbit anti-human CT37 polyclonal IgG purified Ab HPA011284 ( http://www.proteinatlas.org ).

Techniques: Selection, Staining, Control

Journal: The Journal of Immunology Author Choice

Article Title: An Isolated TCR αβ Restricted by HLA-A*02:01/CT37 Peptide Redirecting CD8 + T Cells To Kill and Secrete IFN-γ in Response to Lung Adenocarcinoma Cell Lines

doi: 10.4049/jimmunol.1701054

Figure Lengend Snippet: Constructs features (A), levels of TCRαβ-chain expression in CD8+ T cells transduced with the three different constructs (B), polymerization ability of TCRαβ and CD3 chains encoded in the three constructs and host-encoded CD3 chains (C), and identity of the polymerized CD3 chains (D). Depicted in (A) are the three constructs. The boxes represent the amino acid sequences of the chains indicated inside the boxes. T2A: self-cleavage 2A region of the Thosea asigna virus; P2A: self-cleavage 2A region of the porcine Teschovirus 1; CD3: in construct 3 represent the extracellular and transmembrane domains of the CD3ζ chain, followed by the signaling domains of CD28, 4-1BB, and CD3ζ chains. The lines represent amino acid sequences of linkers: RAKR is the Furin consensus amino acid recognition site; SGSG is a hydrophilic tetrapeptide added to prevent steric hindrance. The arrows represent cleavage sites. In (B), FACS analysis results; transduced CD8+ T cells were stained with FITC–anti-CD8 Ab and PE-HLA-A*02:01/CT37 YLCSGSSYFV peptide complexes. A representative of three experiments is shown. Results using FITC-isotype Ab and PE-control pentamer complexes were negative (data not shown). In (C), biotinylated membrane proteins from CD8+ T cells were immunoprecipitated with an anti-TCRβ Ab and molecular complexes separated by SDS-PAGE, transferred to a membrane, and blots detected by chemiluminescence. A representative of three experiments is shown. In (D), immunoprecipitated proteins obtained as indicated in (C) for construct 3, were blotted using rabbit anti-CD3δ EP4426, anti-CD3γ 134684, anti-CD3ε 133628 Abs, and Armenian hamster anti-CD3ζ H136-968 Abs (Abcam), HRP-conjugated goat anti-rabbit IgG or HPR-conjugated rabbit anti-Armenian hamster IgG, and blots were detected by chemiluminescence using autoradiography films (Kodak BioMax Light). A representative of two experiments is shown.

Article Snippet: Weak IHC staining in healthy kidney and urinary bladder tissues is mentioned in the Human Protein Atlas, using one of two Abs, in this case using the rabbit anti-human CT37 polyclonal IgG purified Ab HPA011284 ( http://www.proteinatlas.org ).

Techniques: Construct, Expressing, Transduction, Virus, Staining, Control, Membrane, Immunoprecipitation, SDS Page, Autoradiography

Journal: The Journal of Immunology Author Choice

Article Title: An Isolated TCR αβ Restricted by HLA-A*02:01/CT37 Peptide Redirecting CD8 + T Cells To Kill and Secrete IFN-γ in Response to Lung Adenocarcinoma Cell Lines

doi: 10.4049/jimmunol.1701054

Figure Lengend Snippet: TCR-transduced CD8+ T cell lysis autologous CD3 cell–depleted PBMCs in a HLA-A*02:01/CT37 peptide YLCSGSSYFV with high functional avidity. (A) Results from [51Cr] release assays (left) at 16:1 E:T ratio. Dashed lines are the results from six experiments with cells isolated from six different HLA-A*02:01–positive donors. The solid line represents the mean values. Effector cells were purified CD8+ T cells transduced with construct 1. Target cells were CD3-depleted autologous PBMCs handled as in Materials and Methods and pulsed with indicated amounts of HLA-A*A02:01–restricted CT37 YLCSGSSYF peptide. [51Cr]-released cytotoxic assay proceeded for 4 h. Each experiment was done in triplicate. In (A), on the right we show the curve obtained with the mean values (in blue). We show the EC50 value extrapolated from this curve. (B) Results from FACS analysis experiments conducted to determine whether silencing of the CD8A chain will affect HLA-A*0201/CT37 YLCSGSSYF peptide complex recognition by the NSCLC_7 CD8+ T cell line. Left, The negative FACS analysis control; middle, graphic showing results of NSCLC_7 CD8+ T cell line transduced with control siRNA; right, results from NSCLC_7 cell line transduced with CT37-specific siRNA.

Article Snippet: Weak IHC staining in healthy kidney and urinary bladder tissues is mentioned in the Human Protein Atlas, using one of two Abs, in this case using the rabbit anti-human CT37 polyclonal IgG purified Ab HPA011284 ( http://www.proteinatlas.org ).

Techniques: Lysis, Functional Assay, Isolation, Purification, Transduction, Construct, Control

Journal: The Journal of Immunology Author Choice

Article Title: An Isolated TCR αβ Restricted by HLA-A*02:01/CT37 Peptide Redirecting CD8 + T Cells To Kill and Secrete IFN-γ in Response to Lung Adenocarcinoma Cell Lines

doi: 10.4049/jimmunol.1701054

Figure Lengend Snippet: Functional analysis of constructs in allogeneic CD8+ T cells. Retroviral transduction of effector (E) allogeneic CD8+ T cells was performed as explained in Materials and Methods. For [51Cr] release assay results presented in (A) and (B), six experiments, with each variable in triplicate, were performed. (A) After two cycles of 10 d stimulation in vitro, E cells were harvested, washed, counted, and plated to be tested at appropriate E:T ratios. Target (T) cells were CD3-depleted autologous PBMCs (HLA-A*02:01–positive) labeled with 200 μCi of Na251CrO4 for 1 h and washed. These cells were then 1) left untreated, 2) pulsed with 100 ng/ml of control CT37-YYLCSGSSYF peptide, or 3) pulsed with 100 ng/ml of CT37–YLCSGSSYFV peptide with high affinity for the HLA-A*02:01 class I allele. [51Cr]-released cytotoxic assay proceeded for 4 h. The p values from Mann–Whitney comparison of two means are presented. In (B), [51Cr]-released cytotoxic assay proceeded at a 16:1 E:T ratio. Target cells (as in A) were pulsed with 100 ng/ml of CT37-YLCSGSSYFV peptide. Cultures proceeded for 4 h as indicated above, without anti–HLA-A2 Ab or in the presence of this Ab at the concentrations indicated. The p values from Kruskal–Wallis comparison of more than two means are presented. (C) IFN-γ–secretion assays proceeded as in (B), but the target cells were not labeled with [51Cr]. The supernatants were harvested and tested with a highly sensitive ELISA kit. The p values from Mann–Whitney comparison of two means resulting from cultures that proceeded without Ab are presented. (D) The results from ELISPOT assay to detect the frequency of Ag-specific IFN-γ–secreting CD8+ T cells are presented. Target cells were pulsed with 100 ng/ml of CT37-YLCSGSSYFV peptide and cultures proceeded for 16 h at a 16:1 E:T ratio. The p values from Mann–Whitney comparison of two means are presented.

Article Snippet: Weak IHC staining in healthy kidney and urinary bladder tissues is mentioned in the Human Protein Atlas, using one of two Abs, in this case using the rabbit anti-human CT37 polyclonal IgG purified Ab HPA011284 ( http://www.proteinatlas.org ).

Techniques: Functional Assay, Construct, Retroviral, Transduction, Release Assay, In Vitro, Labeling, Control, MANN-WHITNEY, Comparison, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot

Journal: The Journal of Immunology Author Choice

Article Title: An Isolated TCR αβ Restricted by HLA-A*02:01/CT37 Peptide Redirecting CD8 + T Cells To Kill and Secrete IFN-γ in Response to Lung Adenocarcinoma Cell Lines

doi: 10.4049/jimmunol.1701054

Figure Lengend Snippet: Features of lung ADC cell lines used (A) and cytotoxic capacity of CD8+ T cells transduced with the three different constructs (B). In (A), total RNA was isolated from cell lines and negative control (A549 cell line) as indicated, and RT-PCR results obtained. The mean of triplicates is shown (Ai). Protein lysates from cell lines indicted and negative control A549 were immunoprecipitated with rabbit polyclonal anti-human CT37 Ab HPA011284, proteins were separated by SDS-PAGE, transferred to a membrane, and CT37 blotted using rabbit polyclonal AP51690PU-N Ab and HRP-conjugated goat anti-rabbit IgG. Blots were detected by chemiluminescence using autoradiography films [Kodak BioMax Light; (Aii)]. In (B), retroviral transduction of effector (E) allogeneic CD8+ T cells was performed as explained in Materials and Methods. For results presented in (Bi)–(Biii), six [51Cr] release assays, with each variable in triplicate, were performed. After 2 cycles of 10 d stimulation in vitro, E cells were harvested, washed, counted, and plated to be tested at 16:1 E:T ratios. Target (T) lung ADC lines HLA-A*A-02:01–positive HCC2935 (Bi), and H1993 (Bii), and HLA-A*02:01–negative line H1299, were labeled with 200 μCi of Na251CrO4 for 1 h and washed. [51Cr]-released cytotoxic assay proceed for 4 h at a 16:1 E:T ratio. Cultures proceeded without or with an anti–HLA-A2 Ab to block the HLA-A*02:01 molecule, and at the indicated concentrations. The HCC2935 cell line carries wild type (wt) TP53 and KRAS genes, and an EGFR 2237_2254del10 (deletion E746-S752). The H1993 line carries a TP53 726C > G (C242W) substitution, and wt EGFR and KRAS genes. The H1299 carries a TP53 gene deletion, and wt EGFR and KRAS genes. Data on TP53 gene mutations was obtained from http://p53.free.fr/Database/Cancer_cell_lines/NSCLC.html. We genotyped mutations in the EGFR and KRAS genes as explained in Materials and Methods. The p values from Mann–Whitney comparison of two means are presented.

Article Snippet: Weak IHC staining in healthy kidney and urinary bladder tissues is mentioned in the Human Protein Atlas, using one of two Abs, in this case using the rabbit anti-human CT37 polyclonal IgG purified Ab HPA011284 ( http://www.proteinatlas.org ).

Techniques: Transduction, Construct, Isolation, Negative Control, Reverse Transcription Polymerase Chain Reaction, Immunoprecipitation, SDS Page, Membrane, Autoradiography, Retroviral, In Vitro, Labeling, Blocking Assay, MANN-WHITNEY, Comparison

Journal: The Journal of Immunology Author Choice

Article Title: An Isolated TCR αβ Restricted by HLA-A*02:01/CT37 Peptide Redirecting CD8 + T Cells To Kill and Secrete IFN-γ in Response to Lung Adenocarcinoma Cell Lines

doi: 10.4049/jimmunol.1701054

Figure Lengend Snippet: Cytotoxic capacity of CD8+ T cells transduced with the three different constructs against HLA-A*02:01–specific peptide under CT37 expression and CT37-expression silencing. Retroviral transduction of effector (E) CD8+ T cells was performed as explained in Materials and Methods. For results presented in (A)–(C), six [51Cr] release assays, with each variable in triplicate, were performed. After 2 cycles of 10 d stimulation in vitro, E cells were harvested, washed, counted, and plated to be tested at 16:1 E:T ratios. Target (T) lung ADC lines HLA-A*A-02:01–positive HCC2935, H1993, and H522 were used. CT37 expression was targeted using CT37-specific siRNA or control siRNA as explained in Materials and Methods. The cell lines were labeled with 200 μCi of Na251CrO4 for 1 h and washed. [51Cr]-released cytotoxic assay proceeded for 4 h. The p values from Mann–Whitney comparison of two means are presented.

Article Snippet: Weak IHC staining in healthy kidney and urinary bladder tissues is mentioned in the Human Protein Atlas, using one of two Abs, in this case using the rabbit anti-human CT37 polyclonal IgG purified Ab HPA011284 ( http://www.proteinatlas.org ).

Techniques: Transduction, Construct, Expressing, Retroviral, In Vitro, Control, Labeling, MANN-WHITNEY, Comparison

Journal: Translational Cancer Research

Article Title: OPN3 enhances the proliferation, migration, and invasion of triple-negative breast cancer cells via the regulation of the TGF-β signaling pathway

doi: 10.21037/tcr-24-1374

Figure Lengend Snippet: Upregulation of OPN3 in BC correlates with patient diagnosis in the TCGA database. (A-G) bcGenExMiner v5.0 (A-C), Sento academic (D,E), and GEPIA2.0 (F,G) databases depicting OPN3 expression in TNBC, non-TNBC, and different BC subtypes, red represents tumor tissue, black represents normal tissue. (H,I) Kaplan-Meier Plotter online site plotted Kaplan-Meier plots for OS (H) and RFS (I) of BC patients based on OPN3 expressions (high vs. low, log-rank test P=0.002, P≤0.001). *, P≤0.05; ***, P≤0.001. OPN3 , Opsin3; TNBC, triple-negative breast cancer; IHC, immunohistochemistry; BC, breast cancer; TCGA, The Cancer Genome Atlas; BRCA, breast invasive carcinoma; TPM, transcripts per million; HR, hazard ratio; CI, confidence interval; OS, overall survival; RFS, recurrence free survival.

Article Snippet: These specimens were stained with a diluted OPN3 primary antibody (1:200, Affinity, Biosciences LTD, Jiangsu, China) and kept for 60 min at 20–25 °C, followed by dropwise addition of MaxVision TM HRP reagent (Fuzhou Meixin Co., Ltd., Fuzhou, China, KIT-5006).

Techniques: Biomarker Discovery, Expressing, Immunohistochemistry

Journal: Translational Cancer Research

Article Title: OPN3 enhances the proliferation, migration, and invasion of triple-negative breast cancer cells via the regulation of the TGF-β signaling pathway

doi: 10.21037/tcr-24-1374

Figure Lengend Snippet: Upregulation of OPN3 in TNBC cells and tissues correlates with the prognosis of patients, OPN3 knockdown suppressed TNBC cell propagation, dissemination, and invasion and enhanced apoptosis in vitro . (A-C) Expression of OPN3 in normal epithelial cells, TNBC, and non-TNBC cell proteins, and mRNAs was observed via WB and qRT-PCR. (D) Comparison of OPN3 expression differences in TNBC and adjacent paracancerous tissues as per the IHC scores. (E) IHC representative plots of OPN3 in TNBC tumor and adjacent paracancerous tissues (“-” represents negative “+” low, “++” moderate, “+++” high expressions). (F) OPN3 level in BT-549 cells post-transfection with si- OPN3 and si-nc via qRT-PCR analysis. (G) OPN3 protein level was detected by immunofluorescence assay after transfection with si- OPN3 #3 (50×). (H,I) Proliferative ability of BT-549 cells after suppression of OPN3 was analyzed by CCK-8 and clone formation assay. (J,K) BT-549 cell invasive and migrating potential were evaluated through transwell and scratch assays after silencing of OPN3 (100×). (L) Flow cytometry was employed to evaluate the apoptotic potential of BT-549 cells after the suppression of OPN3 . Data are illustrated as mean ± SD. *, P≤0.05; **, P≤0.01; ***, P≤0.001; ****, P≤0.0001 depicted as significant variations between groups. (I,J) Crystal violet staining method. OPN3 , Opsin3; IHC, immunohistochemistry; TNBC, triple-negative breast cancer; WB, Western blotting; qRT-PCR, quantitative real-time polymerase chain reaction; CCK-8, Cell Counting Kit-8; DAPI, 4’,6-diamidino-2-phenylindole dihydrochloride.

Article Snippet: These specimens were stained with a diluted OPN3 primary antibody (1:200, Affinity, Biosciences LTD, Jiangsu, China) and kept for 60 min at 20–25 °C, followed by dropwise addition of MaxVision TM HRP reagent (Fuzhou Meixin Co., Ltd., Fuzhou, China, KIT-5006).

Techniques: Knockdown, In Vitro, Expressing, Quantitative RT-PCR, Comparison, Transfection, Immunofluorescence, CCK-8 Assay, Tube Formation Assay, Flow Cytometry, Staining, Immunohistochemistry, Western Blot, Real-time Polymerase Chain Reaction, Cell Counting

Journal: Translational Cancer Research

Article Title: OPN3 enhances the proliferation, migration, and invasion of triple-negative breast cancer cells via the regulation of the TGF-β signaling pathway

doi: 10.21037/tcr-24-1374

Figure Lengend Snippet: Clinicopathological features of TNBC patients and their associations with OPN3 expression

Article Snippet: These specimens were stained with a diluted OPN3 primary antibody (1:200, Affinity, Biosciences LTD, Jiangsu, China) and kept for 60 min at 20–25 °C, followed by dropwise addition of MaxVision TM HRP reagent (Fuzhou Meixin Co., Ltd., Fuzhou, China, KIT-5006).

Techniques: Biomarker Discovery

Journal: Translational Cancer Research

Article Title: OPN3 enhances the proliferation, migration, and invasion of triple-negative breast cancer cells via the regulation of the TGF-β signaling pathway

doi: 10.21037/tcr-24-1374

Figure Lengend Snippet: Clinicopathologic factors for overall survival in all TNBC patients via univariate and multivariable analyses.

Article Snippet: These specimens were stained with a diluted OPN3 primary antibody (1:200, Affinity, Biosciences LTD, Jiangsu, China) and kept for 60 min at 20–25 °C, followed by dropwise addition of MaxVision TM HRP reagent (Fuzhou Meixin Co., Ltd., Fuzhou, China, KIT-5006).

Techniques:

Journal: Translational Cancer Research

Article Title: OPN3 enhances the proliferation, migration, and invasion of triple-negative breast cancer cells via the regulation of the TGF-β signaling pathway

doi: 10.21037/tcr-24-1374

Figure Lengend Snippet: OPN3 overexpression enhances TNBC cell propagation, migration, and invasion in vitro and in vivo , and inhibits apoptosis, and OPN3 enhances EMT in TNBC cells. (A,B) The expression pattern of OPN3 in BT-549 cells post-transfection with lv- OPN3 and lv-nc was analyzed via WB and qRT-PCR. (C-E) BT-549 cells were infected with lentiviral OPN3 overexpression (lv- OPN3 ) or a control vector (lv-nc), and the BT-549 cells were injected (subcutaneous route) into female nude mice (N=5/group). (C) Images of tumor tissue 22 days after cell injection. (D) Measurement and comparison of tumor weights. (E) Changes in tumor volume were measured every three days after injection. (F,G) The impact of OPN3 overexpression on the proliferative capacity of BT-549 cells was investigated by CCK-8 and clone formation. (H,I) Transwell and wound healing assays reveal the migration and invasive potential of BT-549 cells after OPN3 overexpression (100×). (J) The apoptotic capacity of BT- 549 cells after OPN3 overexpression was examined by flow cytometry. (K) Cellular morphology changes after OPN3 overexpression in BT-549 cells (400×). (L) The relationship between OPN3 and E-cadherin, N-cadherin, Vimentin, and Snail is illustrated in Timer2.0. (M) Immunofluorescence images (50×) of the relationship between high and low expression of OPN3 and EMT markers in BT-549 cells. (N) The expressions of mRNA of EMT-associated genes in BT-549 cells were analyzed via qRT-PCR to determine the effects of OPN3 overexpression or suppression. (O,P) The expression patterns of EMT-related proteins in BT-549 cells with overexpression or silenced of OPN3 were analyzed using WB. Data are illustrated as mean ± SD. *, P≤0.05; **, P≤0.01; ***, P≤0.001; ****, P≤0.0001 indicates substantial variations between groups. (G,H) Crystal violet staining method. OPN3 , Opsin3; TNBC, triple-negative breast cancer; EMT, epithelial-mesenchymal transition; WB, Western blotting; qRT-PCR, quantitative real-time polymerase chain reaction; CCK-8, Cell Counting Kit-8; DAPI, 4’,6-diamidino-2-phenylindole dihydrochloride.

Article Snippet: These specimens were stained with a diluted OPN3 primary antibody (1:200, Affinity, Biosciences LTD, Jiangsu, China) and kept for 60 min at 20–25 °C, followed by dropwise addition of MaxVision TM HRP reagent (Fuzhou Meixin Co., Ltd., Fuzhou, China, KIT-5006).

Techniques: Over Expression, Migration, In Vitro, In Vivo, Expressing, Transfection, Quantitative RT-PCR, Infection, Control, Plasmid Preparation, Injection, Comparison, CCK-8 Assay, Flow Cytometry, Immunofluorescence, Staining, Western Blot, Real-time Polymerase Chain Reaction, Cell Counting

Journal: Translational Cancer Research

Article Title: OPN3 enhances the proliferation, migration, and invasion of triple-negative breast cancer cells via the regulation of the TGF-β signaling pathway

doi: 10.21037/tcr-24-1374

Figure Lengend Snippet: OPN3 enhances EMT via the TGF-β/SMAD2 signaling pathway. (A) A positive association was observed between the levels of OPN3 and those of TGFBR1 and SMAD2 in the GEPIA2.0 database analysis. (B) TGF-β levels in the supernatants of BT-549 cells with either overexpression or silenced of OPN3 were measured using ELISA. (C,D) The mRNA and protein levels of downstream proteins in the TGF-β pathway were evaluated in BT-549 cells overexpressed or silenced OPN3 using qRT-PCR and WB. (E) The levels of EMT-related and TGF-β pathway downstream proteins in BT-549 cells treated withLY2109761 (1 μmol) or TGF-β1 (10 ng/mL) following OPN3 overexpression or silenced were examined by WB. (F) Clone formation assays were carried out to analyze the propagation of BT-549 cells after overexpression or silencing of OPN3 with the addition of LY2109761 (1 μmol) or TGF-β1 (10 ng/mL), respectively (100×). (G,H) Migrating and invasive potential of BT-549 cells were analyzed via transwell and scratch assays after the overexpression or silencing of OPN3 with the addition of LY2109761 (1 μmol) or TGF-β1 (10 ng/mL), respectively (100×). Data are illustrated as mean ± SD. ns, not statistically significant; *, P≤0.05; **, P≤0.01; ***, P≤0.001; ****, P≤0.0001 indicates significant variations between groups. (F,G) Crystal violet staining method; OPN3 , Opsin3; TGF-β, transforming growth factor-beta; EMT, epithelial-mesenchymal transition; ELISA, enzyme-linked immunosorbent assay; WB, Western blotting; qRT-PCR, quantitative real-time polymerase chain reaction.

Article Snippet: These specimens were stained with a diluted OPN3 primary antibody (1:200, Affinity, Biosciences LTD, Jiangsu, China) and kept for 60 min at 20–25 °C, followed by dropwise addition of MaxVision TM HRP reagent (Fuzhou Meixin Co., Ltd., Fuzhou, China, KIT-5006).

Techniques: Over Expression, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Staining, Western Blot, Real-time Polymerase Chain Reaction

Journal: Translational Cancer Research

Article Title: OPN3 enhances the proliferation, migration, and invasion of triple-negative breast cancer cells via the regulation of the TGF-β signaling pathway

doi: 10.21037/tcr-24-1374

Figure Lengend Snippet: Schematic diagram of OPN3 -induced EMT model and promoting invasive metastasis of TNBC cells via TGF-β/SMAD2 signaling pathway. This image was drawn from the Researcher’s Home Online website, image export number: ID:UIPAI918b8. OPN3 , Opsin3; EMT, epithelial-mesenchymal transition; TNBC, triple-negative breast cancer.

Article Snippet: These specimens were stained with a diluted OPN3 primary antibody (1:200, Affinity, Biosciences LTD, Jiangsu, China) and kept for 60 min at 20–25 °C, followed by dropwise addition of MaxVision TM HRP reagent (Fuzhou Meixin Co., Ltd., Fuzhou, China, KIT-5006).

Techniques:

Journal: International Journal of General Medicine

Article Title: Predictive Values of Homeobox Gene A-Antisense Transcript 3 (HOXA-AS3), Cystatin 6 (CST6), and Chromobox Homolog 4 (CBX4) Expressions in Cancer Tissues for Recurrence of Early Colon Cancer After Surgery

doi: 10.2147/IJGM.S436588

Figure Lengend Snippet: ( A ) Representative immunohistochemical staining images of CST6 and CBX4 expressions; ( B ) statistical chart for HOXA-AS3, CST6 and CBX4. ***P<0.001.

Article Snippet: After that, they were incubated with primary antibody against CST6 (Proteintech, USA, 17076-1-AP) and rabbit anti-human CBX4 polyclonal antibody (Elabscience Biotechnology Co., Ltd., China, E-AB-17696) at 4°C overnight, followed by addition of horseradish peroxidase-labeled secondary antibody polymer in appropriate drops and incubation for 50 min in a greenhouse.

Techniques: Immunohistochemical staining, Staining

Journal: International Journal of General Medicine

Article Title: Predictive Values of Homeobox Gene A-Antisense Transcript 3 (HOXA-AS3), Cystatin 6 (CST6), and Chromobox Homolog 4 (CBX4) Expressions in Cancer Tissues for Recurrence of Early Colon Cancer After Surgery

doi: 10.2147/IJGM.S436588

Figure Lengend Snippet: Correlations Between HOXA-AS3, CST6, and CBX4 Expressions and Their Correlations with Recurrence of Early Colon Cancer After Surgery

Article Snippet: After that, they were incubated with primary antibody against CST6 (Proteintech, USA, 17076-1-AP) and rabbit anti-human CBX4 polyclonal antibody (Elabscience Biotechnology Co., Ltd., China, E-AB-17696) at 4°C overnight, followed by addition of horseradish peroxidase-labeled secondary antibody polymer in appropriate drops and incubation for 50 min in a greenhouse.

Techniques: Expressing

Journal: International Journal of General Medicine

Article Title: Predictive Values of Homeobox Gene A-Antisense Transcript 3 (HOXA-AS3), Cystatin 6 (CST6), and Chromobox Homolog 4 (CBX4) Expressions in Cancer Tissues for Recurrence of Early Colon Cancer After Surgery

doi: 10.2147/IJGM.S436588

Figure Lengend Snippet: Effects of HOXA-AS3, CST6, and CBX4 on Recurrence of Early Colon Cancer After Surgery

Article Snippet: After that, they were incubated with primary antibody against CST6 (Proteintech, USA, 17076-1-AP) and rabbit anti-human CBX4 polyclonal antibody (Elabscience Biotechnology Co., Ltd., China, E-AB-17696) at 4°C overnight, followed by addition of horseradish peroxidase-labeled secondary antibody polymer in appropriate drops and incubation for 50 min in a greenhouse.

Techniques: Standard Deviation, Expressing

Journal: International Journal of General Medicine

Article Title: Predictive Values of Homeobox Gene A-Antisense Transcript 3 (HOXA-AS3), Cystatin 6 (CST6), and Chromobox Homolog 4 (CBX4) Expressions in Cancer Tissues for Recurrence of Early Colon Cancer After Surgery

doi: 10.2147/IJGM.S436588

Figure Lengend Snippet: ROC curves of HOXA-AS3, CST6, and CBX4 expressions in predicting recurrence of early colon cancer after surgery.

Article Snippet: After that, they were incubated with primary antibody against CST6 (Proteintech, USA, 17076-1-AP) and rabbit anti-human CBX4 polyclonal antibody (Elabscience Biotechnology Co., Ltd., China, E-AB-17696) at 4°C overnight, followed by addition of horseradish peroxidase-labeled secondary antibody polymer in appropriate drops and incubation for 50 min in a greenhouse.

Techniques:

Journal: International Journal of General Medicine

Article Title: Predictive Values of Homeobox Gene A-Antisense Transcript 3 (HOXA-AS3), Cystatin 6 (CST6), and Chromobox Homolog 4 (CBX4) Expressions in Cancer Tissues for Recurrence of Early Colon Cancer After Surgery

doi: 10.2147/IJGM.S436588

Figure Lengend Snippet: Predictive Values of HOXA-AS3, CST6, and CBX4 Expressions for Recurrence of Early Colon Cancer After Surgery

Article Snippet: After that, they were incubated with primary antibody against CST6 (Proteintech, USA, 17076-1-AP) and rabbit anti-human CBX4 polyclonal antibody (Elabscience Biotechnology Co., Ltd., China, E-AB-17696) at 4°C overnight, followed by addition of horseradish peroxidase-labeled secondary antibody polymer in appropriate drops and incubation for 50 min in a greenhouse.

Techniques: Standard Deviation, Expressing