Journal: OncoTargets and therapy

Article Title: CD47 Overexpression Is Associated with Epstein–Barr Virus Infection and Poor Prognosis in Patients with Nasopharyngeal Carcinoma

doi: 10.2147/OTT.S245023

Figure Lengend Snippet: Associations of Clinicopathological Characteristics with CD47 and LMP1 Expression in 66 Non-Metastatic NPC Patients

Article Snippet: The sections were then washed in Tris-buffered saline (pH7.4) and incubated at 4°C overnight with anti-CD47 antibody (D3O7P, 63000s; 1:50; Cell Signaling Technology, Danvers, MA, USA) or anti-EBV latent membrane protein 1 (LMP1) antibody (1:50, ab78113, Abcam, Cambridge, MA, USA).

Techniques: Expressing

Journal: OncoTargets and therapy

Article Title: CD47 Overexpression Is Associated with Epstein–Barr Virus Infection and Poor Prognosis in Patients with Nasopharyngeal Carcinoma

doi: 10.2147/OTT.S245023

Figure Lengend Snippet: Immunohistochemical staining for CD47 and LMP1 in representative nasopharyngeal carcinoma (NPC) tissue samples. CD47-negative expression ( A ) and LMP1-positive expression ( B ) in # 1,600,231 NPC metastatic lymph node tissue; CD47-positive expression ( C ) and LMP1-negative expression in # 1,704,160 NPC nasopharyngeal tissue, ( D ); CD47-positive expression ( E ) and LMP1-positive expression ( F ) in # 1,508,532 NPC metastatic lymph node tissue.

Article Snippet: The sections were then washed in Tris-buffered saline (pH7.4) and incubated at 4°C overnight with anti-CD47 antibody (D3O7P, 63000s; 1:50; Cell Signaling Technology, Danvers, MA, USA) or anti-EBV latent membrane protein 1 (LMP1) antibody (1:50, ab78113, Abcam, Cambridge, MA, USA).

Techniques: Immunohistochemical staining, Staining, Expressing

Journal: OncoTargets and therapy

Article Title: CD47 Overexpression Is Associated with Epstein–Barr Virus Infection and Poor Prognosis in Patients with Nasopharyngeal Carcinoma

doi: 10.2147/OTT.S245023

Figure Lengend Snippet: Prognostic values of CD47 and LMP1 expression in nasopharyngeal carcinoma (NPC) patients. Kaplan–Meier survival analysis and Log rank test demonstrated the association between CD47-positive expression and both ( A ) median disease-free survival (mDFS) and ( B ) median overall survival (mOS) and the association between LMP1-positive expression and both ( C ) mDFS and ( D ) mOS.

Article Snippet: The sections were then washed in Tris-buffered saline (pH7.4) and incubated at 4°C overnight with anti-CD47 antibody (D3O7P, 63000s; 1:50; Cell Signaling Technology, Danvers, MA, USA) or anti-EBV latent membrane protein 1 (LMP1) antibody (1:50, ab78113, Abcam, Cambridge, MA, USA).

Techniques: Expressing

Journal: OncoTargets and therapy

Article Title: CD47 Overexpression Is Associated with Epstein–Barr Virus Infection and Poor Prognosis in Patients with Nasopharyngeal Carcinoma

doi: 10.2147/OTT.S245023

Figure Lengend Snippet: Univariate and Multivariate Analyses of Prognostic Factors Predicting Disease-Free Survival (DFS) in 66 Non-Metastatic NPC Patients

Article Snippet: The sections were then washed in Tris-buffered saline (pH7.4) and incubated at 4°C overnight with anti-CD47 antibody (D3O7P, 63000s; 1:50; Cell Signaling Technology, Danvers, MA, USA) or anti-EBV latent membrane protein 1 (LMP1) antibody (1:50, ab78113, Abcam, Cambridge, MA, USA).

Techniques: Expressing

Journal: OncoTargets and therapy

Article Title: CD47 Overexpression Is Associated with Epstein–Barr Virus Infection and Poor Prognosis in Patients with Nasopharyngeal Carcinoma

doi: 10.2147/OTT.S245023

Figure Lengend Snippet: Correlation Between the Expression of CD47 and LMP1

Article Snippet: The sections were then washed in Tris-buffered saline (pH7.4) and incubated at 4°C overnight with anti-CD47 antibody (D3O7P, 63000s; 1:50; Cell Signaling Technology, Danvers, MA, USA) or anti-EBV latent membrane protein 1 (LMP1) antibody (1:50, ab78113, Abcam, Cambridge, MA, USA).

Techniques: Expressing

Journal: OncoTargets and therapy

Article Title: CD47 Overexpression Is Associated with Epstein–Barr Virus Infection and Poor Prognosis in Patients with Nasopharyngeal Carcinoma

doi: 10.2147/OTT.S245023

Figure Lengend Snippet: CD47 was highly expressed in EBV-positive nasopharyngeal carcinoma (NPC) cell lines and was associated with EBV infection. ( A ) Protein expression level of CD47 and ( B ) relative expression level of CD47 mRNA in various NPC cell lines. ( C ) Protein expression levels of CD47 and LMP1 and relative expression level of CD47 mRNA in the constructed EBV-positive cell line (CNE-2-EBV+) and the EBV-negative parental cell line (CNE-2). ( D ) Protein expression levels of CD47 and LMP1 and relative expression level of CD47 mRNA in the CNE-2 cell line transfected with control vector or LMP1 plasmid.

Article Snippet: The sections were then washed in Tris-buffered saline (pH7.4) and incubated at 4°C overnight with anti-CD47 antibody (D3O7P, 63000s; 1:50; Cell Signaling Technology, Danvers, MA, USA) or anti-EBV latent membrane protein 1 (LMP1) antibody (1:50, ab78113, Abcam, Cambridge, MA, USA).

Techniques: Infection, Expressing, Construct, Transfection, Control, Plasmid Preparation

Journal: OncoTargets and therapy

Article Title: CD47 Overexpression Is Associated with Epstein–Barr Virus Infection and Poor Prognosis in Patients with Nasopharyngeal Carcinoma

doi: 10.2147/OTT.S245023

Figure Lengend Snippet: EBV upregulates CD47 expression in NPC cells, which is mediated by LMP1. Protein expression levels of CD47 and LMP1 after transfection of ( A ) CNE-2 and CNE2-EBV+ cell lines and ( B ) CNE-2-vector and CNE-2-LMP1 stable cell lines with Mock-siRNA or LMP1-siRNA.

Article Snippet: The sections were then washed in Tris-buffered saline (pH7.4) and incubated at 4°C overnight with anti-CD47 antibody (D3O7P, 63000s; 1:50; Cell Signaling Technology, Danvers, MA, USA) or anti-EBV latent membrane protein 1 (LMP1) antibody (1:50, ab78113, Abcam, Cambridge, MA, USA).

Techniques: Expressing, Transfection, Plasmid Preparation, Stable Transfection

Journal: Cell reports

Article Title: SOX10 requirement for melanoma tumor growth is due, in part, to immune-mediated effects

doi: 10.1016/j.celrep.2021.110085

Figure Lengend Snippet:

Article Snippet: For detection of human SOX10 (#D5V9L) and CD47 (#D3O7P), mouse MITF (#D5G7V), mouse ZEB1 (#E2G6Y), or human and mouse CEACAM1 (#D1P4T) antibodies were purchased from Cell Signaling Technology (Danver, MA).

Techniques: Control, Recombinant, CRISPR, Knockdown, Western Blot, Generated, Software

Journal: Cell reports

Article Title: SOX10 requirement for melanoma tumor growth is due, in part, to immune-mediated effects

doi: 10.1016/j.celrep.2021.110085

Figure Lengend Snippet:

Article Snippet: For detection of human SOX10 (#D5V9L) and CD47 (#D3O7P), mouse MITF (#D5G7V), mouse ZEB1 (#E2G6Y), or human and mouse CEACAM1 (#D1P4T) antibodies were purchased from Cell Signaling Technology (Danver, MA).

Techniques: Control, Recombinant, CRISPR, Knockdown, Western Blot, Generated, Software

Journal: Cell reports

Article Title: SOX10 requirement for melanoma tumor growth is due, in part, to immune-mediated effects

doi: 10.1016/j.celrep.2021.110085

Figure Lengend Snippet: (A) SOX10 expression was probed by western blot in parental mouse melanoma YUMM1.1 cells and Sox10 knockout clones. (B) SOX10 expression was probed by western blot in parental human melanoma MeWo cells and SOX10 knockout clones. (C) RNA was collected from 3 biological replicates of parental and Sox10 knockout clones, RNA-seq was performed, and data were mined for immune checkpoint proteins. Shown are fold changes of mRNA levels in Sox10 knockout clone #1.41 (CR. #1.41) or Sox10 knockout clone #1.51 (CR. #1.51) versus parental. *p < 1.0E -5; #p < 0.01. (D) RNA-seq was performed on parental and SOX10 knockout clones, and data were mined for immune checkpoint proteins. Shown are fold changes of mRNA levels across SOX10 knockout clones #2.1, 2.2, and 2.8 versus parental (CR. #2.1, 2, and 8) or SOX10 knockout clone #4.11 (CR. #4.11). *p < 1.0E -5; #p < 0.01. (E) Differentially expressed genes in YUMM1.1 Sox10 knockout cells compared to those in parental cells were plotted by gene rank against log2 fold change.

Article Snippet: For detection of human SOX10 (#D5V9L) and CD47 (#D3O7P), mouse MITF (#D5G7V), mouse ZEB1 (#E2G6Y), or human and mouse CEACAM1 (#D1P4T) antibodies were purchased from Cell Signaling Technology (Danver, MA).

Techniques: Expressing, Western Blot, Knock-Out, Clone Assay, RNA Sequencing

Journal: Cell reports

Article Title: SOX10 requirement for melanoma tumor growth is due, in part, to immune-mediated effects

doi: 10.1016/j.celrep.2021.110085

Figure Lengend Snippet: (A) Parental mouse melanoma YUMM1.1 cells and Sox10 knockout clones were treated with or without 100 ng/mL IFN γ , and HVEM expression was probed by flow cytometry staining. Mean fluorescence intensity (MFI) was quantified. (B) Parental human melanoma MeWo cells and SOX10 knockout clones were treated with or without 100 ng/mL IFN γ . HVEM expression was probed by flow cytometry staining, and MFI was quantified. (C) Parental mouse melanoma YUMM1.1 cells and Sox10 knockout clones were treated with or without 100 ng/mL IFN γ . CEACAM1 expression was probed by flow cytometry staining, and MFI was quantified. (D) Parental human melanoma MeWo cells and SOX10 knockout clones were treated with or without 100 ng/mL IFN γ . CEACAM1 expression was probed by flow cytometry staining, and MFI was quantified. (E) CEACAM1 expression was probed by western blot in parental mouse melanoma YUMM1.1 cells and Sox10 knockout clones. The arrow indicates a nonspecific band ("n.s."). (F) CEACAM1 expression was probed by western blot in parental human melanoma MeWo cells and SOX10 knockout clones. All data in this figure represent 3 biological replicates; *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: For detection of human SOX10 (#D5V9L) and CD47 (#D3O7P), mouse MITF (#D5G7V), mouse ZEB1 (#E2G6Y), or human and mouse CEACAM1 (#D1P4T) antibodies were purchased from Cell Signaling Technology (Danver, MA).

Techniques: Knock-Out, Clone Assay, Expressing, Flow Cytometry, Staining, Fluorescence, Western Blot

Journal: Cell reports

Article Title: SOX10 requirement for melanoma tumor growth is due, in part, to immune-mediated effects

doi: 10.1016/j.celrep.2021.110085

Figure Lengend Snippet: (A) RNA-seq from the melanoma TCGA dataset was visualized using cBioportal, and the mRNA expression level of HVEM ( TNFRSF14 ) was plotted versus SOX10 expression ( ; ). Shown are values batch normalized from Illumina HiSeq_RNASeqV2. SOX10 mutation type, structural variant, and copy number are indicated for each sample. (B) RNA-seq from the melanoma TCGA dataset was visualized using cBioportal, and the mRNA expression level of CEACAM1 was plotted versus SOX10 express ion ( ; ). Shown are values batch normalized from Illumina HiSeq_RNASeqV2. SOX10 mutation type, structural variant, and copy number are indicated for each sample. (C) Single-cell RNA-seq data of malignant cells were visualized by t-distributed stochastic neighbor embedding (tSNE) plots by using the Single Cell portal ( https://singlecell.broadinstitute.org/single_cell ), whereby each cluster represents a distinct melanoma tumor and each circle represents an individual cell.

Article Snippet: For detection of human SOX10 (#D5V9L) and CD47 (#D3O7P), mouse MITF (#D5G7V), mouse ZEB1 (#E2G6Y), or human and mouse CEACAM1 (#D1P4T) antibodies were purchased from Cell Signaling Technology (Danver, MA).

Techniques: RNA Sequencing, Expressing, Mutagenesis, Variant Assay

Journal: Cell reports

Article Title: SOX10 requirement for melanoma tumor growth is due, in part, to immune-mediated effects

doi: 10.1016/j.celrep.2021.110085

Figure Lengend Snippet: (A) CRISPR-Cas9 was used to knockout Sox10 from the mouse melanoma cell line YUMM1.1. Cell lysates were probed by western blot, and Sox10 knockout was verified in two individual clones. (B) In vitro cell growth of Sox10 knockout clones was evaluated using the IncuCyte live-cell imager. Data are representative of 3 independent experiments, each with 3 technical replicates. (C) YUMM1.1 parental or Sox10 knockout cells were injected into NSG mice. Tumors were measured using digital caliper every 2–3 days. Data were collected from 5 mice per group. (D) Related to (C), mice were sacrificed when tumors exceeded 650 mm 3 in volume. Shown is a Kaplan-Meier survival curve. (E) YUMM1.1 parental or Sox10 knockout clones #1.41 or #1.51 were injected into C57BL/6 mice, and tumors were measured by caliper every 2–3 days. Data were collected from 5–6 mice per group. (F) Related to (E), mice were sacrificed when tumors exceeded 650 mm 3 in volume. Shown is a Kaplan-Meier survival curve. **p < 0.01.

Article Snippet: For detection of human SOX10 (#D5V9L) and CD47 (#D3O7P), mouse MITF (#D5G7V), mouse ZEB1 (#E2G6Y), or human and mouse CEACAM1 (#D1P4T) antibodies were purchased from Cell Signaling Technology (Danver, MA).

Techniques: CRISPR, Knock-Out, Western Blot, Clone Assay, In Vitro, Injection

Journal: Cell reports

Article Title: SOX10 requirement for melanoma tumor growth is due, in part, to immune-mediated effects

doi: 10.1016/j.celrep.2021.110085

Figure Lengend Snippet: (A) HVEM was expressed in YUMM1.1 parental and CR. #1.41 Sox10 knockout cells by lentiviral transduction. HVEM expression was validated by flow cytometry staining. Data are representative of 3 biological replicates. An unstained control is shown in light gray. (B) YUMM1.1 parental, CR. #1.41 Sox10 knockout cells, or HVEM-overexpressing cells were injected into BL6 mice, and tumors were measured by digital caliper every 2–3 days. Data were collected from 7 mice per group. (C) Related to (B), time-to-tumor onset was tracked. Tumors were considered fully formed when they reached ~50 mm 3 . (D) Two individual clones were generated from CRISPR-Cas9 knockout of Ceacam1 in YUMM1.1 cells. CEACAM1 surface expression was evaluated by flow cytometry. Data are representative of 3 biological replicates. An unstained control is shown in light gray. (E) YUMM1.1 parental, CR. #1.8, or CR. #1.30 Ceacam1 knockout cells were injected into BL6 mice, and tumors were measured by caliper every 2–3 days. Data were collected from 10 mice per group. (F) Related to (E), time-to-tumor onset was tracked. Tumors were considered fully formed when they reached ~50 mm 3 .**p < 0.01, ***p < 0.001.

Article Snippet: For detection of human SOX10 (#D5V9L) and CD47 (#D3O7P), mouse MITF (#D5G7V), mouse ZEB1 (#E2G6Y), or human and mouse CEACAM1 (#D1P4T) antibodies were purchased from Cell Signaling Technology (Danver, MA).

Techniques: Knock-Out, Transduction, Expressing, Flow Cytometry, Staining, Control, Injection, Clone Assay, Generated, CRISPR

Journal: Cell reports

Article Title: SOX10 requirement for melanoma tumor growth is due, in part, to immune-mediated effects

doi: 10.1016/j.celrep.2021.110085

Figure Lengend Snippet: (A) YUMM1.1 parental or Sox10 knockout clone #1.51 cells were injected into Rag1 knockout mice, and tumors were measured by caliper every 2–3 days. Data were collected from 6–7 mice per group. (B) BL6 mice were treated with 300 μg of either CD8-depleting antibody or the relevant isotype control 2 days before tumor inoculation, and twice per week throughout the course of the experiment. YUMM1.1 parental (n = 3) or Sox10 knockout clone #1.51 (n = 6) cells were injected into treated mice, and tumors were measured by caliper every 2–3 days. (C) Related to (A), (B), and , and . Tumors were measured every 2–3 days by caliper measurement, and time-to-tumor onset was tracked. Tumors were considered fully formed when they reached 50 mm 3 . (D) Related to (C), mice were sacrificed when tumors exceeded 650 mm 3 in volume. Shown is a Kaplan-Meier survival curve. **p < 0.01, ***p < 0.001.

Article Snippet: For detection of human SOX10 (#D5V9L) and CD47 (#D3O7P), mouse MITF (#D5G7V), mouse ZEB1 (#E2G6Y), or human and mouse CEACAM1 (#D1P4T) antibodies were purchased from Cell Signaling Technology (Danver, MA).

Techniques: Knock-Out, Injection, Control

Journal: Cell reports

Article Title: SOX10 requirement for melanoma tumor growth is due, in part, to immune-mediated effects

doi: 10.1016/j.celrep.2021.110085

Figure Lengend Snippet: (A) Melanoma immunohistochemistry (IHC) samples from TCGA were previously scored for lymphocyte density and lymphocyte distribution. These two scores were summed to give a lymphocyte score. A Spearman’s correlation was conducted to determine the relationship between SOX10 mRNA levels and lymphocyte scores. (B) Gene set enrichment analysis (GSEA) was performed on patient data from the cutaneous melanoma TCGA dataset to determine the relationship between SOX10 mRNA levels and Kyoto Encyclopedia of Genes and Genomes (KEGG) gene pathways. Shown are the pathways significantly negatively correlated with SOX10 expression and normalized enrichment scores (p < 0.05). In bold are immune-related pathways. (C) Shown are enrichment plots, related to (B).

Article Snippet: For detection of human SOX10 (#D5V9L) and CD47 (#D3O7P), mouse MITF (#D5G7V), mouse ZEB1 (#E2G6Y), or human and mouse CEACAM1 (#D1P4T) antibodies were purchased from Cell Signaling Technology (Danver, MA).

Techniques: Immunohistochemistry, Expressing

Journal: Cell reports

Article Title: SOX10 requirement for melanoma tumor growth is due, in part, to immune-mediated effects

doi: 10.1016/j.celrep.2021.110085

Figure Lengend Snippet:

Article Snippet: For detection of human SOX10 (#D5V9L) and CD47 (#D3O7P), mouse MITF (#D5G7V), mouse ZEB1 (#E2G6Y), or human and mouse CEACAM1 (#D1P4T) antibodies were purchased from Cell Signaling Technology (Danver, MA).

Techniques: Control, Recombinant, CRISPR, Knockdown, Western Blot, Generated, Software