Journal: International Journal of Oncology

Article Title: BST2 regulated by the transcription factor STAT1 can promote metastasis, invasion and proliferation of oral squamous cell carcinoma via the AKT/ERK1/2 signaling pathway

doi: 10.3892/ijo.2023.5502

Figure Lengend Snippet: si-RNA sequences.

Article Snippet: The membranes were then incubated overnight with primary antibodies against STAT1 (1:1,000; cat. no. 10144-2-AP; ProteinTech Group, Inc.), phosphorylated (p)-STAT1 (1:1,000; cat. no. ab109461; Abcam), AKT (pan; 1:1,000; cat. no. 4691S; Cell Signaling Technology, Inc.), p-AKT (1:2,000; cat. no. 4060S; Cell Signaling Technology, Inc.), ERK1/2 (1:1,000; cat. no. 11257-1-AP; ProteinTech Group, Inc.), p-ERK1/2 (1:1,000; cat. no. 28733-1-AP-ProteinTech Group, Inc.) or β-actin (1:2,000; cat. no. 20536-1-AP; ProteinTech Group, Inc.) at 4°C.

Techniques: Sequencing, Negative Control

Journal: International Journal of Oncology

Article Title: BST2 regulated by the transcription factor STAT1 can promote metastasis, invasion and proliferation of oral squamous cell carcinoma via the AKT/ERK1/2 signaling pathway

doi: 10.3892/ijo.2023.5502

Figure Lengend Snippet: Sequences of primers used for reverse transcription-quantitative PCR.

Article Snippet: The membranes were then incubated overnight with primary antibodies against STAT1 (1:1,000; cat. no. 10144-2-AP; ProteinTech Group, Inc.), phosphorylated (p)-STAT1 (1:1,000; cat. no. ab109461; Abcam), AKT (pan; 1:1,000; cat. no. 4691S; Cell Signaling Technology, Inc.), p-AKT (1:2,000; cat. no. 4060S; Cell Signaling Technology, Inc.), ERK1/2 (1:1,000; cat. no. 11257-1-AP; ProteinTech Group, Inc.), p-ERK1/2 (1:1,000; cat. no. 28733-1-AP-ProteinTech Group, Inc.) or β-actin (1:2,000; cat. no. 20536-1-AP; ProteinTech Group, Inc.) at 4°C.

Techniques: Sequencing

Journal: International Journal of Oncology

Article Title: BST2 regulated by the transcription factor STAT1 can promote metastasis, invasion and proliferation of oral squamous cell carcinoma via the AKT/ERK1/2 signaling pathway

doi: 10.3892/ijo.2023.5502

Figure Lengend Snippet: STAT1 binds to the BST2 promoter to regulate BST2 expression in oral squamous cell carcinoma. (A) JASPAR and UCSC databases were used to predict the binding sites between STAT1 and the BST2 promoter. (B) mRNA expression levels of STAT1 and BST2 in SCC-15 cell lines were assessed using reverse transcription-quantitative PCR after regulation of STAT1 or BST2. (C) Relative luciferase activity was assessed using the dual-luciferase reporter assay in SCC-15 cells co-transfected with oe-STAT1 or oe-NC and BST2-WT or BST2-MUT. ** P<0.01 and *** P<0.001. BST2, bone marrow stromal antigen 2; oe, overexpression; WT, wildtype; MUT, mutant; NS, not significant; UCSC, University of California Santa Cruz Genome Browser.

Article Snippet: The membranes were then incubated overnight with primary antibodies against STAT1 (1:1,000; cat. no. 10144-2-AP; ProteinTech Group, Inc.), phosphorylated (p)-STAT1 (1:1,000; cat. no. ab109461; Abcam), AKT (pan; 1:1,000; cat. no. 4691S; Cell Signaling Technology, Inc.), p-AKT (1:2,000; cat. no. 4060S; Cell Signaling Technology, Inc.), ERK1/2 (1:1,000; cat. no. 11257-1-AP; ProteinTech Group, Inc.), p-ERK1/2 (1:1,000; cat. no. 28733-1-AP-ProteinTech Group, Inc.) or β-actin (1:2,000; cat. no. 20536-1-AP; ProteinTech Group, Inc.) at 4°C.

Techniques: Expressing, Binding Assay, Real-time Polymerase Chain Reaction, Luciferase, Activity Assay, Reporter Assay, Transfection, Over Expression, Mutagenesis

Journal: International Journal of Oncology

Article Title: BST2 regulated by the transcription factor STAT1 can promote metastasis, invasion and proliferation of oral squamous cell carcinoma via the AKT/ERK1/2 signaling pathway

doi: 10.3892/ijo.2023.5502

Figure Lengend Snippet: STAT1 is highly expressed in HNSC and OSCC cell lines. (A) The mRNA expression levels of STAT1 from the Gene Expression Profiling Interactive Analysis database in different tumors. (B) Differential expression of STAT1 between normal and tumor tissues (tumor tissues, n=519; normal tissues, n=44). (C) Kyoto Encyclopedia of Genes and Genomes pathway analysis performed using the linkedomics database. (D) The protein and mRNA expression levels of STAT1 in different OSCC cell lines. * P<0.05 and *** P<0.001. HNSC, head and neck squamous cell carcinoma; OSCC, oral squamous cell carcinoma; TPM, transcripts per million; FDR, false discovery rate; NS, not significant; T, tumor; N, normal.

Article Snippet: The membranes were then incubated overnight with primary antibodies against STAT1 (1:1,000; cat. no. 10144-2-AP; ProteinTech Group, Inc.), phosphorylated (p)-STAT1 (1:1,000; cat. no. ab109461; Abcam), AKT (pan; 1:1,000; cat. no. 4691S; Cell Signaling Technology, Inc.), p-AKT (1:2,000; cat. no. 4060S; Cell Signaling Technology, Inc.), ERK1/2 (1:1,000; cat. no. 11257-1-AP; ProteinTech Group, Inc.), p-ERK1/2 (1:1,000; cat. no. 28733-1-AP-ProteinTech Group, Inc.) or β-actin (1:2,000; cat. no. 20536-1-AP; ProteinTech Group, Inc.) at 4°C.

Techniques: Expressing

Journal: International Journal of Oncology

Article Title: BST2 regulated by the transcription factor STAT1 can promote metastasis, invasion and proliferation of oral squamous cell carcinoma via the AKT/ERK1/2 signaling pathway

doi: 10.3892/ijo.2023.5502

Figure Lengend Snippet: STAT1 positively regulates BST2/AKT/ERK1/2 and biological behavior of OSCC. (A) Western blotting analysis of the protein expression levels of STAT1, BST2, AKT and ERK1/2 and the extent of AKT and ERK1/2 phosphorylation after using si-RNA or oe plasmid to regulate STAT1 or BST2 in SCC-15 cells. (B) Cell scratch test assay (magnification, ×50), and (C) Transwell assay (magnification, ×100) were used to assess the migration and invasion ability of SCC-15 cells after STAT1 downregulation. (D) Clone formation and (E) cell proliferation assays were performed to assess changes in the viability and proliferation of SCC-15 cells after STAT1 downregulation. * P<0.05, ** P<0.01 and *** P<0.001. BST2, bone marrow stromal antigen 2; si, small interfering; oe, overexpression; NC, negative control; p, phosphorylated; NS, not significant.

Article Snippet: The membranes were then incubated overnight with primary antibodies against STAT1 (1:1,000; cat. no. 10144-2-AP; ProteinTech Group, Inc.), phosphorylated (p)-STAT1 (1:1,000; cat. no. ab109461; Abcam), AKT (pan; 1:1,000; cat. no. 4691S; Cell Signaling Technology, Inc.), p-AKT (1:2,000; cat. no. 4060S; Cell Signaling Technology, Inc.), ERK1/2 (1:1,000; cat. no. 11257-1-AP; ProteinTech Group, Inc.), p-ERK1/2 (1:1,000; cat. no. 28733-1-AP-ProteinTech Group, Inc.) or β-actin (1:2,000; cat. no. 20536-1-AP; ProteinTech Group, Inc.) at 4°C.

Techniques: Western Blot, Expressing, Plasmid Preparation, Wound Healing Assay, Transwell Assay, Migration, Over Expression, Negative Control

Journal: International Journal of Oncology

Article Title: BST2 regulated by the transcription factor STAT1 can promote metastasis, invasion and proliferation of oral squamous cell carcinoma via the AKT/ERK1/2 signaling pathway

doi: 10.3892/ijo.2023.5502

Figure Lengend Snippet: STAT1 and BST2 serve a synergistic role in the biological behavior of oral squamous cell carcinoma. (A) Western blotting analysis of the protein expression levels of STAT1, BST2, AKT, ERK1/2 and the extent of AKT and ERK1/2 phosphorylation after using si-RNA or oe plasmid to regulate STAT1 or BST2 in SCC-15 cells. (B) Cell scratch test assay (magnification, ×50), (C) Transwell assay (magnification, ×100) and (D) clone formation assay were used to assess the migration, invasion and proliferation of SCC-15 cells after using si-RNA or oe plasmid to regulate STAT1 or BST2. * P<0.05, ** P<0.01 and *** P<0.001. BST2, bone marrow stromal antigen 2; si, small interfering; oe, overexpression; NC, negative control; p, phosphorylated; NS, not significant.

Article Snippet: The membranes were then incubated overnight with primary antibodies against STAT1 (1:1,000; cat. no. 10144-2-AP; ProteinTech Group, Inc.), phosphorylated (p)-STAT1 (1:1,000; cat. no. ab109461; Abcam), AKT (pan; 1:1,000; cat. no. 4691S; Cell Signaling Technology, Inc.), p-AKT (1:2,000; cat. no. 4060S; Cell Signaling Technology, Inc.), ERK1/2 (1:1,000; cat. no. 11257-1-AP; ProteinTech Group, Inc.), p-ERK1/2 (1:1,000; cat. no. 28733-1-AP-ProteinTech Group, Inc.) or β-actin (1:2,000; cat. no. 20536-1-AP; ProteinTech Group, Inc.) at 4°C.

Techniques: Western Blot, Expressing, Plasmid Preparation, Wound Healing Assay, Transwell Assay, Tube Formation Assay, Migration, Over Expression, Negative Control

Journal: International Journal of Oncology

Article Title: BST2 regulated by the transcription factor STAT1 can promote metastasis, invasion and proliferation of oral squamous cell carcinoma via the AKT/ERK1/2 signaling pathway

doi: 10.3892/ijo.2023.5502

Figure Lengend Snippet: (A) Photograph of tumors excised from mice, growth curve of subcutaneous tumor volume, and the weight of mice. (B) The protein expression levels of STAT1, BST2, AKT and ERK1/2 and the extent of STAT1, AKT and ERK1/2 phosphorylation in vivo after downregulation of STAT1 (n=3 selected from each group) and mRNA expression levels of STAT1 and BST2. Representative images of (C) hematoxylin and eosin and (D) immunohistochemical staining for STAT1, p-STAT1 and BST2 of oral squamous cell carcinoma tumor samples (magnification, ×200). * P<0.05, ** P<0.01 and *** P<0.001. BST2, bone marrow stromal antigen 2; si, small interfering; oe, overexpression; NC, negative control; p, phosphorylated; NS, not significant.

Article Snippet: The membranes were then incubated overnight with primary antibodies against STAT1 (1:1,000; cat. no. 10144-2-AP; ProteinTech Group, Inc.), phosphorylated (p)-STAT1 (1:1,000; cat. no. ab109461; Abcam), AKT (pan; 1:1,000; cat. no. 4691S; Cell Signaling Technology, Inc.), p-AKT (1:2,000; cat. no. 4060S; Cell Signaling Technology, Inc.), ERK1/2 (1:1,000; cat. no. 11257-1-AP; ProteinTech Group, Inc.), p-ERK1/2 (1:1,000; cat. no. 28733-1-AP-ProteinTech Group, Inc.) or β-actin (1:2,000; cat. no. 20536-1-AP; ProteinTech Group, Inc.) at 4°C.

Techniques: Expressing, In Vivo, Immunohistochemical staining, Staining, Over Expression, Negative Control

Journal: Frontiers in Immunology

Article Title: Lentiviral Mediated ADA2 Gene Transfer Corrects the Defects Associated With Deficiency of Adenosine Deaminase Type 2

doi: 10.3389/fimmu.2022.852830

Figure Lengend Snippet: Lentivirus mediated ADA2 gene transfer restores ADA2 protein expression, enzyme activity and rescues immunophenotype in monocyte derived macrophages (MDM) from DADA2 patients (A) . Representative flow cytometric histogram showing ADA2 expression (MFI, median fluorescence intensity) in MDM obtained from patients with DADA2 and healthy controls. (B) EFS-GFP-ADA2 transduction of macrophage cells derived from patients with DADA2 resulted in restoration of ADA2 protein expression assessed by flow cytometry compared to no change in protein expression in EFS-GFP alone treated cells. (C) ADA2 enzyme activity assessed by an automated spectrophotometric assay also recovered in the culture supernatant of EFS-GFP-ADA2 transduced MDM from DADA2 patients compared to supernatants derived from EFS-GFP transduced cells. (D, E) EFS-GFP-ADA2 transduction of MDM derived from patients also led to a reduction in levels of TNF-α cytokine production in culture supernatants compared to EFS-GFP alone treated cells. Cumulative results and individual changes for each sample assessed are shown. (F, G) There was also suppression of IFN-γ release expression in culture supernatants and downregulation of p-STAT1 expression in EFS-GFP-ADA2 transduced MDM compared to EFS-GFP treated cells. (H) There was significant improvement in CD62E expression (MFI) on HUVEC incubated with culture supernatants from EFS-GFP-ADA2 transduced MDM from patients with DADA2 compared to co incubation with supernatants from EFS-GFP treated MDM. Dotted line represents levels observed in healthy control samples. MDM, monocyte derived macrophages; MFI, median fluorescence intensity; TNFα, tumour necrosis factor-α; IL, interleukin; EFS, elongation factor 1α short; IFN, interferon; GFP, green fluorescent protein; HUVEC, human umbilical vein endothelial cells; INF, interferon; UT, untransduced: ADA2, adenosine deaminase 2.

Article Snippet: Cells were stained with BV421-anti-STAT1 pY701 (BD Bioscience, cat: 562985, 1:50) and cell surface markers APC-CD68 (Miltenyi Biotec, cat: 160-109-462, REA613, 1:30).

Techniques: Expressing, Activity Assay, Derivative Assay, Fluorescence, Transduction, Flow Cytometry, Spectrophotometric Assay, Incubation

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Role of KCa3.1 Channels in Macrophage Polarization and Its Relevance in Atherosclerotic Plaque Instability.

doi: 10.1161/ATVBAHA.116.308461

Figure Lengend Snippet: Figure 2. The role of KCa3.1 in M1 and M2 macrophage polarization in vitro. Macrophage colony-stimulating factor (M-CSF)–stimulated human macrophages were cotreated with vehicle or 100 nmol/L TRAM-34 during interferon (IFN)-γ and lipopolysaccharide LPS-induced M1 polarization and interleukin (IL)-4-induced M2 polarization. A, Flow cytometry analysis of cell surface markers for M1 (CD80 and CD197) and M2 (CD163 and CD206) macrophages during M1 and M2 polarization with or without TRAM-34. The flow cytometry results are expressed as MFI (n=3 in each group). B, Real-time polymerase chain reaction was performed to further evaluate the transcript expression of genes related to M1 and M2 macrophage polarization (n=5–6 in each group). C, Western blotting was conducted on cell lysates with antibodies for P-STAT1, P-STAT3, and P-STAT6; total STAT1, STAT3, and STAT6; and GAPDH. The relative densities for the phosphorylated STAT compared with total STAT are shown as histograms (n=4 in each group). Statistical significance values (P) are indi- cated. MFI indicates mean fluorescence intensity; and STAT, signal transducer and activator of transcription.

Article Snippet: The antibodies used targeted KCa3.1 (Abcam, cat. no. ab83740), P-701-STAT1 (Cell Signaling, cat. no. 7649), STAT1 (Cell Signaling, cat. no. 9172), P-705-STAT3 (Cell Signaling, cat. no. 9145), STAT3 (Cell Signaling, cat. no. 4904), STAT 6 (Cell Signaling, cat. no.5397), P-641-STAT6 (Cell Signaling, cat. no. 9361) and GAPDH (Santa Cruz, cat. no. sc-365062).

Techniques: In Vitro, Flow Cytometry, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Fluorescence

Journal: Cancer Management and Research

Article Title: Expression of Carboxypeptidase X M14 Family Member 2 Accelerates the Progression of Hepatocellular Carcinoma via Regulation of the gp130/JAK2/Stat1 Pathway

doi: 10.2147/CMAR.S228984

Figure Lengend Snippet: Effect of CPXM2 on the proliferation and metastasis of hepatic stellate cells in vitro. ( A ) Examination of the effect of CPXM2 on the phosphorylation levels of JAK2 and Stat1 in LX2 cells using Western blot analysis. ( B ) Relative protein expression level of CPXM2 and the phosphorylation status of JAK2 and Stat1 in LX2 cells. **P<0.01. ( C ) Examination of CPXM2 in LX2 cells using immunofluorescence. The CPXM2 protein was stained red (labeled with PE-conjugated secondary antibody), and the nucleus was stained blue (stained with DAPI). Scale bar, 20 µm. ( D ) Growth curves of LX2 cells transfected with a CPXM2 overexpression plasmid determined by Cell Counting Kit-8 assay. **P<0.01. ( E ) A plate colony formation assay was used to determine the effect of CPXM2 on the proliferative capacity of LX2 cells. **P<0.01. ( F ) Matrigel invasion assay was used to examine the effect of CPXM2 on the invasive capacity of LX2 cells in vitro (left), and corresponding statistical analysis of invasive cells (right). Scale bar, 20 µm; **P<0.01. ( G ) Wound-healing assay to detect the effect of CPXM2 on the migratory ability of LX2 cells in vitro (left), and the corresponding statistical analysis (right). **P<0.01. Abbreviations: OD, optical density; CPXM2, carboxypeptidase X, M14 family member 2; Stat1, signal transducer and activator of transcription 1; JAK2, Janus kinase 2.

Article Snippet: Then, the separated proteins were electro-transferred to polyvinylidene fluoride (PVDF) membranes and blocked with 5% skimmed milk diluted in TBS-Tween 20 buffer (TBST) at room temperature (RT) for 2 h. Subsequently, the PVDF membranes were probed with primary antibodies against human gp130 (cat. no. 9145, Cell Signaling Technology, Inc), Stat1 (cat. no. 9139, Cell Signaling Technology, Inc), phospho- Stat1 (cat. no. 9167, Cell Signaling Technology, Inc), phospho-JAK2 (cat. no. 68790, Cell Signaling Technology, Inc), JAK2 (cat. no. 13531, Cell Signaling Technology, Inc), CPXM2 (cat. no. ab201077, Abcam, USA) or β-actin (cat. no. ab8226, Abcam, USA) at a dilution of 1:1000 at 4°C overnight.

Techniques: In Vitro, Phospho-proteomics, Western Blot, Expressing, Immunofluorescence, Staining, Labeling, Transfection, Over Expression, Plasmid Preparation, Cell Counting, Colony Assay, Invasion Assay, Wound Healing Assay

Journal: Cancer Management and Research

Article Title: Expression of Carboxypeptidase X M14 Family Member 2 Accelerates the Progression of Hepatocellular Carcinoma via Regulation of the gp130/JAK2/Stat1 Pathway

doi: 10.2147/CMAR.S228984

Figure Lengend Snippet: The effects of shRNA-mediated silencing of CPXM2 expression in HCC cells. ( A ) Western blotting was used to examine the effects of CPXM2 silencing and the activation of the JAK2/Stat1 signaling pathway in Huh1 cells. ( B ) Corresponding statistical analysis of the activation of the JAK2/Stat1 signaling pathway. **P<0.01 vs scramble. ( C ) Growth curves of Huh1 cells determined using a Cell Counting Kit-8 assay. **P<0.01 vs scramble. ( D ) A colony formation assay was used to explore the proliferative capacity of Huh1 cells. **P<0.01 vs scramble. ( E ) A Matrigel invasion assay to examine the impact of CPXM2 silencing on the invasive ability of Huh1 cells in vitro, and corresponding statistical analysis of the number of invading cells. Scale bar, 20 µm. **P<0.01 vs scramble. ( F ) Wound-healing assay to determine the migratory ability of Huh1 cells in vitro (left), and the corresponding statistical analysis (right). **P<0.01 vs scramble. Abbreviations: shRNA, short hairpin RNA; HCC, hepatocellular carcinoma; CPXM2, carboxypeptidase X, M14 family member 2; Stat1, signal transducer and activator of transcription 1; JAK2, Janus kinase 2; p-, phosphorylated.

Article Snippet: Then, the separated proteins were electro-transferred to polyvinylidene fluoride (PVDF) membranes and blocked with 5% skimmed milk diluted in TBS-Tween 20 buffer (TBST) at room temperature (RT) for 2 h. Subsequently, the PVDF membranes were probed with primary antibodies against human gp130 (cat. no. 9145, Cell Signaling Technology, Inc), Stat1 (cat. no. 9139, Cell Signaling Technology, Inc), phospho- Stat1 (cat. no. 9167, Cell Signaling Technology, Inc), phospho-JAK2 (cat. no. 68790, Cell Signaling Technology, Inc), JAK2 (cat. no. 13531, Cell Signaling Technology, Inc), CPXM2 (cat. no. ab201077, Abcam, USA) or β-actin (cat. no. ab8226, Abcam, USA) at a dilution of 1:1000 at 4°C overnight.

Techniques: shRNA, Expressing, Western Blot, Activation Assay, Cell Counting, Colony Assay, Invasion Assay, In Vitro, Wound Healing Assay

Journal: Cancer Management and Research

Article Title: Expression of Carboxypeptidase X M14 Family Member 2 Accelerates the Progression of Hepatocellular Carcinoma via Regulation of the gp130/JAK2/Stat1 Pathway

doi: 10.2147/CMAR.S228984

Figure Lengend Snippet: shRNA was used to silence gp130 expression in CPXM2-expressing LX2 cells. ( A ) Western blotting was used to examine the effects of gp130 silencing on the phosphorylation status of JAK2 and Stat1 in CPXM2-expressing LX2 cells. **P<0.01 vs scramble. ( B ) Quantitative analysis of JAK2/Stat1 phosphorylation levels. **P<0.01 vs scramble. ( C ) Growth curves of LX2 cells determined using a Cell Counting Kit-8 assay. **P<0.01 vs scramble. ( D ) A colony formation assay was used to determine the proliferative capacity of LX2 cells with gp130 silenced. **P<0.01 vs scramble. ( E ) A Matrigel invasion assay was used to examine the impact of gp130 silencing on the invasiveness of LX2 cells in vitro (left), and corresponding statistical analysis of the invasiveness cells (right). Scale bar, 20 µm. **P<0.01 vs scramble. ( F ) A wound-healing assay was used to examine the migratory ability of the LX2 cell line in vitro (left), and corresponding statistical analysis (right). **P<0.01 vs scramble. Abbreviations: shRNA, short hairpin RNA; Stat1, signal transducer and activator of transcription 1; JAK2, Janus kinase 2; p-, phosphorylated; gp130, glycoprotein 130; CPXM2, carboxypeptidase X, M14 family member 2.

Article Snippet: Then, the separated proteins were electro-transferred to polyvinylidene fluoride (PVDF) membranes and blocked with 5% skimmed milk diluted in TBS-Tween 20 buffer (TBST) at room temperature (RT) for 2 h. Subsequently, the PVDF membranes were probed with primary antibodies against human gp130 (cat. no. 9145, Cell Signaling Technology, Inc), Stat1 (cat. no. 9139, Cell Signaling Technology, Inc), phospho- Stat1 (cat. no. 9167, Cell Signaling Technology, Inc), phospho-JAK2 (cat. no. 68790, Cell Signaling Technology, Inc), JAK2 (cat. no. 13531, Cell Signaling Technology, Inc), CPXM2 (cat. no. ab201077, Abcam, USA) or β-actin (cat. no. ab8226, Abcam, USA) at a dilution of 1:1000 at 4°C overnight.

Techniques: shRNA, Expressing, Western Blot, Phospho-proteomics, Cell Counting, Colony Assay, Invasion Assay, In Vitro, Wound Healing Assay