psin ef1 puro vector  (Addgene inc)

Journal: International journal of medical sciences

Article Title: OCT4 promotes lung cancer progression through upregulation of VEGF-correlated chemokine-1.

doi: 10.7150/ijms.102505

Figure Lengend Snippet: Figure 1. Expression of OCT4 and VCC-1 in clinical lung adenocarcinoma tissues and lung cancer cell lines. (A-C) Immunohistochemical staining (A) and quantification (B, C) of OCT4 and VCC-1 expression in lung adenocarcinoma patients (Stage I, n = 3; Stage II, n = 3; Stage III, n = 3) and normal lung tissues (Normal), (Scale bars, 100 μm for OCT4; 20 μm for VCC-1). (D) Positive correlation between OCT4 and VCC-1 intensities (r = 0.4789, p = 0.0126, Pearson’s correlation coefficient). (E) Expression of OCT4 and VCC-1 in six lung cancer cell lines and HEL299 normal lung fibroblasts detected by immunoblot analysis. Expression of β-actin served as the loading control.

Article Snippet: Construction of OCT4 and VCC-1 expression vectors and generation of lentiviral vectors encoding OCT4 or VCC-1 shRNAs The lentiviral vector encoding human OCT4 (pSin-EF2-OCT4-Pur, Addgene plasmid 16579) was obtained from Addgene (http://www.addgene.org).

Techniques: Expressing, Immunohistochemical staining, Staining, Western Blot, Control

Journal: International journal of medical sciences

Article Title: OCT4 promotes lung cancer progression through upregulation of VEGF-correlated chemokine-1.

doi: 10.7150/ijms.102505

Figure Lengend Snippet: Figure 2. Overexpression of OCT4 increases, whereas knockdown of OCT4 decreases, VCC-1 expression in lung cancer cells. (A-C) Detection of OCT4 and VCC-1 expression. H1299 cells were transfected with 4 µg of pSin-EF2-OCT4-Pur (OCT4) or pSin-EF2-GFP-Pur (GFP), or mock-transfected (A), or with 1, 2, and 4 µg of pSin-EF2-OCT4-Pur or pSin-EF2-GFP-Pur (4 µg) (B), or transduced with lentiviral vectors expressing shRNAs specific to OCT4 (#1 or #2) or luciferase (Luc) (C). After 48 h, levels of OCT4 and VCC-1 mRNA were examined by qPCR (A, n = 4), and their protein levels were detected by immunoblotting (B, C, n = 3). Expression of β-actin served as the loading control. Representative immunoblots from three independent experiments (left panels) and quantitative analysis of OCT4 (middle panels) and VCC-1 (right panels) are shown. Ratios between the intensity of the bands corresponding to the indicated protein and those corresponding to β-actin were calculated, and ratios of control cells were

Article Snippet: Construction of OCT4 and VCC-1 expression vectors and generation of lentiviral vectors encoding OCT4 or VCC-1 shRNAs The lentiviral vector encoding human OCT4 (pSin-EF2-OCT4-Pur, Addgene plasmid 16579) was obtained from Addgene (http://www.addgene.org).

Techniques: Over Expression, Knockdown, Expressing, Transfection, Transduction, Luciferase, Western Blot, Control

Journal: International journal of medical sciences

Article Title: OCT4 promotes lung cancer progression through upregulation of VEGF-correlated chemokine-1.

doi: 10.7150/ijms.102505

Figure Lengend Snippet: Figure 3. OCT4 and VCC-1 promote TGF-β production in lung cancer cells. (A-C) Detection of TGF-β production by ELISA. H1299 cells were transfected with pCMV-Tag2B (Vector, 6 µg), pCMV-Tag2B-OCT4 (OCT4, 1, 2, 4, and 6 µg), pCMV-Tag2B-VCC-1 (VCC-1, 1, 2, 4, and 6 µg), or mock-transfected (A, B). A549 cells were transduced with lentiviral vectors expressing shRNAs specific to VCC-1 (#1 to #4) or luciferase (Luc) (C) for 48 h. Dose-dependent overexpression of OCT and VCC-1 in H1299 cells transfected with Flag-tagged OCT4 and VCC-1 expression vectors were verified by immunoblotting with the anti-Flag antibody, respectively (lower panels, A, B). The culture medium was analyzed for TGF-β production by ELISA (upper panels, A-C, n = 3). (D) IL-4 and VCC-1 proteins enhance TGF-β production. THP-1 cells were treated with PMA (5 ng/ml) for 48 h, and stimulated with recombinant IL-4 (90 ng/ml) or VCC-1 (5 nM) proteins for 24 h. Levels of TGF-β in the culture medium were determined by ELISA at 48 h post-treatment (n = 3). (E) Detection of VEGF in H1299 cells that had been transfected with pCMV-Tag2B-OCT4 (OCT4), pCMV-Tag2B (Vector), or mock-transfected for 48 h. The culture medium was analyzed for VEGF production by ELISA (n = 3). Note that overexpression of OCT4 does not affect VEGF expression.

Article Snippet: Construction of OCT4 and VCC-1 expression vectors and generation of lentiviral vectors encoding OCT4 or VCC-1 shRNAs The lentiviral vector encoding human OCT4 (pSin-EF2-OCT4-Pur, Addgene plasmid 16579) was obtained from Addgene (http://www.addgene.org).

Techniques: Enzyme-linked Immunosorbent Assay, Transfection, Plasmid Preparation, Transduction, Expressing, Luciferase, Over Expression, Western Blot, Recombinant

Journal: International journal of medical sciences

Article Title: OCT4 promotes lung cancer progression through upregulation of VEGF-correlated chemokine-1.

doi: 10.7150/ijms.102505

Figure Lengend Snippet: Figure 4. Lung cancer cells overexpressing OCT4 or VCC-1 attract the migration of macrophage-like THP-1 cells. (A, B) H1299 cells plated in the lower wells in the Boyden chambers were transfected with pCMV-Tag2B (Vector, 6 µg), pCMV-Tag2B-OCT4 (OCT4, 1, 2, 4, and 6 µg), pCMV-Tag2B-VCC-1 (VCC-1, 1, 2, 4, and 6 µg), or

Article Snippet: Construction of OCT4 and VCC-1 expression vectors and generation of lentiviral vectors encoding OCT4 or VCC-1 shRNAs The lentiviral vector encoding human OCT4 (pSin-EF2-OCT4-Pur, Addgene plasmid 16579) was obtained from Addgene (http://www.addgene.org).

Techniques: Migration, Transfection, Plasmid Preparation

Journal: International journal of medical sciences

Article Title: OCT4 promotes lung cancer progression through upregulation of VEGF-correlated chemokine-1.

doi: 10.7150/ijms.102505

Figure Lengend Snippet: Figure 5. Knockdown of VCC-1 in A549 lung cancer cells decreases tumor growth in a human tumor xenograft model. (A) Cell proliferative assay of VCC-1-knockdown (shVCC-1-1 or shVCC-1-2), shRNA control (shLuc), and parental A549 cells (n = 4). (B) Tumor volumes of mice bearing VCC-1-knockdown (shVCC-1-1 or shVCC-1-2) or control A549 tumors. Groups of four NOD/SCID mice were subcutaneously inoculated with 1 × 106 cells of VCC-1-knockdown or control A549 cells. Tumor volumes of the mice were monitored and measured to elucidate the influence of VCC-1 on tumor development. (C) A schematic representation of the OCT4-VCC-1 axis involved in lung cancer progression. OCT4 overexpression in lung cancer cells upregulates VCC-1, which drives tumor aggressiveness through TGF-β secretion and tumor-associated macrophage (TAM) recruitment. OCT4 overexpression in lung cancer cells also promotes M2 macrophage polarization by increasing macrophage colony-stimulating factor (M-CSF) production and enhancing tumor migration, growth, and metastasis. The impact of OCT4 on the upregulation of M-CSF (the pathways in gray color) has been described in our previous paper [4].

Article Snippet: Construction of OCT4 and VCC-1 expression vectors and generation of lentiviral vectors encoding OCT4 or VCC-1 shRNAs The lentiviral vector encoding human OCT4 (pSin-EF2-OCT4-Pur, Addgene plasmid 16579) was obtained from Addgene (http://www.addgene.org).

Techniques: Knockdown, shRNA, Control, Over Expression, Migration

Journal: International journal of medical sciences

Article Title: OCT4 promotes lung cancer progression through upregulation of VEGF-correlated chemokine-1.

doi: 10.7150/ijms.102505

Figure Lengend Snippet: Figure 2. Overexpression of OCT4 increases, whereas knockdown of OCT4 decreases, VCC-1 expression in lung cancer cells. (A-C) Detection of OCT4 and VCC-1 expression. H1299 cells were transfected with 4 µg of pSin-EF2-OCT4-Pur (OCT4) or pSin-EF2-GFP-Pur (GFP), or mock-transfected (A), or with 1, 2, and 4 µg of pSin-EF2-OCT4-Pur or pSin-EF2-GFP-Pur (4 µg) (B), or transduced with lentiviral vectors expressing shRNAs specific to OCT4 (#1 or #2) or luciferase (Luc) (C). After 48 h, levels of OCT4 and VCC-1 mRNA were examined by qPCR (A, n = 4), and their protein levels were detected by immunoblotting (B, C, n = 3). Expression of β-actin served as the loading control. Representative immunoblots from three independent experiments (left panels) and quantitative analysis of OCT4 (middle panels) and VCC-1 (right panels) are shown. Ratios between the intensity of the bands corresponding to the indicated protein and those corresponding to β-actin were calculated, and ratios of control cells were

Article Snippet: Construction of OCT4 and VCC-1 expression vectors and generation of lentiviral vectors encoding OCT4 or VCC-1 shRNAs The lentiviral vector encoding human OCT4 (pSin-EF2-OCT4-Pur, Addgene plasmid 16579) was obtained from Addgene (http://www.addgene.org).

Techniques: Over Expression, Knockdown, Expressing, Transfection, Transduction, Luciferase, Western Blot, Control

Journal: International journal of medical sciences

Article Title: OCT4 promotes lung cancer progression through upregulation of VEGF-correlated chemokine-1.

doi: 10.7150/ijms.102505

Figure Lengend Snippet: Figure 3. OCT4 and VCC-1 promote TGF-β production in lung cancer cells. (A-C) Detection of TGF-β production by ELISA. H1299 cells were transfected with pCMV-Tag2B (Vector, 6 µg), pCMV-Tag2B-OCT4 (OCT4, 1, 2, 4, and 6 µg), pCMV-Tag2B-VCC-1 (VCC-1, 1, 2, 4, and 6 µg), or mock-transfected (A, B). A549 cells were transduced with lentiviral vectors expressing shRNAs specific to VCC-1 (#1 to #4) or luciferase (Luc) (C) for 48 h. Dose-dependent overexpression of OCT and VCC-1 in H1299 cells transfected with Flag-tagged OCT4 and VCC-1 expression vectors were verified by immunoblotting with the anti-Flag antibody, respectively (lower panels, A, B). The culture medium was analyzed for TGF-β production by ELISA (upper panels, A-C, n = 3). (D) IL-4 and VCC-1 proteins enhance TGF-β production. THP-1 cells were treated with PMA (5 ng/ml) for 48 h, and stimulated with recombinant IL-4 (90 ng/ml) or VCC-1 (5 nM) proteins for 24 h. Levels of TGF-β in the culture medium were determined by ELISA at 48 h post-treatment (n = 3). (E) Detection of VEGF in H1299 cells that had been transfected with pCMV-Tag2B-OCT4 (OCT4), pCMV-Tag2B (Vector), or mock-transfected for 48 h. The culture medium was analyzed for VEGF production by ELISA (n = 3). Note that overexpression of OCT4 does not affect VEGF expression.

Article Snippet: Construction of OCT4 and VCC-1 expression vectors and generation of lentiviral vectors encoding OCT4 or VCC-1 shRNAs The lentiviral vector encoding human OCT4 (pSin-EF2-OCT4-Pur, Addgene plasmid 16579) was obtained from Addgene (http://www.addgene.org).

Techniques: Enzyme-linked Immunosorbent Assay, Transfection, Plasmid Preparation, Transduction, Expressing, Luciferase, Over Expression, Western Blot, Recombinant