lentiviral vectors encoding oct4  (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: BMC Cancer

Article Title: Oct4 upregulates osteopontin via Egr1 and is associated with poor outcome in human lung cancer

doi: 10.1186/s12885-019-6014-5

Figure Lengend Snippet: Oct4 binds to the Egr1 promoter to transactivate Egr1 in lung cancer cells. a , H1299 cells that had been cotransfected with pGL2-Egr1p and pTCY-Renilla for 24 h were transduced with LV.Oct4 or LV.Null (left panel). A549 cells stably overexpressing Oct4 and vector control cells were cotransfected with pGL2-Egr1p and pTCY-Renilla (right panel). After 48 h, cell lysates were harvested, and their firefly and Renilla luciferase activities were determined. The ratio of firefly luciferase activity to Renilla luciferase activity of vector control cells was set to 100, and the values are presented as relative promoter activities. b , Schematic representation of two deletion constructions containing different lengths of the Egr1 promoter (left panel). Numbering is relative to the translational start site at + 1. H1299 cells were cotransfected with full-length or different deletion constructs of the Egr1 promoter and pTCY-Renilla. After 24 h, the cells were transfected with pCMV-tag2B-Oct4 or the control vector pCMV-tag2B. Cell lysates were assessed for firefly and Renilla luciferase activities 48 h later. RLU, relative luciferase unit. c , ChIP analysis showing direct binding of Oct4 to the Oct4 response element (ORE)-containing region in the Egr1 promoter. Cross-linked chromatin of A549 cells was immunoprecipitated with anti-Oct4 or anti-IgG antibody combined with protein G agarose beads, followed by PCR amplification of the Egr1 promoter region encompassing the ORE (between − 309 to − 413 bp) region. Data are mean ± SEM. *, p < 0.05; ***, p < 0.001

Article Snippet: The lentiviral vector pSin-EF2-Oct4-Pur encoding Oct4 was purchased from Addgene (Cambridge, MA, USA), and the control vector pSin-EF2-Pur encoding no transgenes was described previously 18 .

Techniques: Transduction, Stable Transfection, Plasmid Preparation, Luciferase, Activity Assay, Construct, Transfection, Binding Assay, Immunoprecipitation, Amplification

Journal: BMC Cancer

Article Title: Oct4 upregulates osteopontin via Egr1 and is associated with poor outcome in human lung cancer

doi: 10.1186/s12885-019-6014-5

Figure Lengend Snippet: Oct4, Egr1, and OPN are overexpressed in lung cancer and Egr1 expression is correlated with shorter survival. a , Representative staining for Oct4, Egr1, and OPN in human lung tumors graded as low and high expression. b , Numbers of tumors expressing high or low levels of Egr1 in tumors with high or low Oct4 expression. c . Numbers of tumors expressing high or low levels of OPN in tumors with high or low Egr1 expression. d , Tumor recurrence rates between high- and low- expression of Oct4, Egr1, and OPN in lung cancer patients, none of them reached statistical significance (Oct4: p = 0.273; Egr1: 0.261; OPN: 0.253). e - g , Kaplan-Meier curves for disease-free survivals in patients with high (red line) and low (black line) Oct4 ( e ), Egr1 ( f ), and OPN ( g ) expression

Article Snippet: The lentiviral vector pSin-EF2-Oct4-Pur encoding Oct4 was purchased from Addgene (Cambridge, MA, USA), and the control vector pSin-EF2-Pur encoding no transgenes was described previously 18 .

Techniques: Expressing, Staining

Journal: BMC Cancer

Article Title: Oct4 upregulates osteopontin via Egr1 and is associated with poor outcome in human lung cancer

doi: 10.1186/s12885-019-6014-5

Figure Lengend Snippet: Overexpression of Oct4 enhances Egr1 expression, OPN production, and migratory capability of human lung cancer cells. a and b , H1299 cells (left panel) were transiently transfected with 6 μg of pSin-EF2-Oct4-Pur or the control plasmid pSin-EF2-Pur, and their RNA ( a ) and protein ( b ) levels of Oct4 and Egr1 were analyzed at 48 post-transfection by RT-PCR ( a ) and immunoblot ( b ) analysis. A549 cells (right panel) stably overexpressing Oct4 and vector control cells were examined for Oct4 and Egr1 expression by RT-PCR ( a ) and immunoblot ( b ) analysis. Expression of GAPDH ( a ) and β-actin ( b ) served as the loading control. c , The levels of OPN in the supernatants of Oct4-overexpressing and control A549 cells were quantified by ELISA. d , Migratory capabilities of A549-Oct4 and A549-vector cells were detected by a Boyden chamber assay. Cells that migrated through the membrane of the lower surface in the Boyden chamber were stained with Giemsa solution and visualized by light microscopy and photographed (left panel), and three fields in each membrane were counted (right panel). Data are mean ± SEM. **, p < 0.01

Article Snippet: The lentiviral vector pSin-EF2-Oct4-Pur encoding Oct4 was purchased from Addgene (Cambridge, MA, USA), and the control vector pSin-EF2-Pur encoding no transgenes was described previously 18 .

Techniques: Over Expression, Expressing, Transfection, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Western Blot, Stable Transfection, Enzyme-linked Immunosorbent Assay, Boyden Chamber Assay, Staining, Light Microscopy

Journal: BMC Cancer

Article Title: Oct4 upregulates osteopontin via Egr1 and is associated with poor outcome in human lung cancer

doi: 10.1186/s12885-019-6014-5

Figure Lengend Snippet: Oct4-overexpressing lung tumors express higher levels of Egr1 in a mouse tumor model, and knockdown of Oct4 reduces the migratory capability of lung cancer cells. a , Immunohistochemical staining for Oct4, Egr1 and OPN in the tumors excised at day 60 from NOD/SCID mice that had been inoculated with 5 × 10 6 of A549-Oct4 cells via the tail vein. b , Lung tissues of tumor-bearing mice were harvested, and the number of metastatic nodules was counted. Data are mean ± SEM. *, p < 0.05; **, p < 0.01

Article Snippet: The lentiviral vector pSin-EF2-Oct4-Pur encoding Oct4 was purchased from Addgene (Cambridge, MA, USA), and the control vector pSin-EF2-Pur encoding no transgenes was described previously 18 .

Techniques: Immunohistochemical staining, Staining

Journal: BMC Cancer

Article Title: Oct4 upregulates osteopontin via Egr1 and is associated with poor outcome in human lung cancer

doi: 10.1186/s12885-019-6014-5

Figure Lengend Snippet: Knockdown of Egr1 reduced the levels of OPN secreted into the culture medium and decreased the migratory capability. a , Levels of OPN in the supernatants of Egr1 knockdown and control A549-Oct4 cells were quantified by ELISA. b , Migratory capabilities of Egr1 knockdown or control A549-Oct4 cells via lentivirus-mediated delivery of shRNA for 48 h. Cells that migrated through the membrane to the lower surface in the Boyden chamber were stained with Giemsa solution and visualized by light microscopy and photographed (left panel), and three fields in each membrane were counted (right panel). Data are mean ± SEM. *, p < 0.05; **, p < 0.01

Article Snippet: The lentiviral vector pSin-EF2-Oct4-Pur encoding Oct4 was purchased from Addgene (Cambridge, MA, USA), and the control vector pSin-EF2-Pur encoding no transgenes was described previously 18 .

Techniques: Enzyme-linked Immunosorbent Assay, shRNA, Staining, Light Microscopy