psin ef2 oct4 pur 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

Journal: Cell reports

Article Title: Hyperpolarization-activated cyclic nucleotide-gated cation channel 3 promotes HCC development in a female-biased manner

doi: 10.1016/j.celrep.2023.113157

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: pSin-EF2 vector , Yu et al. , Addgene Cat#16579; RRID:Addgene_16579.

Techniques: Virus, Recombinant, Lysis, Extraction, SYBR Green Assay, DNA Extraction, Plasmid Preparation, CCK-8 Assay, Activity Assay, Enzyme-linked Immunosorbent Assay, Reporter Assay, RNA Sequencing, Software

Journal: Cells

Article Title: The Pro-Survival Oct4/Stat1/Mcl-1 Axis Is Associated with Poor Prognosis in Lung Adenocarcinoma Patients

doi: 10.3390/cells10102642

Figure Lengend Snippet: Oct4 is highly expressed in human lung adenocarcinoma and is correlated with Stat1 expression. ( A , B ) Comparison of Oct4 ( A ) and Stat1 ( B ) expression in a normal human lung and lung adenocarcinoma (AC). ( C ) Correlation of Oct4 and Stat1 was analyzed by Pearson correlation coefficient in human lung adenocarcinoma. Data were obtained from Oncomine.org (accession no. GSE31210). Data are the mean ± S.E.M. ( D , E ) Kaplan–Meier analysis of relapse-free survival in lung cancer patients according to the expression levels of Oct4 ( D ) and Stat1 ( E ). H, high expression; L, low expression. ( F ) Kaplan–Meier analysis of relapse-free survival in lung cancer patients with high (H) or low (L) expression levels of both Oct4 and Stat1. Data were obtained from the Kaplan–Meier plotter database (Affymetrix ID: 208286_x_at (Oct4) and M97935_MB_at (Stat1); cut-off value, 190 (Oct4) and 360 (Stat1)). Statistical differences were analyzed by the log-rank test. * p < 0.05, ** p < 0.01 and *** p < 0.001.

Article Snippet: The lentiviral vector pSin-EF2-Oct4-Pur was purchased from Addgene (Cambridge, MA, USA) and the coding region of Oct4 was removed to generate the control vector pSin-EF2-Pur.

Techniques: Expressing

Journal: Cells

Article Title: The Pro-Survival Oct4/Stat1/Mcl-1 Axis Is Associated with Poor Prognosis in Lung Adenocarcinoma Patients

doi: 10.3390/cells10102642

Figure Lengend Snippet: Oct4 induces Stat1 expression in lung adenocarcinoma cells. ( A ) A549 and H1299 cells were cotransfected with pFRL2-Stat1p and pSin-EF2-Oct4-Pur and then cultured for 48 h. Luciferase activity was determined by a chemiluminescence analyzer. ( B ) RNA levels of Oct4 and Stat1 were detected by quantitative real-time RT-PCR in A549 and H1299 cells transduced with lentiviral vectors encoding Oct4. ( C ) Detection of Stat1 expression by immunoblotting in Oct4-overexpressing A549 and H1299 cells. Data are the mean ± S.E.M. ** p < 0.01 and *** p < 0.001.

Article Snippet: The lentiviral vector pSin-EF2-Oct4-Pur was purchased from Addgene (Cambridge, MA, USA) and the coding region of Oct4 was removed to generate the control vector pSin-EF2-Pur.

Techniques: Expressing, Cell Culture, Luciferase, Activity Assay, Quantitative RT-PCR, Transduction, Western Blot

Journal: Cells

Article Title: The Pro-Survival Oct4/Stat1/Mcl-1 Axis Is Associated with Poor Prognosis in Lung Adenocarcinoma Patients

doi: 10.3390/cells10102642

Figure Lengend Snippet: Oct4 directly binds to the Stat1 promoter to modulate Stat1 expression. ( A ) A549 cells were transfected with pSin-EF2-Oct4-Pur and different deletion forms of the Stat1 promoter, termed from -585 to +1545, from +364 to +1545, from +598 to +1545 and from +1064 to +1545, were transfected into A549 cells. Total cell lysates were collected at 48 h after transfection and their luciferase activities were determined by a chemiluminescence analyzer. ( B ) ChIP assay was performed in A549 cells using anti-Oct4 antibody. Normal IgG served as negative control. ( C ) The transactivation activity of wild-type and mutant Stat1 promoters was determined in Oct4-overexpressing A549 cells. Data are the mean ± S.E.M. ** p < 0.01 and *** p < 0.001.

Article Snippet: The lentiviral vector pSin-EF2-Oct4-Pur was purchased from Addgene (Cambridge, MA, USA) and the coding region of Oct4 was removed to generate the control vector pSin-EF2-Pur.

Techniques: Expressing, Transfection, Luciferase, Negative Control, Activity Assay, Mutagenesis

Journal: Cells

Article Title: The Pro-Survival Oct4/Stat1/Mcl-1 Axis Is Associated with Poor Prognosis in Lung Adenocarcinoma Patients

doi: 10.3390/cells10102642

Figure Lengend Snippet: Oct4 increases Mcl-1 expression. ( A ) Detection of total and phospho-Stat1 by immunoblotting in Oct4-overexpressing A549 cells. ( B ) Stat1 transactivation activity was determined using luciferase assay in Oct4-overexpressing cells. ( C ) Mcl-1 expression was examined in Oct4-overexpressing A549 cells. Data are the mean ± S.E.M. *** p < 0.001.

Article Snippet: The lentiviral vector pSin-EF2-Oct4-Pur was purchased from Addgene (Cambridge, MA, USA) and the coding region of Oct4 was removed to generate the control vector pSin-EF2-Pur.

Techniques: Expressing, Western Blot, Activity Assay, Luciferase

Journal: Cells

Article Title: The Pro-Survival Oct4/Stat1/Mcl-1 Axis Is Associated with Poor Prognosis in Lung Adenocarcinoma Patients

doi: 10.3390/cells10102642

Figure Lengend Snippet: Inhibition of Stat1 sensitizes lung cancer cells to cisplatin-induced apoptosis. ( A ) Detection of Mcl-1 by immunoblotting in A549 cells transduced with lentiviral vectors expressing shStat1. ( B ) Stat1-knockdown cells were treated with cisplatin (5 μg/mL) for 24 h. Cleavage of caspase-3 was examined by immunoblotting. ( C ) A549 cells were treated with the Stat1 inhibitor fludarabine (Flu, 2.5 μg/mL) for 24 h. Detection of Mcl-1, as well as total and phospho-Stat1, by immunoblotting. ( D ) Cleavage of caspase-3 was detected in Flu-treated A549 cells.

Article Snippet: The lentiviral vector pSin-EF2-Oct4-Pur was purchased from Addgene (Cambridge, MA, USA) and the coding region of Oct4 was removed to generate the control vector pSin-EF2-Pur.

Techniques: Inhibition, Western Blot, Transduction, Expressing

Journal: Cells

Article Title: The Pro-Survival Oct4/Stat1/Mcl-1 Axis Is Associated with Poor Prognosis in Lung Adenocarcinoma Patients

doi: 10.3390/cells10102642

Figure Lengend Snippet: Oct4 suppresses apoptosis through Stat1. ( A ) Oct4-overexpressing A549 and H1299 cells were transduced with lentiviral vectors expressing shStat1. Mcl-1 expression was examined by immunoblotting in the transduced cells. ( B ) After treatment with cisplatin (5 μg/mL), cleavage of caspase-3 was detected in the transduced cells. ( C , D ) Apoptotic cells were stained using TUNEL assay (C) and counted (D) in the transduced cells treated with cisplatin. Scale bar = 50 μm; original magnification, ×200. (E) Oct4-overexpressing A549 cells were treated with fludarabine (Flu, 2.5 μg/mL) for 24 h and cleaved caspase-3 was detected by immunoblotting. Data are the mean ± S.E.M. ** p < 0.01 and *** p < 0.001.

Article Snippet: The lentiviral vector pSin-EF2-Oct4-Pur was purchased from Addgene (Cambridge, MA, USA) and the coding region of Oct4 was removed to generate the control vector pSin-EF2-Pur.

Techniques: Transduction, Expressing, Western Blot, Staining, TUNEL Assay

Journal: Cells

Article Title: The Pro-Survival Oct4/Stat1/Mcl-1 Axis Is Associated with Poor Prognosis in Lung Adenocarcinoma Patients

doi: 10.3390/cells10102642

Figure Lengend Snippet: Oct4-promoted tumor growth is attenuated by reducing Stat1 expression. ( A ) NOD/SCID mice were subcutaneously inoculated with A549-Oct4 or A549-vector cells ( n = 9). The tumor volume was measured every 3 days. ( B ) Detection of Oct4 and Stat1 by immunohistochemical staining in tumor tissues. Scale bar = 50 μm; original magnification, ×200. ( C ) Tumor-bearing mice were intratumorally treated with 1 × 10 9 LPs of lentiviral vectors expressing shRNA specific to Stat1 at day 21 ( n = 9 for each group). Tumor growth was monitored three times a week. ( D ) Detection of Oct4 and Stat1 by immunohistochemical staining in tumor tissues. Scale bar = 50 μm; original magnification, ×200. ( E ) Tumor-bearing mice were intratumorally treated with fludarabine (Flu) at day 14 ( n = 8 for each group). ( F ) Detection of phospho-Stat1 by immunoblotting in tumor tissues. Data are the mean ± S.E.M. * p < 0.05 and *** p < 0.001.

Article Snippet: The lentiviral vector pSin-EF2-Oct4-Pur was purchased from Addgene (Cambridge, MA, USA) and the coding region of Oct4 was removed to generate the control vector pSin-EF2-Pur.

Techniques: Expressing, Plasmid Preparation, Immunohistochemical staining, Staining, shRNA, Western Blot