Journal: Gene Therapy

Article Title: Efficacy of combining ING4 and TRAIL genes in cancer-targeting gene virotherapy strategy: first evidence in preclinical hepatocellular carcinoma

doi: 10.1038/gt.2017.86

Figure Lengend Snippet: Validation of TRAIL and ING4 gene expression in human HCC cells treated with Ad-ΔB/TRAIL and/or Ad-ΔB/ING4. ( a ) Schematic representation of genomic structure of generated oncolytic adenovirus (Ad-ΔB) armed with human ING4 gene (Ad-ΔB/ING4) or human TRAIL gene (Ad-ΔB/TRAIL). ING4 or TRAIL gene was incorporated in the E3 region of the viral backbone under the transcriptional control of the human cytomegalovirus (CMV) promoter. Next, viral replication and gene expression pattern of TRAIL and ING4 was validated on human HCC HuH7 cells treated with PBS, Ad-ΔB/TRAIL, Ad-ΔB/ING4 or Ad-ΔB/TRAIL + ING4 at a MOI of 5 for each virus. The harvested cells and their supplements were analyzed by qRT-PCR to measure the relative mRNA of TRAIL ( b ) and ING4 ( d ), ELISA; to measure TRAIL protein ( c ) and western blot ( e and f ); to measure the expression level of ING4, TRAIL and Ad production by measuring E1A protein. Data of ( b – d ) are represented as mean±s.e. *** P <0.001 versus PBS.

Article Snippet: Thereafter, the membranes were incubated overnight at 4 °C with rabbit polyclonal primary antibodies (Santa Cruz Biotechnology, Inc.) against ING4, Ad E1A, TRAIL and β-actin.

Techniques: Biomarker Discovery, Gene Expression, Generated, Control, Virus, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing

Journal: Gene Therapy

Article Title: Efficacy of combining ING4 and TRAIL genes in cancer-targeting gene virotherapy strategy: first evidence in preclinical hepatocellular carcinoma

doi: 10.1038/gt.2017.86

Figure Lengend Snippet: In vitro effect of mono- and combined therapy with Ad-ΔB/TRAIL and Ad-ΔB/ING4 on human HCC and normal liver cells. ( a ) Quantitative results of methylthiazolyltetrazolium (MTT) assay showing the inhibitor effects on viability of human HCC (Hep3B) cells treated with 5 and 10 MOI per vector. ( b ) Quantitative results of MTT assay showing the inhibitor effects on viability of human HCC (HuH7) cells treated with 10 and 20 MOI per vector. Data are represented as mean±s.e. *** P <0.001 versus PBS. ( c ) Representatives of crystal violet staining assay demonstrating the cytotoxic effects on human HCC (Hep3B and HuH7) cells, and on normal human liver (WRL68) cells, at the indicated MOIs of each vector.

Article Snippet: Thereafter, the membranes were incubated overnight at 4 °C with rabbit polyclonal primary antibodies (Santa Cruz Biotechnology, Inc.) against ING4, Ad E1A, TRAIL and β-actin.

Techniques: In Vitro, MTT Assay, Plasmid Preparation, Staining

Journal: Gene Therapy

Article Title: Efficacy of combining ING4 and TRAIL genes in cancer-targeting gene virotherapy strategy: first evidence in preclinical hepatocellular carcinoma

doi: 10.1038/gt.2017.86

Figure Lengend Snippet: Antitumor effect of mono- and combined therapy with Ad-ΔB/TRAIL and Ad-ΔB/ING4 on orthotopic mouse of human HCC model. The model was established by intrahepatic implantation of 2 × 10 6 human HCC Hep3B/fluc cells into the liver lobes of athymic nude mice. As described in Methodology Part , confirmed positive HCC-bearing mice ( n =40) were randomly and equally assigned into four groups and systemically treated with PBS alone (Group 1), Ad-ΔB/TRAIL (Group 2), Ad-ΔB/ING4 (Group 3) and Ad-ΔB/TRAIL + ING4 (Group 4) at a dosage regimen of 1 × 10 10 VP in 200 μl PBS of each virus; repeated three times every other day. At day 3 post-treatment, mice of each group were further subdivided into two subgroups: the first subgroup (4 mice/group) was killed and their blood and liver tumor tissues were harvested and used for biochemical, histopathological and mechanistic explorations, while the second one (6 mice/group) was maintained and monitored by in vivo bioluminescence imaging (BLI) at days 0, 7, 14, 21, 28, and 35 post-treatment to determine the tumor response rate and the therapeutic effect of each tested treatment strategy. ( a ) Photographed BLI showing tumor growth and its response to each applied treatment. ( b ) BLI signals were calculated after background subtraction in total flux photons/s from a body region of interest. Data presented as mean±s.e. * P <0.05, ** P <0.01, *** P <0.001.

Article Snippet: Thereafter, the membranes were incubated overnight at 4 °C with rabbit polyclonal primary antibodies (Santa Cruz Biotechnology, Inc.) against ING4, Ad E1A, TRAIL and β-actin.

Techniques: Virus, In Vivo, Imaging

Journal: Gene Therapy

Article Title: Efficacy of combining ING4 and TRAIL genes in cancer-targeting gene virotherapy strategy: first evidence in preclinical hepatocellular carcinoma

doi: 10.1038/gt.2017.86

Figure Lengend Snippet: Validation of viral replication and TRAIL and ING4 gene expression in the harvested tumor tissues at day 3 post-treatment. The harvested liver tumor tissues of HCC-bearing mice treated with PBS, Ad-ΔB/TRAIL, Ad-ΔB/ING4 or Ad-ΔB/TRAIL+ ING4 were analyzed by qRT-PCR to measure the relative mRNA levels of TRAIL ( a ) and ING4 ( c ), while the expressed proteins of TRAIL ( b ) and ING4 ( d ) were measured using ELISA and western blot assay, respectively. The expression of Ad E1A protein (as an indicator of viral production) was also assessed by western blot assay ( d ). Data points of (a–c) are mean expression±s.e. *** P <0.001 versus PBS.

Article Snippet: Thereafter, the membranes were incubated overnight at 4 °C with rabbit polyclonal primary antibodies (Santa Cruz Biotechnology, Inc.) against ING4, Ad E1A, TRAIL and β-actin.

Techniques: Biomarker Discovery, Gene Expression, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing

Journal: Gene Therapy

Article Title: Efficacy of combining ING4 and TRAIL genes in cancer-targeting gene virotherapy strategy: first evidence in preclinical hepatocellular carcinoma

doi: 10.1038/gt.2017.86

Figure Lengend Snippet: Histopathological, immunological, antiangiogenic and TUNEL findings in the harvested tumor tissues at day 3 post-treatment. The harvested liver tumor tissues of HCC-bearing mice treated with PBS, Ad-ΔB/TRAIL, Ad-ΔB/ING4 or Ad-ΔB/TRAIL + ING4 were subjected to histopathological (H & E staining), immunohistochemical (IHC) staining and in situ apoptosis detection by TUNEL assay; and for ELISA assay for intratumoral levels of IFN-γ and VEGF. ( a ) Representative photographs of H & E staining (panel i); IHC staining of CD31-positive vascular endothelial cells (panel ii); and TUNEL staining of apoptotic cells (panel iii) in the harvested tumor tissues of different groups. Original magnification: × 400. ( b – d ) are relative intratumoral levels of IFN-γ, VEGF and microvascular density, respectively. Results are mean ± s.e. * P <0.05, *** P <0.001 versus PBS. ( e ) Tumor tissues were stained with anti-DX5 (green) and anti-IFN-γ (red) antibodies to assess intratumoral infiltration of NK cells and their activation. Original magnification, × 200. ( f ) The mean intensity of DX5-positive cells was quantified from three independent fields within five different microscope images for each experimental group. Data are representative of three independent experiments. * P <0.05, ** P <0.01 or *** P <0.001 versus PBS-treated group. ( g ) The mean intensity of IFN-γ-positive cells was quantified from three independent fields within five different microscope images for each experimental group. Data are representative of three independent experiments. ** P <0.01 or *** P <0.001 versus PBS-treated group. ( h ) The percentage of DX5 + IFN-γ + NK cells was assessed by ImageJ software. The mean intensity of DX5 + IFN-γ + NK cells was quantified from three independent fields within five different microscope images for each experimental group. Data are representative of three independent experiments. ** P <0.01 or *** P <0.001 versus PBS-treated group.

Article Snippet: Thereafter, the membranes were incubated overnight at 4 °C with rabbit polyclonal primary antibodies (Santa Cruz Biotechnology, Inc.) against ING4, Ad E1A, TRAIL and β-actin.

Techniques: TUNEL Assay, Staining, Immunohistochemical staining, Immunohistochemistry, In Situ, Enzyme-linked Immunosorbent Assay, Activation Assay, Microscopy, Software

Journal: Gene Therapy

Article Title: Efficacy of combining ING4 and TRAIL genes in cancer-targeting gene virotherapy strategy: first evidence in preclinical hepatocellular carcinoma

doi: 10.1038/gt.2017.86

Figure Lengend Snippet: In vivo biochemical findings

Article Snippet: Thereafter, the membranes were incubated overnight at 4 °C with rabbit polyclonal primary antibodies (Santa Cruz Biotechnology, Inc.) against ING4, Ad E1A, TRAIL and β-actin.

Techniques: In Vivo

Journal: Gene Therapy

Article Title: Efficacy of combining ING4 and TRAIL genes in cancer-targeting gene virotherapy strategy: first evidence in preclinical hepatocellular carcinoma

doi: 10.1038/gt.2017.86

Figure Lengend Snippet: In vitro apoptotic findings and expression pattern of caspases-8 and-3 in HCC cells and tumor tissues. ( a ) In vitro flow cytometric analysis of apoptosis induction in HCC cells treated with Ad-ΔB/TRAIL, Ad-ΔB/ING4 or Ad-ΔB/TRAIL + ING4 by using Annexin V and propidium iodide (PI) fluorescence staining assay. Each scatter plot demonstrates the percentage of early and late apoptotic cells. *** P <0.001 versus PBS. Next, western blotting analysis of human HCC cells ( b ) and harvested tumor tissues ( c ) treated with PBS, Ad-ΔB/TRAIL, Ad-ΔB/ING4 or Ad-ΔB/TRAIL + ING4 showing the expression pattern of caspase-8 and cleaved caspase-3.

Article Snippet: Thereafter, the membranes were incubated overnight at 4 °C with rabbit polyclonal primary antibodies (Santa Cruz Biotechnology, Inc.) against ING4, Ad E1A, TRAIL and β-actin.

Techniques: In Vitro, Expressing, Fluorescence, Staining, Western Blot

Journal: Pathobiology : journal of immunopathology, molecular and cellular biology

Article Title: Inhibitor of growth 4 induces growth suppression and apoptosis in glioma U87MG.

doi: 10.1159/000218334

Figure Lengend Snippet: Fig. 2. The transcription level of ING4 mRNA by RT-PCR in various grades of glioma tissues: glioblastoma (lane a); as- trocytoma (lane b); normal brain tissues (lane c).

Article Snippet: Materials and Methods Materials and Cell Line Therm RT-PCR/plat Taq (23191-050), Lipofectamine 2000 (LS11668-027), G-418 (INALCO1758-1811), TRIzol and MTT reagent were purchased from Invitrogen (Carlsbad, Calif., USA), In Situ Cell Death Detection Kit (11684817910) from Roche (Nutley, N.J., USA), SABC (goat-IgG)-POD kit from BOSTER (Wuhan, China), goat polyclonal antibody against ING4 (ab3714) from Abcam Inc. (Cambridge, Mass., USA), and the antibodies against Bcl-2 (SC-23960), Bax (SC-70407), Cyt-c (SC-81752), caspase 3 (SC-7148), PARP (SC-56197), Cox-2 (SC-7951), SKP2 (SC-74477), p27 (SC-1641) from Santa Cruz Inc. (Santa Cruz, Calif., USA).

Techniques: Reverse Transcription Polymerase Chain Reaction

Journal: Pathobiology : journal of immunopathology, molecular and cellular biology

Article Title: Inhibitor of growth 4 induces growth suppression and apoptosis in glioma U87MG.

doi: 10.1159/000218334

Figure Lengend Snippet: Fig. 3. ING4 expression was determined by indirect IF staining: strong stain and aggregation of the nucleus in U87MG/ pcDNA3.1-ING4 ( a ) and weak stain in U87MG/pcDNA3.1 ( c ). b , d The nucleus of U87MG/pcDNA3.1-ING4 and U87MG/ pcDNA3.1, respectively, counterstained with Hoechst.

Article Snippet: Materials and Methods Materials and Cell Line Therm RT-PCR/plat Taq (23191-050), Lipofectamine 2000 (LS11668-027), G-418 (INALCO1758-1811), TRIzol and MTT reagent were purchased from Invitrogen (Carlsbad, Calif., USA), In Situ Cell Death Detection Kit (11684817910) from Roche (Nutley, N.J., USA), SABC (goat-IgG)-POD kit from BOSTER (Wuhan, China), goat polyclonal antibody against ING4 (ab3714) from Abcam Inc. (Cambridge, Mass., USA), and the antibodies against Bcl-2 (SC-23960), Bax (SC-70407), Cyt-c (SC-81752), caspase 3 (SC-7148), PARP (SC-56197), Cox-2 (SC-7951), SKP2 (SC-74477), p27 (SC-1641) from Santa Cruz Inc. (Santa Cruz, Calif., USA).

Techniques: Expressing, Staining

Journal: Pathobiology : journal of immunopathology, molecular and cellular biology

Article Title: Inhibitor of growth 4 induces growth suppression and apoptosis in glioma U87MG.

doi: 10.1159/000218334

Figure Lengend Snippet: Fig. 4. ING4 expression was determined by Western blot analysis: the level of ING4 was significantly higher in U87MG/pcDNA3.1- ING4 (lane a) than U87MG/pcDNA3.1 (lane b) and U87MG (lane c).

Article Snippet: Materials and Methods Materials and Cell Line Therm RT-PCR/plat Taq (23191-050), Lipofectamine 2000 (LS11668-027), G-418 (INALCO1758-1811), TRIzol and MTT reagent were purchased from Invitrogen (Carlsbad, Calif., USA), In Situ Cell Death Detection Kit (11684817910) from Roche (Nutley, N.J., USA), SABC (goat-IgG)-POD kit from BOSTER (Wuhan, China), goat polyclonal antibody against ING4 (ab3714) from Abcam Inc. (Cambridge, Mass., USA), and the antibodies against Bcl-2 (SC-23960), Bax (SC-70407), Cyt-c (SC-81752), caspase 3 (SC-7148), PARP (SC-56197), Cox-2 (SC-7951), SKP2 (SC-74477), p27 (SC-1641) from Santa Cruz Inc. (Santa Cruz, Calif., USA).

Techniques: Expressing, Western Blot

Journal: Pathobiology : journal of immunopathology, molecular and cellular biology

Article Title: Inhibitor of growth 4 induces growth suppression and apoptosis in glioma U87MG.

doi: 10.1159/000218334

Figure Lengend Snippet: Fig. 6. Cell cycle analysis of transfected U87MG by FCM. Increased cell percentages in G1 phase and decreased cell percentages in S phase were found in U87MG/pcDNA3.1-ING4 compared with U87MG/pcDNA3.1.

Article Snippet: Materials and Methods Materials and Cell Line Therm RT-PCR/plat Taq (23191-050), Lipofectamine 2000 (LS11668-027), G-418 (INALCO1758-1811), TRIzol and MTT reagent were purchased from Invitrogen (Carlsbad, Calif., USA), In Situ Cell Death Detection Kit (11684817910) from Roche (Nutley, N.J., USA), SABC (goat-IgG)-POD kit from BOSTER (Wuhan, China), goat polyclonal antibody against ING4 (ab3714) from Abcam Inc. (Cambridge, Mass., USA), and the antibodies against Bcl-2 (SC-23960), Bax (SC-70407), Cyt-c (SC-81752), caspase 3 (SC-7148), PARP (SC-56197), Cox-2 (SC-7951), SKP2 (SC-74477), p27 (SC-1641) from Santa Cruz Inc. (Santa Cruz, Calif., USA).

Techniques: Cell Cycle Assay, Transfection

Journal: Pathobiology : journal of immunopathology, molecular and cellular biology

Article Title: Inhibitor of growth 4 induces growth suppression and apoptosis in glioma U87MG.

doi: 10.1159/000218334

Figure Lengend Snippet: Fig. 10. The expression of ING4 in nude mice xenografts by immunohistochemical technique: U87MG/ pcDNA3.1-ING4 ( a ), U87MG/pcDNA3.1 ( b ) and HE staining ( c ).

Article Snippet: Materials and Methods Materials and Cell Line Therm RT-PCR/plat Taq (23191-050), Lipofectamine 2000 (LS11668-027), G-418 (INALCO1758-1811), TRIzol and MTT reagent were purchased from Invitrogen (Carlsbad, Calif., USA), In Situ Cell Death Detection Kit (11684817910) from Roche (Nutley, N.J., USA), SABC (goat-IgG)-POD kit from BOSTER (Wuhan, China), goat polyclonal antibody against ING4 (ab3714) from Abcam Inc. (Cambridge, Mass., USA), and the antibodies against Bcl-2 (SC-23960), Bax (SC-70407), Cyt-c (SC-81752), caspase 3 (SC-7148), PARP (SC-56197), Cox-2 (SC-7951), SKP2 (SC-74477), p27 (SC-1641) from Santa Cruz Inc. (Santa Cruz, Calif., USA).

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: Pathobiology : journal of immunopathology, molecular and cellular biology

Article Title: Inhibitor of growth 4 induces growth suppression and apoptosis in glioma U87MG.

doi: 10.1159/000218334

Figure Lengend Snippet: Fig. 8. The apoptosis of transfected U87MG was determined by TUNEL assay: more apoptotic cells were observed in U87MG/ pcDNA3.1-ING4 ( a ) compared with U87MG/pcDNA3.1 ( b ).

Article Snippet: Materials and Methods Materials and Cell Line Therm RT-PCR/plat Taq (23191-050), Lipofectamine 2000 (LS11668-027), G-418 (INALCO1758-1811), TRIzol and MTT reagent were purchased from Invitrogen (Carlsbad, Calif., USA), In Situ Cell Death Detection Kit (11684817910) from Roche (Nutley, N.J., USA), SABC (goat-IgG)-POD kit from BOSTER (Wuhan, China), goat polyclonal antibody against ING4 (ab3714) from Abcam Inc. (Cambridge, Mass., USA), and the antibodies against Bcl-2 (SC-23960), Bax (SC-70407), Cyt-c (SC-81752), caspase 3 (SC-7148), PARP (SC-56197), Cox-2 (SC-7951), SKP2 (SC-74477), p27 (SC-1641) from Santa Cruz Inc. (Santa Cruz, Calif., USA).

Techniques: Transfection, TUNEL Assay

Journal: Pathobiology : journal of immunopathology, molecular and cellular biology

Article Title: Inhibitor of growth 4 induces growth suppression and apoptosis in glioma U87MG.

doi: 10.1159/000218334

Figure Lengend Snippet: Fig. 9. The size of nude mice xenografts by subcutaneous inoculation with U87MG, U87MG/pcDNA3.1 and U87MG/ pcDNA3.1-ING4, respectively. The size of xenografts in U87MG/pcDNA3.1- ING4 group was significantly smaller than those in the control tumors.

Article Snippet: Materials and Methods Materials and Cell Line Therm RT-PCR/plat Taq (23191-050), Lipofectamine 2000 (LS11668-027), G-418 (INALCO1758-1811), TRIzol and MTT reagent were purchased from Invitrogen (Carlsbad, Calif., USA), In Situ Cell Death Detection Kit (11684817910) from Roche (Nutley, N.J., USA), SABC (goat-IgG)-POD kit from BOSTER (Wuhan, China), goat polyclonal antibody against ING4 (ab3714) from Abcam Inc. (Cambridge, Mass., USA), and the antibodies against Bcl-2 (SC-23960), Bax (SC-70407), Cyt-c (SC-81752), caspase 3 (SC-7148), PARP (SC-56197), Cox-2 (SC-7951), SKP2 (SC-74477), p27 (SC-1641) from Santa Cruz Inc. (Santa Cruz, Calif., USA).

Techniques: Control

Journal: Pathobiology : journal of immunopathology, molecular and cellular biology

Article Title: Inhibitor of growth 4 induces growth suppression and apoptosis in glioma U87MG.

doi: 10.1159/000218334

Figure Lengend Snippet: Fig. 11. The level of ING4 in nude mice xenografts by RT-PCR analysis: U87MG/pcDNA3.1 (lane a) and U87MG/pcDNA3.1- ING4 (lane b).

Article Snippet: Materials and Methods Materials and Cell Line Therm RT-PCR/plat Taq (23191-050), Lipofectamine 2000 (LS11668-027), G-418 (INALCO1758-1811), TRIzol and MTT reagent were purchased from Invitrogen (Carlsbad, Calif., USA), In Situ Cell Death Detection Kit (11684817910) from Roche (Nutley, N.J., USA), SABC (goat-IgG)-POD kit from BOSTER (Wuhan, China), goat polyclonal antibody against ING4 (ab3714) from Abcam Inc. (Cambridge, Mass., USA), and the antibodies against Bcl-2 (SC-23960), Bax (SC-70407), Cyt-c (SC-81752), caspase 3 (SC-7148), PARP (SC-56197), Cox-2 (SC-7951), SKP2 (SC-74477), p27 (SC-1641) from Santa Cruz Inc. (Santa Cruz, Calif., USA).

Techniques: Reverse Transcription Polymerase Chain Reaction

Journal: Pathobiology : journal of immunopathology, molecular and cellular biology

Article Title: Inhibitor of growth 4 induces growth suppression and apoptosis in glioma U87MG.

doi: 10.1159/000218334

Figure Lengend Snippet: Fig. 12. The expression of proteins in- volved in cell cycle and apoptosis includ- ing p27, SKP2, Bcl-2, Bax, Cyt-c, caspase 3, PARP and Cox-2 by Western blot analysis: U87MG/pcDNA3.1-ING4 (lane a) and U87MG/pcDNA3.1 (lane b).

Article Snippet: Materials and Methods Materials and Cell Line Therm RT-PCR/plat Taq (23191-050), Lipofectamine 2000 (LS11668-027), G-418 (INALCO1758-1811), TRIzol and MTT reagent were purchased from Invitrogen (Carlsbad, Calif., USA), In Situ Cell Death Detection Kit (11684817910) from Roche (Nutley, N.J., USA), SABC (goat-IgG)-POD kit from BOSTER (Wuhan, China), goat polyclonal antibody against ING4 (ab3714) from Abcam Inc. (Cambridge, Mass., USA), and the antibodies against Bcl-2 (SC-23960), Bax (SC-70407), Cyt-c (SC-81752), caspase 3 (SC-7148), PARP (SC-56197), Cox-2 (SC-7951), SKP2 (SC-74477), p27 (SC-1641) from Santa Cruz Inc. (Santa Cruz, Calif., USA).

Techniques: Expressing, Western Blot

Journal: Pathobiology : journal of immunopathology, molecular and cellular biology

Article Title: Inhibitor of growth 4 induces growth suppression and apoptosis in glioma U87MG.

doi: 10.1159/000218334

Figure Lengend Snippet: Fig. 13. The expression of p27, SKP2, Bcl-2, Bax, Cyt-c, caspase 3, PARP and Cox-2 in nude mice xenografts by immunohistochemical technique: xenografts in the U87MG/pcDNA3.1-ING4 and the U87MG/pcDNA3.1 group.

Article Snippet: Materials and Methods Materials and Cell Line Therm RT-PCR/plat Taq (23191-050), Lipofectamine 2000 (LS11668-027), G-418 (INALCO1758-1811), TRIzol and MTT reagent were purchased from Invitrogen (Carlsbad, Calif., USA), In Situ Cell Death Detection Kit (11684817910) from Roche (Nutley, N.J., USA), SABC (goat-IgG)-POD kit from BOSTER (Wuhan, China), goat polyclonal antibody against ING4 (ab3714) from Abcam Inc. (Cambridge, Mass., USA), and the antibodies against Bcl-2 (SC-23960), Bax (SC-70407), Cyt-c (SC-81752), caspase 3 (SC-7148), PARP (SC-56197), Cox-2 (SC-7951), SKP2 (SC-74477), p27 (SC-1641) from Santa Cruz Inc. (Santa Cruz, Calif., USA).

Techniques: Expressing, Immunohistochemical staining

Journal: Anatomical record (Hoboken, N.J. : 2007)

Article Title: ING4 induces cell growth inhibition in human lung adenocarcinoma A549 cells by means of Wnt-1/beta-catenin signaling pathway.

doi: 10.1002/ar.20685

Figure Lengend Snippet: Fig. 8. Western blot analysis for determining the expression of cell proliferation-regulating proteins cyclinD1, p27, SKP2, Cox2, and activation of the Wnt-1/b-catenin pathway in A549 clones with or without exogenous ING4 gene: A549 clones with pcDNA3.1-ING4 (lane 1); A549 clones with pcDNA3.1 (lane 2).

Article Snippet: The Therm RT-PCR/plat Taq, QIAGEN Plasmid Maxi Kit, QIAqick Gel Extraction Kit, Lipofectamine 2000, and G-418 were purchased from Invitrogen Corporation (Carlsbad, CA); the goat polyclonal antibody against ING4 (ab3714) from Abcam Inc. (Cambridge, MA); and the goat polyclonal antibody against Wnt-1, mouse monoclonal antibodies against b-catenin, p27, Cox2, and GAPDH, and rabbit monoclonal antibodies against SKP2 and cyclinD1 from Santa Cruz Biotechnology Inc. (Santa Cruz, CA).

Techniques: Western Blot, Expressing, Activation Assay, Clone Assay

Journal: Anatomical record (Hoboken, N.J. : 2007)

Article Title: ING4 induces cell growth inhibition in human lung adenocarcinoma A549 cells by means of Wnt-1/beta-catenin signaling pathway.

doi: 10.1002/ar.20685

Figure Lengend Snippet: Fig. 8. Western blot analysis for determining the expression of cell proliferation-regulating proteins cyclinD1, p27, SKP2, Cox2, and activation of the Wnt-1/b-catenin pathway in A549 clones with or without exogenous ING4 gene: A549 clones with pcDNA3.1-ING4 (lane 1); A549 clones with pcDNA3.1 (lane 2).

Article Snippet: The Therm RT-PCR/plat Taq, QIAGEN Plasmid Maxi Kit, QIAqick Gel Extraction Kit, Lipofectamine 2000, and G-418 were purchased from Invitrogen Corporation (Carlsbad, CA); the goat polyclonal antibody against ING4 (ab3714) from Abcam Inc. (Cambridge, MA); and the goat polyclonal antibody against Wnt-1, mouse monoclonal antibodies against b-catenin, p27, Cox2, and GAPDH, and rabbit monoclonal antibodies against SKP2 and cyclinD1 from Santa Cruz Biotechnology Inc. (Santa Cruz, CA).

Techniques: Western Blot, Expressing, Activation Assay, Clone Assay