Journal: Oncotarget

Article Title: Transcriptional coactivator CBP upregulates hTERT expression and tumor growth and predicts poor prognosis in human lung cancers

doi:

Figure Lengend Snippet: (A) Lung cancer cells were co-transfected with the plasmids of hTERT promoter driven-luciferase and pCDNA3.1-CBP or pCDNA3.1-Lac Z for 48 h followed by a dual-luciferase assay. The relative luciferase intensity per mg protein was calculated in the treated cells. (B) Lung cancer cells were co-transfected with hTERT promoter driven-luciferase and CBP siRNA or control siRNA for 48 h followed by a dual-luciferase assay. The relative luciferase intensity per mg protein was calculated in the treated cells. (C) Up-regulation of hTERT protein expression by the overexpression of CBP. H1299 cells were treated with pcDNA3.1-CBP for 48 h, and the expression of CBP itself in the nucleus and hTERT protein in the cytoplasm were analyzed by Western blot in lung cancer cells. (D, E) Downregulation of hTERT protein expression by the knockdown of CBP expression. H1299 cells were transfected with a CBP-specific siRNA for 48 h, the expression of CBP itself in the nucleus and hTERT protein in the cytoplasm were analyzed by Western blot (D), and the telomerase activity was assessed (E). All of the measurements represent the means ± SE of three independent experiments. *, P < 0.05, significant differences between treatment groups and DMSO control groups.

Article Snippet: To inhibit CBP expression, H1299 cells were transfected with CBP specific siRNA (10μmol/L, Santa Cruz, sc-29244, siRNA-1 and OriGene, SR300976, siRNA-2); nonspecific siRNA (10μmol/L, Santa Cruz, sc-44230).

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

Journal: Oncotarget

Article Title: Transcriptional coactivator CBP upregulates hTERT expression and tumor growth and predicts poor prognosis in human lung cancers

doi:

Figure Lengend Snippet: ( A ) H1299 cells were transfected with CBP overexpression vector pcDNA3.1-CBP. At different time points after transfection, cell viability was measured by MTT assay. ( B ) H1299 cells were treated with CBP specific siRNA or inhibitor. At different time points after treatment, cell viability was measured by MTT assay. The mean and SE obtained from three independent experiments are plotted (*, P < 0.05,**, P < 0.01). (C) The nude mice containing xenografts of human lung cancer were intratumorally treated with non-specific control siRNA or CBP-specific siRNA, and the tumor volumes ± SE were calculated at different days after treatment. (N=5; *, P < 0.05). (D) Immunohistochemistry of CBP and hTERT from tumor xenografts in nonspecific control siRNA- and CBP-specific siRNA-treated nude mice (400× magnification).

Article Snippet: To inhibit CBP expression, H1299 cells were transfected with CBP specific siRNA (10μmol/L, Santa Cruz, sc-29244, siRNA-1 and OriGene, SR300976, siRNA-2); nonspecific siRNA (10μmol/L, Santa Cruz, sc-44230).

Techniques: Transfection, Over Expression, Plasmid Preparation, MTT Assay, Control, Immunohistochemistry

Journal: Oncotarget

Article Title: Transcriptional coactivator CBP upregulates hTERT expression and tumor growth and predicts poor prognosis in human lung cancers

doi:

Figure Lengend Snippet: (A) The nuclear extracts of human lung normal and cancer cells were prepared for immunoprecipitation using an antibody against Sp1 or AP-2β and then evaluated by immunoblot using antibody against CBP. (B) Human lung cancer H1299 cells grown on chamber slides were cultivated for 24 h, and the subcellular localization and the colocalization of CBP with Sp1 or AP-2β were examined by confocal microscopy analysis with a confocal microscope. More than 100 cells were inspected per experiment, and cells with typical morphology were presented. (C) Streptavidin-agarose bead pulldown assay with hTERT promoter (-378 to +60) as probes was done. Sp1 was tested in the pulled down proteins by immunoblot using antibody against Sp1. (D) Chromatin immunoprecipitation assays were done using antibody against Sp1. PCR products of hTERT promoter (-378 to +60) were separated on 1% agarose gels. The last lane represents the IgG control. (E) Immunoprecipitation was performed using antibody against Sp1. The acetylated Sp1 was determined by immunoblot using the antibody against acetylation. (F) Immunoprecipitation was performed in human lung cancer cells (H1299) treated by non-specific siRNA or CBP specific siRNA or inhibitor using antibody against Sp1. The acetylated Sp1 was tested by immunoblot using antibody against acetylation. (G) Streptavidin-agarose bead pulldown assay with hTERT promoter (-378 to +60) as probes was done in lung cancer cells (H1299) treated by non-specific siRNA or CBP specific siRNA or CBP-specific inhibitor. The level of Sp1 in the pulled down proteins was determined by immunoblot. Densitometric analysis was used to analyze quantitatively the binding activity and acetylation level of Sp1 proteins.

Article Snippet: To inhibit CBP expression, H1299 cells were transfected with CBP specific siRNA (10μmol/L, Santa Cruz, sc-29244, siRNA-1 and OriGene, SR300976, siRNA-2); nonspecific siRNA (10μmol/L, Santa Cruz, sc-44230).

Techniques: Immunoprecipitation, Western Blot, Confocal Microscopy, Microscopy, Chromatin Immunoprecipitation, Control, Binding Assay, Activity Assay

Journal: Frontiers in Oncology

Article Title: Distinct cholangiocarcinoma cell migration in 2D monolayer and 3D spheroid culture based on galectin-3 expression and localization

doi: 10.3389/fonc.2022.999158

Figure Lengend Snippet: Migration-related expression in CCA cells in 2D and 3D culture systems. (A) CCA cells were grown as monolayers and spheroids for 6 days. Cells were harvested and lysed. Protein expression was determined by Western blotting. Migration-related proteins were compared between the two culture systems in each cell line. β-actin was used as an internal control. (B) Expressions of galectin-3 was quantified as relative intensity to β-actin. The data represent means and ± standard error. ** p < 0.01, NS represents not significant.

Article Snippet: The galectin-3 shRNA plasmid (sc-155994-SH) containing a pool of three to five lentiviral vector plasmids each encoding galectin-3-specific 19-25 nucleotides (plus hairpin) shRNAs, and scramble shRNA plasmid-A (sc-108060) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Migration, Expressing, Western Blot

Journal: Frontiers in Oncology

Article Title: Distinct cholangiocarcinoma cell migration in 2D monolayer and 3D spheroid culture based on galectin-3 expression and localization

doi: 10.3389/fonc.2022.999158

Figure Lengend Snippet: Localization of galectin-3 in 2D and 3D CCA cells. CCA cells were cultured as monolayer and spheroids for 10 days before harvesting for the immunofluorescence. CCA cells were fixed, permeabilized and blocked before staining with galectin-3 antibody (green). TRITC-phalloidin and Hoechst 33342 were used as counterstains for F-actin (red) and nuclei (blue), respectively. The tumor spheroid images were taken under a confocal microscope using 60x and 120x magnifications. The scale bars in 2D and 3D systems indicate 25 µm and 10 µm, respectively.

Article Snippet: The galectin-3 shRNA plasmid (sc-155994-SH) containing a pool of three to five lentiviral vector plasmids each encoding galectin-3-specific 19-25 nucleotides (plus hairpin) shRNAs, and scramble shRNA plasmid-A (sc-108060) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Cell Culture, Immunofluorescence, Staining, Microscopy

Journal: Frontiers in Oncology

Article Title: Distinct cholangiocarcinoma cell migration in 2D monolayer and 3D spheroid culture based on galectin-3 expression and localization

doi: 10.3389/fonc.2022.999158

Figure Lengend Snippet: Effect of galectin-3 on 2D and 3D KKU-213A cell migration. (A) The relative galectin-3 mRNA level of KKU-213A knockdown cells compared to the control. (B) Representative galectin-3 immunoblots of KKU-213A cells subjected to gal-3 and scrambled shRNA. β-actin was used as the loading control. (C) The relative galectin-3 protein expression of KKU-213A knockdown cells compared to the control. (D) The morphology of galectin-3 knockdown cells in 2D and 3D cultures. The 2D culture images were taken before cells reached confluence and 3D culture images were taken on day 6. (E) Control and galectin-3 knockdown KKU-213A cells were grown as monolayer or tumor spheroids for 4 days. rGal-3 was treated during the migration assay. The migration area of KKU-213A monolayer and tumor spheroids were collected at 0 h and 12 h. The scale bars represent 100 µm. (F, G) Bars represent the relative migration of galectin-3 knockdown cells with and without the treatment of rGal-3 in (F) 2D and (G) 3D conditions compared to the control cells. The data represent means and ± standard error. ** p < 0.01.

Article Snippet: The galectin-3 shRNA plasmid (sc-155994-SH) containing a pool of three to five lentiviral vector plasmids each encoding galectin-3-specific 19-25 nucleotides (plus hairpin) shRNAs, and scramble shRNA plasmid-A (sc-108060) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Migration, Western Blot, shRNA, Expressing

Journal: Frontiers in Oncology

Article Title: Distinct cholangiocarcinoma cell migration in 2D monolayer and 3D spheroid culture based on galectin-3 expression and localization

doi: 10.3389/fonc.2022.999158

Figure Lengend Snippet: Kaplan-Meier survival curve of CCA patients according to galectin-3 expression. The cumulative (A) overall survival ( P = 0.54) and (B) disease-free survival ( P = 0.41) plot stratified by the level of galectin-3 expression.

Article Snippet: The galectin-3 shRNA plasmid (sc-155994-SH) containing a pool of three to five lentiviral vector plasmids each encoding galectin-3-specific 19-25 nucleotides (plus hairpin) shRNAs, and scramble shRNA plasmid-A (sc-108060) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Expressing

Journal: Oncotarget

Article Title: Transcriptional coactivator CBP upregulates hTERT expression and tumor growth and predicts poor prognosis in human lung cancers.

doi: 10.18632/oncotarget.2430

Figure Lengend Snippet: Figure 2: The effect of CBP on hTERT promoter activity, hTERT protein expression and telomerase activity. (A) Lung cancer cells were co-transfected with the plasmids of hTERT promoter driven-luciferase and pCDNA3.1-CBP or pCDNA3.1-Lac Z for 48 h followed by a dual-luciferase assay. The relative luciferase intensity per mg protein was calculated in the treated cells. (B) Lung cancer cells were co-transfected with hTERT promoter driven-luciferase and CBP siRNA or control siRNA for 48 h followed by a dual-luciferase assay. The relative luciferase intensity per mg protein was calculated in the treated cells. (C) Up-regulation of hTERT protein expression by the overexpression of CBP. H1299 cells were treated with pcDNA3.1-CBP for 48 h, and the expression of CBP itself in the nucleus and hTERT protein in the cytoplasm were analyzed by Western blot in lung cancer cells. (D, E) Downregulation of hTERT protein expression by the knockdown of CBP expression. H1299 cells were transfected with a CBP-specific siRNA for 48 h, the expression of CBP itself in the nucleus and hTERT protein in the cytoplasm were analyzed by Western blot (D), and the telomerase activity was assessed (E). All of the measurements represent the means ± SE of three independent experiments. *, P < 0.05, significant differences between treatment groups and DMSO control groups.

Article Snippet: To inhibit CBP expression, H1299 cells were transfected with CBP specific siRNA (10μmol/L, Santa Cruz, sc-29244, siRNA-1 and OriGene, SR300976, siRNA-2); nonspecific siRNA (10μmol/L, Santa Cruz, sc-44230).

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

Journal: Oncotarget

Article Title: Transcriptional coactivator CBP upregulates hTERT expression and tumor growth and predicts poor prognosis in human lung cancers.

doi: 10.18632/oncotarget.2430

Figure Lengend Snippet: Figure 3: The effect of CBP on lung tumor growth in vitro and in vivo. (A) H1299 cells were transfected with CBP overexpression vector pcDNA3.1-CBP. At different time points after transfection, cell viability was measured by MTT assay. (B) H1299 cells were treated with CBP specific siRNA or inhibitor. At different time points after treatment, cell viability was measured by MTT assay. The mean and SE obtained from three independent experiments are plotted (*, P < 0.05,**, P < 0.01). (C) The nude mice containing xenografts of human lung cancer were intratumorally treated with non-specific control siRNA or CBP-specific siRNA, and the tumor volumes ± SE were calculated at different days after treatment. (N=5; *, P < 0.05). (D) Immunohistochemistry of CBP and hTERT from tumor xenografts in nonspecific control siRNA- and CBP-specific siRNA-treated nude mice (400x magnification).

Article Snippet: To inhibit CBP expression, H1299 cells were transfected with CBP specific siRNA (10μmol/L, Santa Cruz, sc-29244, siRNA-1 and OriGene, SR300976, siRNA-2); nonspecific siRNA (10μmol/L, Santa Cruz, sc-44230).

Techniques: In Vitro, In Vivo, Transfection, Over Expression, Plasmid Preparation, MTT Assay, Control, Immunohistochemistry

Journal: Oncotarget

Article Title: Transcriptional coactivator CBP upregulates hTERT expression and tumor growth and predicts poor prognosis in human lung cancers.

doi: 10.18632/oncotarget.2430

Figure Lengend Snippet: Figure 6: The interaction of CBP with Sp1 and AP-2 and the acetylationt of Sp1 by CBP in lung cancer cells. (A) The nuclear extracts of human lung normal and cancer cells were prepared for immunoprecipitation using an antibody against Sp1 or AP-2β and then evaluated by immunoblot using antibody against CBP. (B) Human lung cancer H1299 cells grown on chamber slides were cultivated for 24 h, and the subcellular localization and the colocalization of CBP with Sp1 or AP-2β were examined by confocal microscopy analysis with a confocal microscope. More than 100 cells were inspected per experiment, and cells with typical morphology were presented. (C) Streptavidin-agarose bead pulldown assay with hTERT promoter (-378 to +60) as probes was done. Sp1 was tested in the pulled down proteins by immunoblot using antibody against Sp1. (D) Chromatin immunoprecipitation assays were done using antibody against Sp1. PCR products of hTERT promoter (-378 to +60) were separated on 1% agarose gels. The last lane represents the IgG control. (E) Immunoprecipitation was performed using antibody against Sp1. The acetylated Sp1 was determined by immunoblot using the antibody against acetylation. (F) Immunoprecipitation was performed in human lung cancer cells (H1299) treated by non-specific siRNA or CBP specific siRNA or inhibitor using antibody against Sp1. The acetylated Sp1 was tested by immunoblot using antibody against acetylation. (G) Streptavidin-agarose bead pulldown assay with hTERT promoter (-378 to +60) as probes was done in lung cancer cells (H1299) treated by non-specific siRNA or CBP specific siRNA or CBP-specific inhibitor. The level of Sp1 in the pulled down proteins was determined by immunoblot. Densitometric analysis was used to analyze quantitatively the binding activity and acetylation level of Sp1 proteins.

Article Snippet: To inhibit CBP expression, H1299 cells were transfected with CBP specific siRNA (10μmol/L, Santa Cruz, sc-29244, siRNA-1 and OriGene, SR300976, siRNA-2); nonspecific siRNA (10μmol/L, Santa Cruz, sc-44230).

Techniques: Immunoprecipitation, Western Blot, Confocal Microscopy, Microscopy, Chromatin Immunoprecipitation, Control, Binding Assay, Activity Assay