Journal: Scientific Reports

Article Title: TGFβ1 secreted by cancer-associated fibroblasts induces epithelial-mesenchymal transition of bladder cancer cells through lncRNA-ZEB2NAT

doi: 10.1038/srep11924

Figure Lengend Snippet: A : TGFβ1 in conditional mediums secreted by 5637 (CTRL), NF and CAF cells were quantified by ELISA. B : The mRNA expression levels of TGFβ1 in CAFs, NFs and three bladder cancer cell lines by qRT-PCR using β-actin gene as the normalization control. C : The expression of phosphorylated Smad2 and total Smad2 protein in the CAF-CM treated bladder cancer cell lines by immunoblotting. β-actin protein was used as the loading control. D : The expression levels of TGFβ1 and TGFβRII in bladder cancer cell lines cultured with CAF-CM were detected by qRT-PCR using β-actin gene as the normalization control. *** P < 0.001.

Article Snippet: The concentrations of TGFβ1 in different media were measured using human TGFβ1 ELISA kit (BOSTER, EK0513, Wuhan, China), according to the manufacturer’s instructions.

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Quantitative RT-PCR, Control, Western Blot, Cell Culture

Journal: Scientific Reports

Article Title: TGFβ1 secreted by cancer-associated fibroblasts induces epithelial-mesenchymal transition of bladder cancer cells through lncRNA-ZEB2NAT

doi: 10.1038/srep11924

Figure Lengend Snippet: A : Summary of altered expression of lncRNAs in the CAF-CM-treated 5637 and J82 cells, comparing to the NF-CM-treated ones. 1.5-fold of relative mRNA level using qRT-PCR, normalized by β-actin gene, was used as the threshold for significant changes. B and C : ZEB2NAT expression levels in the CAF-CM treated 5637 and J82 cells upon the treatments of a TGFβ1 neutralizing antibody ( B ) and a TGFβRI inhibitor (SB-431542) ( C ), respectively. D : Exogenous expression of ZEB2NAT lncRNA in 5637 cells, detected by qRT-PCR using β-actin gene as the normalization control. E – G : Effects of ZEB2NAT lncRNAs overexpression on cell invasion by the Transwell invasion assay ( E , F ) and protein levels of E-cadherin, ZEB1 and ZEB2 by immunoblotting ( G ) in 5637 cells. H: Knockdown of ZEB2NAT by RNAi using two siRNAs targeting different regions of the lncRNA (siZEB2NAT-1 and siZEB2NAT-2) in 5637 cells. The relative ZEB2NAT expression levels were at 48 hours after siRNA transfection and determined by qRT-PCR using β-actin gene as the normalization control. I , J , K : Effects of ZEB2NAT lncRNAs knockdown on cell invasion by the Transwell invasion assay ( I , J ) and protein levels of E-cadherin and ZEB2 by immunoblotting ( K ) in the CAF-CM treated 5637 cells. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The concentrations of TGFβ1 in different media were measured using human TGFβ1 ELISA kit (BOSTER, EK0513, Wuhan, China), according to the manufacturer’s instructions.

Techniques: Expressing, Quantitative RT-PCR, Control, Over Expression, Transwell Invasion Assay, Western Blot, Knockdown, Transfection

Journal: Scientific Reports

Article Title: TGFβ1 secreted by cancer-associated fibroblasts induces epithelial-mesenchymal transition of bladder cancer cells through lncRNA-ZEB2NAT

doi: 10.1038/srep11924

Figure Lengend Snippet: The expression levels of ZEB2NAT ( A ) and TGFβ1 transcripts ( B ), and ZEB2 protein ( C ) in 30 human bladder cancer tissues (Tumor or T) and the paired normal tissues (Normal or N) from the same patient. Relative ZEB2NAT and TGFβ1 mRNA levels were assessed by qRT-PCR and normalized by β-actin gene. D – F : The correlations among ZEB2NAT, TGFβ1 and ZEB2 were decided by Pearson correlation analysis. ΔCT values in qRT-PCR were used as the expression levels of ZEB2NAT and TGFβ1 transcripts. ZEB2 protein bands on Western blot were quantified by Image J software. P < 0.05 was considered statistically significant.

Article Snippet: The concentrations of TGFβ1 in different media were measured using human TGFβ1 ELISA kit (BOSTER, EK0513, Wuhan, China), according to the manufacturer’s instructions.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Software

Journal: Neoplasia (New York, N.Y.)

Article Title: Overcoming Resistance to AC0010, a Third Generation of EGFR Inhibitor, by Targeting c-MET and BCL-2

doi: 10.1016/j.neo.2018.11.004

Figure Lengend Snippet: Elucidation of mechanisms of AC0010 resistance by RNAseq profiling. Total RNA was isolated and subjected to RNAseq profiling as detailed in the Materials and Methods section. Shown are genes in p53 and TGFβ pathways enriched in H1975-P1 cells (A), and apoptosis and NFκB pathways in H1975-AVR1 cells (B). Real-time PCR analysis to confirm increased expression of c-MET and BCL-2 in H1975-P1-R1 and H1975-AVR1-R2 cells, respectively (C). Western blot analysis of the levels of c-MET and BCL-2 in H1975-P1-R1 and H1975-AVR1-R2 cells as well as the phosphorylation status of c-MET, EGFR, and their downstream molecules (AKT1 and ERK) (D).

Article Snippet: The expression of c-MET, BCL-2, and CDH11 was measured using the TaqMan Gene Expression Assay (Hs01565584_m1, Hs00608023_m1, and Hs00901479_m1, Applied Biosystems).

Techniques: Isolation, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Phospho-proteomics

Journal: Neoplasia (New York, N.Y.)

Article Title: Overcoming Resistance to AC0010, a Third Generation of EGFR Inhibitor, by Targeting c-MET and BCL-2

doi: 10.1016/j.neo.2018.11.004

Figure Lengend Snippet: Overcoming AC0010 resistance by targeting c-MET in cell culture model. (A) H1975-P1-R1 cells were transfected with siRNA targeting c-MET, along with the control siRNA, and the sensitivity of transfected cells to AC0010 was determined by WST-1 assay. Western blot analysis was performed to show c-MET knockdown. (B) Growth curve of H1975-P1-R1 cells treated with various concentrations of crizotinib. (C) Growth curve of H1975-P1-R1 cells treated with various concentrations of AC0010 in combination with 2 μM crizotinib or 0.5 μM crizotinib ( n = 3). (D) Growth curve of H1975-P1-R1 cells treated with 1 μM AC0010 in combination with various concentrations of crizotinib ( n = 3). (E) Clonogenic survival assays of H1975-P1-R1 cells treated with AC0010, criztonib, or the combination with indicated concentrations ( n = 3). (F) Western blot analysis of the phosphorylation status of c-MET, EGFR, and their downstream molecules (AKT1 and ERK) in H1975-P1-R1 cells treated with indicated drugs and concentrations.

Article Snippet: The expression of c-MET, BCL-2, and CDH11 was measured using the TaqMan Gene Expression Assay (Hs01565584_m1, Hs00608023_m1, and Hs00901479_m1, Applied Biosystems).

Techniques: Cell Culture, Transfection, Control, WST-1 Assay, Western Blot, Knockdown, Phospho-proteomics

Journal: Neoplasia (New York, N.Y.)

Article Title: Overcoming Resistance to AC0010, a Third Generation of EGFR Inhibitor, by Targeting c-MET and BCL-2

doi: 10.1016/j.neo.2018.11.004

Figure Lengend Snippet: Overcoming AC0010 resistance by targeting c-MET in in vivo xenograft model. (A) Inhibition of H1975-P1-R1 tumor growth by AC0010 or crizotinib, alone or in combination, for a period of 14 days. Tumor growth curves are plotted as mean ± SEM ( n = 8). (B) Tumor weights of each group were analyzed using SPSS 17.0 software; t test statistical analysis. (C) Mouse body weights of each group are plotted as mean ± SEM ( n = 8). (D) H1975-P1-R1 tumor issues from mice treated with vehicle, AC0010, crizotinib, or AC0010 combined with crizotinib for 14 days were isolated and subjected to Western blotting analysis using indicated Abs.

Article Snippet: The expression of c-MET, BCL-2, and CDH11 was measured using the TaqMan Gene Expression Assay (Hs01565584_m1, Hs00608023_m1, and Hs00901479_m1, Applied Biosystems).

Techniques: In Vivo, Inhibition, Software, Isolation, Western Blot

Journal: Cell Death & Disease

Article Title: PTTG2 silencing results in induction of epithelial-to-mesenchymal transition and apoptosis

doi: 10.1038/cddis.2013.48

Figure Lengend Snippet: Characterization of Pttg2-depleted HCT116 cells. ( a ) Schematic diagram showing the sequences and location of shPttg1 and shPttg2. Multiple sequence alignments were made using the Clustal W software ( www.clustal.org ). ( b ) Relative quantification of Pttg1 and Pttg2 mRNA levels in Pttg1 and Pttg2-silenced HCT116 wild-type and HCT116 pttg1 −/− cells by real-time PCR. Expression of each gene is shown as mean±S.E.M. of three independent experiments. Data are shown as 2 −ΔCT normalized to HPRT1. ( c ) Analysis of PTTG1 and PTTG2 protein levels 72 h after shRNA-Pttg1 or shRNA-Pttg2 treatment in HCT116 wild-type and HCT116 pttg1 −/− cells. β -Actin was used as loading control. Densitometric analysis for western blots of PTTG1 and PTTG2 protein expression (% of control) in control and shRNA-treated (shPttg1 or shPttg2) wild-type and Pttg1 −/− HCT116 cells

Article Snippet: Expression of Pttg2 and Pttg3 was measured using the Applied BiosystemsTaqMan Gene Expression Assays Hs00747713_sH and Hs00607485_s1.

Techniques: Sequencing, Software, Quantitative Proteomics, Real-time Polymerase Chain Reaction, Expressing, shRNA, Control, Western Blot

Journal: Cell Death & Disease

Article Title: PTTG2 silencing results in induction of epithelial-to-mesenchymal transition and apoptosis

doi: 10.1038/cddis.2013.48

Figure Lengend Snippet: Pttg1 and Pttg2 show different biochemical properties. ( a ) PTTG2 does not interact with Separase. Extracts of HEK293T cells expressing N-terminally Flag-tagged separase, C-terminally Myc-tagged PTTG2, or both were used for immunoprecipitation with anti-Myc antibodies. The immunoprecipitates were analyzed by immunoblotting with anti-FLAG and anti-Myc antibodies. The asterisks indicate the immunoglobulin light chains. As positive control, the interaction between PTTG1 and separase was tested using Flag-separase and Myc-tagged PTTG1. ( b ) PTTG2 lacks transactivation capacity. The ORF of Pttg2 was fused to the GAL4 DNA-binding domain (pGAL4-DBD-Pttg2) and tested for transcription activation in HEK293T cells cotransfected with a GAL4 site-dependent reporter plasmid driving expression of the luciferase gene (pGAL4-4RE-Luc). pGAL4-PTTG1 and pGAL4-VP16 were used as positive controls. Luciferase values from at least three independent transfections are plotted as fold activation over control

Article Snippet: Expression of Pttg2 and Pttg3 was measured using the Applied BiosystemsTaqMan Gene Expression Assays Hs00747713_sH and Hs00607485_s1.

Techniques: Expressing, Immunoprecipitation, Western Blot, Positive Control, Binding Assay, Activation Assay, Plasmid Preparation, Luciferase, Transfection, Control

Journal: Cell Death & Disease

Article Title: PTTG2 silencing results in induction of epithelial-to-mesenchymal transition and apoptosis

doi: 10.1038/cddis.2013.48

Figure Lengend Snippet: Silencing of Pttg2 impairs cell adhesion and proliferative capacity of HCT116 cells. ( a ) Phase-contrast images ( × 10 objective) of Pttg2-silenced HCT116 cells 72 h (upper panels) and 1 week post-infection (PI) (lower panels). Inserts show close-up views of representative cell culture areas. Pttg2-depleted cells show rounder morphology than control cells, indicating a defect in cell adhesion properties. ( b ) Spheroid formation of Pttg2 and Pttg1-silenced HCT116 (wt) cells in suspension. Seventy-two hours post-infection, cells were transferred to poly-HEMA-coated plates at 6 × 10 4 cells/ml. After 48 h in suspension, cells were photographed at 10 × . Control and shPttg1-treated cells formed dense spheroids whereas shPttg2-silenced cells adhere to each other very weakly. Representative areas of each culture were selected. ( c ) Percentage of BrdU-positive cells after shRNA-Pttg2 treatment. HCT116 cells were infected and assayed for BrdU incorporation 72 h post-infection. Bars represent the mean±S.E.M. ( d ) Analysis of mitotic index in Pttg2-depleted HCT116 cells. Cells were treated with shRNA-Pttg2 lentivirus and incubated with nocodazole 1 μ M 72 h post-infection, for 24 h. Percentage of H3P-positive (mitotic) cells was measured by flow cytometry. Bars represent the mean±S.E.M. ( e ) Percentage of cell death following shRNA Pttg2 or shRNA Pttg1 treatment of HCT116 cells growing under adherent or suspension conditions. Seventy-two hours post-infection, cells were transferred onto control or poly-HEMA-coated (PH) plates in the absence or presence of methylcellulose (MC) and the levels of apoptosis was determined 48 h later by measuring the percentage of cells containing a subG1 DNA content by flow cytometry. Results represent the means of three independent experiments±S.E.M. ( f ) p21 induction in Pttg2-depleted HCT116 cells. HCT116 cells treated with shRNA-Pttg2 lentivirus for 72 h were harvested and the levels of p21 determined by immunoblotting using specific p21 antibodies. β -Actin was used as loading control

Article Snippet: Expression of Pttg2 and Pttg3 was measured using the Applied BiosystemsTaqMan Gene Expression Assays Hs00747713_sH and Hs00607485_s1.

Techniques: Infection, Cell Culture, Control, Suspension, shRNA, BrdU Incorporation Assay, Incubation, Flow Cytometry, Western Blot

Journal: Cell Death & Disease

Article Title: PTTG2 silencing results in induction of epithelial-to-mesenchymal transition and apoptosis

doi: 10.1038/cddis.2013.48

Figure Lengend Snippet: Defective adhesion in Pttg2-depleted HCT116 cells results in p53- and p21-dependent apoptosis. ( a ) Phase-contrast images of Pttg2-silenced HCT116 cells after z-VAD treatment. A round morphology was still observed in Pttg2-depleted cells, suggesting a defective cell adhesion. ( b ) Apoptosis can be rescued in Pttg2-silenced HCT116 cells after caspase inhibition treatment. The percentage of apoptotic cells (subG1) after PTTG2 depletion was drastically reduced after treatment with the caspase inhibitor z-VAD. Bars represent the mean±S.E.M. ( c ) Levels of apoptotic indicators in Pttg2-silenced HCT116 cells. Cells were harvested after 72 h post-infection and assayed for p53, p21, BIM and cleaved-Capase 3 levels by western blot using specific antibodies. β -Actin was used as loading control. ( d , e ) Role of p53 in Pttg2-dependent anoikis. ( d ) Phase-contrast images of Pttg2-depleted HCT116 p53 −/− cells. The absence of PTTG2 in HCT116 p53 −/− results in cell rounding similar to wild-type HCT116 cells. ( e ) FACS analysis of Pttg2-depleted HCT116 p53 −/− cells. In the absence of PTTG2, the percentage of apoptotic cells (subG1) was partially reduced in HCT116 p53 −/− cells compared with HCT116 wild-type cells. Bars represent the mean±S.E.M. ( f ) The induction of p21 in Pttg2-treated cells occurs in a p53-dependent manner. Western blot analysis of p21 levels in HCT116 p53 −/− cells 72 h post-infection with shPttg2 or control lentivirus. β -Actin was used as loading control. ( g ) Role of p21 in anoikis in Pttg2-depleted cells. The percentage of apoptotic cells in HCT116 p21 −/− following treatment with shPttg2 was significantly reduced compared with HCT116 wild-type cells. Bars represent the mean±S.E.M.

Article Snippet: Expression of Pttg2 and Pttg3 was measured using the Applied BiosystemsTaqMan Gene Expression Assays Hs00747713_sH and Hs00607485_s1.

Techniques: Inhibition, Infection, Western Blot, Control

Journal: Cell Death & Disease

Article Title: PTTG2 silencing results in induction of epithelial-to-mesenchymal transition and apoptosis

doi: 10.1038/cddis.2013.48

Figure Lengend Snippet: Loss of cell adhesion in shPttg2-treated cells precedes the onset of apoptosis. ( a ) Spheroid formation of Pttg2-depleted cells after z-VAD treatment. Wild-type HCT116 cells were infected for 72 h in the presence or absence of the caspases inhibitor z-VAD and the cell–cell adhesion capacity assayed on poly-HEMA (PH)-coated plates. The experiments were repeated three times, and representative areas of each culture are shown. ( b ) Cell-cycle profile of Pttg2-silenced HCT116 cells growing under poly-HEMA conditions after z-VAD treatment. The percentage of apoptotic cells (subG1) was significantly reduced in the presence of z-VAD, whereas the other cell-cycle phases (G1, S and G2/M) were not modified. Data are representative of two independent experiments. ( c ) Phase-contrast images to assess multicellular aggregates formation in shPttg2-treated HCT116 p53 −/− cells growing on poly-HEMA-coated plates. Images show a representative region of each condition. ( d ) Analysis of the cell-cycle profile of Pttg2-depleted HCT116 p53 −/− cells growing under poly-HEMA conditions. ShPttg2-treated cells lacking p53 showed a reduced percentage of cell death (subG1). The other cell-cycle phases (G1, S and G2/M) remained unaltered. Data are representative of two independent experiments

Article Snippet: Expression of Pttg2 and Pttg3 was measured using the Applied BiosystemsTaqMan Gene Expression Assays Hs00747713_sH and Hs00607485_s1.

Techniques: Infection, Modification

Journal: Cell Death & Disease

Article Title: PTTG2 silencing results in induction of epithelial-to-mesenchymal transition and apoptosis

doi: 10.1038/cddis.2013.48

Figure Lengend Snippet: Pttg2-depleted cells exhibit cytoskeletal defects. Microtubule cytoskeleton of PTTG2 depleted cells. Confocal images of Pttg2-silenced HCT116 cells stably expressing Histone H2B (H2B)-cherry fusion gene. Seventy-two hours post-infection, cells were fixed with methanol at −20 °C and stained for α -tubulin (green). Scale bars, 25 μ m

Article Snippet: Expression of Pttg2 and Pttg3 was measured using the Applied BiosystemsTaqMan Gene Expression Assays Hs00747713_sH and Hs00607485_s1.

Techniques: Stable Transfection, Expressing, Infection, Staining

Journal: Cell Death & Disease

Article Title: PTTG2 silencing results in induction of epithelial-to-mesenchymal transition and apoptosis

doi: 10.1038/cddis.2013.48

Figure Lengend Snippet: Pttg2 depletion results in deregulated E-cadherin and vimentin levels and induction of the epithelial-to-mesenchymal transition. ( a ) Summary of gene expression profile according to Gene Ontology. Pie charts show the percentage of genes included in each category (molecular function, biological process and cellular component) and subcategories with a P -value <0.01. The GO analysis was performed using DAVID v.6.7 program. GO analysis of differentially upregulated or downregulated genes are shown in upper and lower panels, respectively. ( b ) Validation of E-Cadherin downregulation in Pttg2-silenced cells by quantitative PCR. HCT116 cells were infected with shPttg2 or control lentivirus and assayed for E-cadherin mRNA levels 72 h post-infection. Values were normalized using the hprt1 gene as an internal control. Columns represent the mean values of 2–3 independent experiments ±S.E.M. ( c ) Western blot analysis of E-Cadherin repression in Pttg2-depleted HCT116 cells. The expression levels of the E-Cadherin protein in shPttg2 cells was compared with control cells expressing an empty lentiviral plasmid. Cells were grown either as a monolayer (attached) or in suspension for 48 h on poly-HEMA-coated plates (+PH) and total lysates analyzed by immunoblotting. ( d ) Vimentin mRNA induction in shPttg2-treated cells. HCT116 cells were infected with shPttg2 or control lentivirus and assayed for vimentin levels 72 h post-infection by quantitative PCR. Values were normalized using the hprt1 gene as an internal control. Columns represent the mean values of 2–3 independent experiments±S.E.M.

Article Snippet: Expression of Pttg2 and Pttg3 was measured using the Applied BiosystemsTaqMan Gene Expression Assays Hs00747713_sH and Hs00607485_s1.

Techniques: Gene Expression, Biomarker Discovery, Real-time Polymerase Chain Reaction, Infection, Control, Western Blot, Expressing, Plasmid Preparation, Suspension