Journal: Journal of Biological Chemistry

Article Title: T Cell Factor 4 Is a Pro-catabolic and Apoptotic Factor in Human Articular Chondrocytes by Potentiating Nuclear Factor κB Signaling

doi: 10.1074/jbc.m113.453985

Figure Lengend Snippet: FIGURE 1. Effects of TCF/LEF members on MMP mRNA expression. A, human primary chondrocytes were infected with adenoviruses expressing GFP,TCF4,andLEF1.ActivationofthecanonicalWntpathwaybyTCF4orLEF1 overexpressionwasvalidatedbyaluciferasereporterassay.*,p0.05;**,p 0.01;n3donors(leftpanel).MMPmRNAexpressionwasmeasuredbyqPCR. *, p 0.05; **, p 0.01; n 3 donors (right panel). Error bars, S.D. B, human chondrocytes were infected with lentiviruses expressing GFP, and FLAG- tagged TCF3 variant 1 (TCF3.E12) and TCF3 variant 2 (TCF3.E47). Overexpres- sion of TCF3 was validated by Western blotting using an anti-FLAG antibody (left panel). MMP mRNA expression was measured by qPCR in human chon- drocytesoverexpressingTCF3variants.**,p0.01;n4donors(rightpanel). C,humanchondrocyteswereinfectedwithlentivirusesexpressingscrambled shRNA (shScr) and shRNA against TCF4 or LEF1. Knockdown of TCF4 and LEF1 protein levels was validated by Western blotting (left panel). MMP mRNA expressionwasmeasuredbyqPCR.*,p0.05;**,p0.01;n3donors(right panel).

Article Snippet: Human TCF3 variant1 (E12) and variant 2 (E47) ORF sequences (Origene) were cloned in to the lentiviral vector pBOB (Addgene plasmid 12335) (11). shRNA sequences against human TCF4 and LEF1 were cloned into the pLKO.1TRC cloning vector (Addgene plasmid 10878) (12).

Techniques: Expressing, Infection, Variant Assay, Western Blot, shRNA, Knockdown

Journal: bioRxiv

Article Title: A novel role for the tumor suppressor gene ITF2 in lung tumorigenesis and chemotherapy response

doi: 10.1101/517169

Figure Lengend Snippet: (A) Viability curves of A2780 cell lines transfected with pCMV6 (S-Ø and R-Ø) and with the overexpression vector (R-ITF2). Each experimental group was exposed to six different CDDP concentrations for 48h. Data were normalized to each untreated control, set to 100%. The data represent the mean ± SD of at least three independent experiments performed in quadruplicate at each drug concentration for each cell line analyzed. (B) Viability of A2780 cell lines transfected with pCMV6 (R-Ø) and with the overexpression vectors (R-ITF2). (C) Relative expression levels of ITF2 measured by quantitative RT-PCR, represented in Log10 scale; In each experimental group, the sensitive cell line transfected with pCMV6 plasmid was used as a calibrator. Each bar represents the combined relative expression of two independent experiments measured in triplicate. (D) β-catenin transcriptional activity was measured in A2780 cells after ITF2 overexpression and treatment with LiCl (10mM) for 24 hours, transfecting with Super8xTopFlash (Top) or Super8xFopFlash (Fop). The results show the fold induction of the Top/Fop ratio with respect to untreated cells (=1). Values represent the mean of three independent experiments measured by triplicate ± SD. (E) Expression analysis of the downstream gene DKK1 regulated by ITF2 in A2780 cell line transfected with pCMV6 (S-Ø and R-Ø) and with the overexpression vector (R-ITF2) for 24 and 72 hours. Representative images of DKK1 and GAPDH measured by RT-PCR. (F), Expression levels of DKK1 measured by qRT-PCR. Each assay was performed at least three times to confirm the results. *** p < 0.001; * p < 0.05 (Students T-test).

Article Snippet: A Myc-DDK-tagged ORF clone of TCF4 and the negative control pCMV6 were used for in transient transfection (RC224345; OriGene, Rockville, Maryland, USA) using previously described methodology .

Techniques: Transfection, Over Expression, Plasmid Preparation, Concentration Assay, Expressing, Quantitative RT-PCR, Activity Assay, Reverse Transcription Polymerase Chain Reaction

Journal: bioRxiv

Article Title: A novel role for the tumor suppressor gene ITF2 in lung tumorigenesis and chemotherapy response

doi: 10.1101/517169

Figure Lengend Snippet: (A) Correlation between ITF2 expression and HOXD9 in tumor and non-tumor samples from the complete cohort of 25 NSCLC patients. Pearson coefficient was used for linear correlation of the quantitative variables. (B-C) Effect of ITF2 overexpression on HOXD9 levels (B) Validation of the transfection efficiency of ITF2 at mRNA levels. Relative expression levels of ITF2 measured by qRT-PCR, in the cell line H23R, at 24 and 72 hours after transfectionrepresented in Log10 the 2-ΔΔCt. (C) Relative expression levels of HOXD9 measured by quantitative RT-PCR after ITF2 overexpression. For both (B) and (C) the resistant cell line transfected with pCMV6 plasmid was used as a calibrator (R-Ø). H23R cells were also transfected with ITF2 cDNA (R-ITF2). Each bar represents the combined relative expression of two independent experiments measured in triplicate. *** p < 0.001; ** p < 0.01 (Students T-test). (D) Correlation between ITF2 and HOXD9 expression levels with the overall survival analyzed in NSCLC patients selected form the RNA-seq analysis. Data represents the quantitative expression levels of the two genes measured by qRT-PCR and represented as 2-ΔΔCt for ITF2 (referred to the NLM) and 2-ΔCt for HOXD9 . (E) Survival analysis in NSCLC samples according to the mean of HOXD9 . LogRank, Breslow and Tarone-Ware test were used for comparisons and p < 0.05 was considered as a significant change in OS. (F-H) Survival analysis in 1,926 patients from the Kaplan Meier online tool (F) and the TCGA data sets of lung Adenocarcinoma (G, High=23; No Change=203) and SCC (H, High=28; No Change=441) for HOXD9 .

Article Snippet: A Myc-DDK-tagged ORF clone of TCF4 and the negative control pCMV6 were used for in transient transfection (RC224345; OriGene, Rockville, Maryland, USA) using previously described methodology .

Techniques: Expressing, Over Expression, Transfection, Quantitative RT-PCR, Plasmid Preparation, RNA Sequencing Assay