Journal: PLoS ONE

Article Title: Knockdown of Human TCF4 Affects Multiple Signaling Pathways Involved in Cell Survival, Epithelial to Mesenchymal Transition and Neuronal Differentiation

doi: 10.1371/journal.pone.0073169

Figure Lengend Snippet: SH-SY5Y cells were transfected with siRNA oligonucleotides targeting TCF4 (KD1 and KD2) or GAPDH. After 72h, RNA and protein were extracted to assess the knockdown efficiency. (A) Primers complimentary to a constitutive exon present in all TCF4 isoforms (exon 13) were used to measure overall transcript abundance by qPCR in three biological replicates. TCF4 knockdown efficiency was similar with both KD1 and KD2 whereas the control GAPDH KD did not affect TCF4 transcript levels. (B) Western blots of protein lysates prepared from siRNA-treated SH-SY5Y cells demonstrated that KD1 and KD2 reduced TCF4 levels whilst GAPDH knockdown had no apparent effect. α-tubulin was used as a loading control and for normalization. (C) LI-COR quantitation of TCF4 protein levels in siRNA-treated cells. The levels of TCF4 in each experiment were quantified and normalized to α-tubulin as described previously [54,55]. In agreement with the qPCR results, siRNA treatment reduced TCF4 levels to approximately 20% of wild-type compared to mock or GAPDH KD-treated cells.

Article Snippet: Cells were labeled at room temperature with Alexa Fluor 488 phalloidin (165nM, Invitrogen), Hoechst (1µg/ml, Invitrogen) and an anti-TCF4 polyclonal antibody (Covalab, UK) for 1h followed by Alexa Fluor 546 goat anti-rabbit IgG (Invitrogen) for a further 30min.

Techniques: Transfection, Western Blot, Quantitation Assay

Journal: PLoS ONE

Article Title: Knockdown of Human TCF4 Affects Multiple Signaling Pathways Involved in Cell Survival, Epithelial to Mesenchymal Transition and Neuronal Differentiation

doi: 10.1371/journal.pone.0073169

Figure Lengend Snippet: Cells were treated with siRNAs for 72h after which cell viability and caspase activity were measured (A). In addition to the siRNA treatment groups, untransfected SH-SY5Y cells were exposed to staurosporine (1µM in DMSO) and vehicle for 3h to induce apoptosis. Knockdown of TCF4 leads to a significant reduction in cell viability ( P = 2.8x10 -16 , blue bars). Furthermore, TCF4-knockdown is also associated with an increase in caspase 3/7 activity compared to controls ( P = 1.3x10 -3 , red bars). Although GAPDH knockdown is associated with reduced cell viability and elevated caspase 3/7 activity compared to mock-treated cells, both assays showed statistical significant differences between the control groups (mock and GAPDH) compared to TCF4-knockdown (TCF4 KD1 and KD2) supporting the microarray data. As expected, staurosporine treatment also reduced cell viability and increased caspase 3/7 activity in untransfected cells. Western blot analysis of caspase 3 processing after 72h knockdown shows that caspase 3 cleavage products are detected in siRNA treated cells (B). β-actin was used as a loading control for all treatment groups.

Article Snippet: Cells were labeled at room temperature with Alexa Fluor 488 phalloidin (165nM, Invitrogen), Hoechst (1µg/ml, Invitrogen) and an anti-TCF4 polyclonal antibody (Covalab, UK) for 1h followed by Alexa Fluor 546 goat anti-rabbit IgG (Invitrogen) for a further 30min.

Techniques: Activity Assay, Microarray, Western Blot

Journal: PLoS ONE

Article Title: Knockdown of Human TCF4 Affects Multiple Signaling Pathways Involved in Cell Survival, Epithelial to Mesenchymal Transition and Neuronal Differentiation

doi: 10.1371/journal.pone.0073169

Figure Lengend Snippet: Gene expression changes associated with TGF-β and Notch signaling pathways in TCF4-knockdown cells.

Article Snippet: Cells were labeled at room temperature with Alexa Fluor 488 phalloidin (165nM, Invitrogen), Hoechst (1µg/ml, Invitrogen) and an anti-TCF4 polyclonal antibody (Covalab, UK) for 1h followed by Alexa Fluor 546 goat anti-rabbit IgG (Invitrogen) for a further 30min.

Techniques: Expressing

Journal: PLoS ONE

Article Title: Knockdown of Human TCF4 Affects Multiple Signaling Pathways Involved in Cell Survival, Epithelial to Mesenchymal Transition and Neuronal Differentiation

doi: 10.1371/journal.pone.0073169

Figure Lengend Snippet: Gene expression changes associated with the IGF signaling pathway in TCF4-knockdown cells.

Article Snippet: Cells were labeled at room temperature with Alexa Fluor 488 phalloidin (165nM, Invitrogen), Hoechst (1µg/ml, Invitrogen) and an anti-TCF4 polyclonal antibody (Covalab, UK) for 1h followed by Alexa Fluor 546 goat anti-rabbit IgG (Invitrogen) for a further 30min.

Techniques: Expressing, Transduction, Inhibition

Journal: PLoS ONE

Article Title: Knockdown of Human TCF4 Affects Multiple Signaling Pathways Involved in Cell Survival, Epithelial to Mesenchymal Transition and Neuronal Differentiation

doi: 10.1371/journal.pone.0073169

Figure Lengend Snippet: Altered expression of several mental retardation (MR) genes in TCF4-knockdown cells.

Article Snippet: Cells were labeled at room temperature with Alexa Fluor 488 phalloidin (165nM, Invitrogen), Hoechst (1µg/ml, Invitrogen) and an anti-TCF4 polyclonal antibody (Covalab, UK) for 1h followed by Alexa Fluor 546 goat anti-rabbit IgG (Invitrogen) for a further 30min.

Techniques: Expressing