Research article
Wnt pathway reprogramming during human embryonal carcinoma differentiation and potential for therapeutic targeting
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* Corresponding author: Sarah J Freemantle Sarah.Freemantle@dartmouth.edu
1 Department of Pharmacology and Toxicology, Dartmouth Medical School, Hanover, NH 03755, USA
2 Department of Medicine, Dartmouth Medical School, Hanover, NH 03755, USA
3 Norris Cotton Cancer Center, Dartmouth-Hitchcock Medical Center, Lebanon, NH, 03756, USA
BMC Cancer 2009, 9:383 doi:10.1186/1471-2407-9-383
Published: 29 October 2009Additional files
Additional file 1:
Primers used in these analyses. Primer sequences used for human genes analyzed.
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Additional file 2:
Gene expression changes in NT2/D1 cells treated with RA. Raw Wnt array data from NT2/D1 cells treated +/- 10 μM RA for 96 hours.
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Additional file 3:
Gene expression changes in NT2/D1 cells treated with siRNA to POU5F1. Raw Wnt array data from NT2/D1 cells treated with siRNA to POU5F1 or a control siRNA for 96 hours.
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Additional file 4:
Effect of differentiation on PORCN, FRAT2, and FZD5 expression. NT2/D1 cells were treated with RA or transfected with siRNA for POU5F1 to induce differentiation. A real-time PCR assay was used to determine expression of PORCN, FRAT2, and FZD5, respectively. Expression is displayed as percent of control. Graphs depict an average of at least two RNA samples with real-time RT-PCR assays performed in triplicate. Error bars represent standard deviation. a) PORCN expression. b) FRAT2 expression. c) FZD5 expression. (**p < 0.005, ***p < 0.0005).
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Additional file 5:
Knock-down of FZD5 and FZD7 expression with siRNA does not impact the levels of activated JNK. NT2/D1 cells were transfected with siRNA and cell lysates harvested after 72 hours. Lysates were blotted with antibodies specific for the activated form of JNK (phospho-JNK), total JNK and β-actin.
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Additional file 6:
Knock-down of WNT5A leads to a reduction in colony formation. NT2/D1 cells were infected with lentiviral shRNAs to knockdown WNT5A expression, and results were compared to a control shRNA. Graphs depict a representative experiment performed in triplicate. Error bars represent standard deviation. a) WNT5A expression was measured using a real-time PCR assay. b) The ability of the cells to form colonies when plated at a low density is displayed. (* p < 0.05).
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