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Figure 5. Pkp3-catenin regulation of ETV1 reporters or target genes. HEK 293T cells were transfected with expression plasmids for Myc-xETV1 or HA-xPkp3 in the indicated combinations. (A) Pkp3's functional relationship with ETV1 was tested using an established luciferase reporter for Ets family member transcriptional activity harboring a fes gene promoter fragment in triplicate (Fes3xWT-Luc, [57]). Labelled p-values indicate changes that are statistically significant. (B) Pkp3's functional relationship with ETV1 was tested using a second established ETV1 reporter construct harboring a native matrix metalloproteinase-1 (MMP1) promoter fragment (MMP1-Luc,[48]-[50]). (C) MMP1(−87/−88)-Luc is a negative-control construct containing a non-functional ETV1 consensus binding site. Labelled p-values indicate changes that are statistical significant. Performed in parallel to Figure 4B. (D) To assess Pkp3 and ETV1 activity in a cell-free (in vitro) context, we use the Fes3xWT-Luc reporter and in vitro translated the ETV1 and Pkp3 proteins (TnT Wheat Germ Extract). (E) The indicated morpholinos (40 ng) and mRNAs (0.5 ng) were injected into one-cell (cleavage) stage Xenopus embryos followed by RNA extraction at late neurula stages, DNAse treatment, reverse transcription, and real-time PCR to assess Pkp3- depletion effects upon ETV1 target-gene transcription. The co-injection of ETV1 mRNA with Pkp3 morpholino (MO) significantly rescued the impact of Pkp3-catenin depletion. The ETV1 gene targets assessed are components of the dopamine synthesis and transport pathways and include: DDC = dopa decarboxylase; TH = tyrosine hydroxylase; SLC = solute carrier family 18 member 2; and GCH = GTP cyclohydrolase I. All real-time PCR was repeated more than three times with samples tested in triplicate with similar outcomes. Error bars represent the standard deviation from three independent experiments. Differences between negative control (SC MO+β-gal) and Pkp3 knockdown (Pkp3 MO1+ β-gal) are all statistically significant based on the Student's T-Test, demonstrating p-values <0.05. Differences between Pkp3 knockdown (Pkp3 MO1+ β-gal) and its rescue with ectopic ETV1 (Pkp3 MO1+ ETV1) are all statistically significant based on the Student's T-Test, demonstrating p-values <0.05. Image published in: Munoz WA et al. (2014) Image reproduced on Xenbase with permission of the publisher and the copyright holder. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution license Larger Image Printer Friendly View

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