Figure 5

P0071 mRNA isoform localization in MDA-MB-231 and MCF7 cells. (A) Boyden chamber analysis of MCF7 cells. Protrusions of MCF7 cells migrated through a 1 μm micro porous membrane after 24 h assay time. α-Tubulin (green) and DAPI staining (blue) is shown without and after wipe (wipe) to remove cellular material present on the upper side of membranes. Examples of protrusions present on the Boyden chamber membrane lower side are indicated by arrowheads. Scale bar 100 μm. (B) RT-PCR analysis of mRNA localization in MCF7 and MDA-MB-231 cells. The RNA localization ratio was calculated as the relative expression in the protrusion fraction compared to the cell body fraction. The localization ratio was normalized to the RNA localization level of ARPC3 mRNA given the value 1. Pan-p0071 indicates detection of p0071 cDNA from exon 6 to exon 7. Student’s 2-Tailed t-test: *, p < 0.05; **, p < 0.005. (C) Alternative spliced p0071 mRNA isoforms. Schematic drawing of p0071 mRNA isoforms involving the 3′-region of the p0071 gene detected at UCSC Genome Browser (NCBI36/hg18 assembly). pA indicates poly-adenylation signals. Exon 19a and exon 20a represents novel p0071 exon sequences where exon 19a is a 3′-extension of exon 19. Alternative splicing is indicated above the exons and the consensus p0071 splicing below. The four p0071 mRNA isoforms are schematically shown with exons 1 to 16 only indicated. Asterisk above exons show translational stop codons. (D) Expression level analysis of p0071 mRNA isoforms in MCF7 cells relative to MDA-MB-231 cells. Expression of p0071 mRNA isoforms was detected by RT-qPCR, normalized to GAPDH expression, and the expression ratio between MCF7 and MDA-MB-231 cells calculated by division. RT-qPCR experiments were performed minimum three independent times and analyzed with Student’s 2-Tailed t-test: *, p < 0.05; **, p < 0.005.