Figure 1
Trans-migration of MDA-MB-231 protrusions in a modified Boyden chamber assay. (A) Immunofluorescence image showing the typical morphology of human breast cancer MDA-MB-231 cells. The cellular cytoplasm was stained for α-Tubulin (α-Tub, green) and nuclei by DAPI (Blue). Scale bar 20 μm. (B) Schematic drawing showing the principle of the Boyden chamber set-up for isolation of migrating protrusions. Cells were grown on a 1 μm pore size micro porous membrane allowing trans-migration of only the cell protrusions. The lower side of the membrane is coated with ECM protein. (C) Protrusions of MDA-MB-231 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. (D) Histone H3 is absent from the Boyden chamber lower side protrusion fraction. Western blotting was performed with histone H3 antibody and α-Tubulin antibody on cell body fraction (CF) and protrusion fraction (PF) protein samples originating from pooled material from three independent Boyden chambers.
Figure 2
RNA in MDA-MB-231 protrusions. (A-B) Ethidium bromide (EtBr) mediated visualization of RNA in protrusions of MDA-MB-231 cells grown in Boyden chamber. (A) RNase treated cells (+ RNase) are shown for control of RNA specific EtBr staining. The left panels show DAPI staining, central pictures EtBr staining and merged pictures are shown in the right panels. Arrowheads exemplify EtBr staining in cell protrusions. Scale bar 20 μm. (B) EtBr mediated visualization of RNA localization in protrusions migrated through the 1 μm pore size membrane in the Boyden chamber. The cell bodies on the upper side of the membrane seen in (A) were removed by wiping and DAPI staining was performed in parallel to the pictures shown in (A) (left panel). EtBr staining was used to visualize cell material at the membrane lower side (arrowheads, right panel). Exposure times were increased compared to (A) visualizing also the 1 μm membrane pores. Scale bar 20 μm. (C) Determination of the relative RNA localization ratio in MDA-MB-231 protrusions by RT-qPCR analyses of candidate genes. The RNA localization ratio was calculated as the relative expression level in the protrusions fraction compared to the cell body fraction. The localization ratio of RNA in protrusions was normalized to the RNA localization ratio of ARPC3 given the value 1.
Table 1
MDA-MB-231 protrusion localized mRNA
| Accession number | Name | tpm CF | tpm PF | Loc. ratio |
|---|---|---|---|---|
| NM_001184942 | C9orf25 | 12.07 | 31.49 | 2.61 |
The 100 most localized mRNA are shown with their hierarchical order of the mRNA localization ratio (Loc ratio). Expression values are shown as transcripts per million reads (tpm) from cell body fraction (CF) and cell protrusion fraction (PF).
Figure 3
DRS transcriptome analysis of purified RNA from MDA-MB-231 protrusions. (A) Comparison of DRS and RT-qPCR analyses. RNA localization ratios were normalized to ARPC3 mRNA given the value 1. (B) Annotation analysis of the 100 most MDA-MB-231 protrusion localized mRNA compared to the total group of mRNAs expressed with ≥ 5 tpm. Annotations were made using the IPA Ingenuity platform. (C) RT-qPCR confirmation analysis of selected MDA-MB-231 protrusion localized RNA identified by DRS. RNA localization ratios are normalized to ARPC3 mRNA. (D) Western blot analysis. Western blotting was performed on pooled material from three independent Boyden chamber experiments representing cell body fraction (CF) and protrusion fraction (PF). α-Tubulin Western blot analysis was used to equalize the loaded protein amounts. Subsequent western blot analyses were performed with antibodies for Histone H3, ZEB1, ANP32B and ACTB.
Figure 4
Characterization of p0071 mRNA and protein localization. (A) DRS and RT-PCR analysis of p0071 mRNA localization in MDA-MB-231 cell protrusions. The expression level of ARPC3 and p0071 mRNA in PF and CF were determined by RT-qPCR and the p0071 mRNA localization ratio normalized to ARPC3 mRNA given the value 1. p0071 mRNA was detected by primers recognizing all p0071 mRNA isoforms (pan-p0071). (B) Protein localization of p0071 in MDA-MB-231 and MCF7 cells. α–Tubulin (left panels) and p0071 (right panels) were visualized by immunofluorescence analysis in MDA-MB-231 cells (upper panels) and MCF7 cells (lower panels). Cells were counterstained with DAPI to visualize nuclei. A representative p0071 staining in a cell protrusion is indicated by arrowhead. Scale bar 20 μm (left panels) and 10 μm (right panels).
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.