Journal: Molecular Biology of the Cell

Article Title: Tissue-specific and SRSF1-dependent splicing of fibronectin, a matrix protein that controls host cell invasion

doi: 10.1091/mbc.E13-03-0142

Figure Lengend Snippet: Expression of ECM components and adhesion molecules by endometrium and dermis fibroblasts. (A) RNAs were prepared from two independent cultures for each of four matching sets of dermis and endometrium fibroblasts. The relative expression levels of extracellular matrix components and adhesion molecules were determined by RT-qPCR. Data obtained for each endometrium (•) and dermis (◯) primary cell line are plotted, and the corresponding median bars are indicated. The ribosomal phosphoprotein RPLP0 and actin were used as controls. (B) Protein fractions corresponding to the culture supernatants (Sup) and the ECM and cellular proteins (ECM + C) from matching sets of endometrium (E) and dermis (D) fibroblasts were analyzed by Western blot (left). The fibronectin (FN) and actin amount in the ECM+C fraction was quantified with the Odyssey system (LI-COR, Lincoln, NE). The fibronectin amounts relative to actin are plotted with an indication of the median value for both the endometrium (•) and dermis (◯) cultures (right).

Article Snippet: When appropriate, the supplementation of dermal fibroblasts with fibronectin (human plasma FN; BD Biosciences, San Jose, CA) was done at the time of fibroblast seeding with the addition of 50 μg of fibronectin/35-mm dish.

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: Molecular Biology of the Cell

Article Title: Tissue-specific and SRSF1-dependent splicing of fibronectin, a matrix protein that controls host cell invasion

doi: 10.1091/mbc.E13-03-0142

Figure Lengend Snippet: Endometrium and dermis fibroblasts express different fibronectin-splicing isoforms, leading to different matrix patterns. (A) Fibronectin alternative splicing in endometrium and dermis fibroblasts was tested by RT-PCR using primers encompassing the alternatively spliced EDA, EDB, and IIICS regions, respectively. The PCR products were analyzed by gel electrophoresis (DNA size markers in base pairs). The inclusion/exclusion of the exons was further confirmed by sequencing of the DNA from the bands marked with asterisks. Right, PSI values determined for the three FN exons, EDA, EDB, and IIICS, for endometrium (E) and dermis (D) fibroblasts obtained from four different donors. (B) Protein samples corresponding to the insoluble matrix fractions (deoxycholate extraction) from matching sets of endometrium and dermis fibroblasts, as well as of the dermis fibroblasts supplemented with plasma fibronectin for 3 d (+FN), were analyzed by Western blot, using an intermediate filament, lamin A/C, as a loading control. The total and EDA-containing fibronectin amounts were quantified with the Odyssey system. The fibronectin amounts relative to lamin A are plotted. (C) Increasing amounts of plasma fibronectin were added to dermis cultures for 8 h. The protein fraction corresponding to the culture supernatant (top) and the ECM and cell content (bottom) were analyzed for their content in total FN and in EDA+ FN. The amounts of FN protein were quantified and are shown in the graph. For total FN: 5 μg, p = 0.0481 (*); 10 μg, p = 0.0440 (*); 25 μg, p = 0.00891 (**); 50 μg, p = 0.0120 (*). No significant differences were found for the content in EDA+ FN, with p ranging from 0.6025 to 0.9237. (D) Endometrium and dermis fibroblasts were seeded in the same conditions as for the cocultures. For the fibronectin supplementation conditions (dermis+ FN), pFN was added to dermis fibroblasts at the time of the culture. Four days later, cultures were fixed and stained for total fibronectin (FN total), EDA-containing fibronectin (FN– EDA), and Hoechst (shown in the overlays). Confocal microscopy sections. Bar, 100 μm; higher magnification, 20 μm. Arrows show FN that is presumably EDA–, as it was bound by the anti-FN antibody (stained in red) and not by the anti–FN EDA antibody.

Article Snippet: When appropriate, the supplementation of dermal fibroblasts with fibronectin (human plasma FN; BD Biosciences, San Jose, CA) was done at the time of fibroblast seeding with the addition of 50 μg of fibronectin/35-mm dish.

Techniques: Reverse Transcription Polymerase Chain Reaction, Nucleic Acid Electrophoresis, Sequencing, Western Blot, Staining, Confocal Microscopy

Journal: Molecular Biology of the Cell

Article Title: Tissue-specific and SRSF1-dependent splicing of fibronectin, a matrix protein that controls host cell invasion

doi: 10.1091/mbc.E13-03-0142

Figure Lengend Snippet: Fibronectin supplementation of the dermis cell layer enhances trophoblast interstitial migration. Cocultures were set up between human trophoblast explants and endometrial fibroblasts, dermal fibroblasts, or dermal fibroblasts supplemented with plasma fibronectin. After 16 h of coculture, phase-contrast pictures were taken (T0) and again 24 h later (T24h). (A) Phase-contrast micrographs show interstitial EVCT migration for cocultures on endometrium and fibronectin-supplemented dermis fibroblasts and superficial EVCT mass migration (refringent packed EVCTs) in dermis coculture conditions. (B) The type of EVCT migration (superficial vs. interstitial), as well as the occurrence of trophoblast villi movement, were analyzed in the three coculture conditions. Data points corresponding to each of the independent cultures are plotted, and the median values are indicated. Two-tailed p values were determined with the Mann–Whitney rank sum test. For villi movement, ** p = 0.007, * p = 0.036; for EVCT migration, ** p = 0.002, * p = 0.015.

Article Snippet: When appropriate, the supplementation of dermal fibroblasts with fibronectin (human plasma FN; BD Biosciences, San Jose, CA) was done at the time of fibroblast seeding with the addition of 50 μg of fibronectin/35-mm dish.

Techniques: Migration, Two Tailed Test, MANN-WHITNEY

Journal: Molecular Biology of the Cell

Article Title: Tissue-specific and SRSF1-dependent splicing of fibronectin, a matrix protein that controls host cell invasion

doi: 10.1091/mbc.E13-03-0142

Figure Lengend Snippet: Fibronectin supplementation of a synthetic 3D collagen I matrix promotes isolated trophoblast cell migration. Trophoblastic villi were embedded within a 3D collagen I matrix (coll I) supplemented with plasma fibronectin when indicated (coll I/Fn). At day 4, phase-contrast pictures of the cultures were taken. Three types of EVCT migration were identified for coll I/Fn cultures (A; schematized in B): “isolated” migration, corresponding to the migration of single EVCTs in all directions; “packed” cell migration; and “no migration,” for EVCTs showing a low capacity to migrate away from the trophoblastic villi. Bar, 100 μm. (B) Statistical analysis of EVCT migration in coll I and coll I/Fn 3D culture conditions. Data were obtained with villi isolated from four placentas (>100 trophoblastic villi analyzed in each condition). For each placenta, the number of villi giving rise to each type of EVCT migration was rated and is expressed as a percentage of the total number of villi for this placenta. The median value is indicated for 3D cultures performed in collagen I (•) or in collagen I supplemented with plasma fibronectin (◯).

Article Snippet: When appropriate, the supplementation of dermal fibroblasts with fibronectin (human plasma FN; BD Biosciences, San Jose, CA) was done at the time of fibroblast seeding with the addition of 50 μg of fibronectin/35-mm dish.

Techniques: Isolation, Migration

Journal: Molecular Biology of the Cell

Article Title: Tissue-specific and SRSF1-dependent splicing of fibronectin, a matrix protein that controls host cell invasion

doi: 10.1091/mbc.E13-03-0142

Figure Lengend Snippet: Silencing of SRSF1 in endometrium reduces inclusion of fibronectin EDA exon. (A) For silencing experiments, transfections were performed with two SRSF1-targeting siRNAs (siRNA1 and siRNA2), as well as with a scrambled siRNA (siRNA scr), which was used as a control. Data obtained with cells recovered 3 d after the siRNA transfection (similar data were obtained with two rounds of 3-d transfections, with cells collected at day 6). Total protein extracts were analyzed by Western blotting for total FN, FN containing the EDA domain, SRSF1, and, as a loading control, γ-tubulin. Relative protein values were normalized to γ-tubulin concentrations. (B) Fibronectin alternative splicing in each condition was tested by RT-PCR, using primers encompassing the alternatively spliced EDA and EDB domains. PCR products were analyzed by gel electrophoresis. (A, B) Mean values with SEM. The p values were determined by a paired t test: for FN EDA, p = 0.013 (*); for FN EDB, p = 0.058 (ns). (C) In addition, control and siRNA-treated RNA samples ( n = 4 per group) were compared at the Sherbrooke RNomics platform to determine PSI for 46 alternative splicing events, including the EDA and EDB exon alternative splicings. The results are presented as a heat map showing the unsupervised hierarchical clustering of the change in splicing (ΔPSI = PSI value of the control endometrium − PSI value of the SRSF1-depleted endometrium). Splicing changes (ΔPSI values) are represented in shades of bright green (exon skipping upon SRSF1 depletion) to red (exon inclusion), as schematized in the color key histogram.

Article Snippet: When appropriate, the supplementation of dermal fibroblasts with fibronectin (human plasma FN; BD Biosciences, San Jose, CA) was done at the time of fibroblast seeding with the addition of 50 μg of fibronectin/35-mm dish.

Techniques: Transfection, Western Blot, Reverse Transcription Polymerase Chain Reaction, Nucleic Acid Electrophoresis

Journal: Molecular Biology of the Cell

Article Title: Tissue-specific and SRSF1-dependent splicing of fibronectin, a matrix protein that controls host cell invasion

doi: 10.1091/mbc.E13-03-0142

Figure Lengend Snippet: Mechanisms for the different capacities of endometrium and dermis tissues to support trophoblast invasion. 1) Endometrium fibroblasts produce higher concentrations of the SR protein SRSF1 than dermis fibroblasts, which leads 2) to higher inclusion rates of the EDA exon in the fibronectin produced by endometrium and consequently 3) to an endometrium extracellular matrix that contains more and thicker fibronectin bundles than dermis. 4) This results in a better capacity for the endometrium tissue than for dermis to support trophoblast invasion.

Article Snippet: When appropriate, the supplementation of dermal fibroblasts with fibronectin (human plasma FN; BD Biosciences, San Jose, CA) was done at the time of fibroblast seeding with the addition of 50 μg of fibronectin/35-mm dish.

Techniques: Produced