Journal: eLife

Article Title: Coalescent RNA-localizing and transcriptional activities of SAM68 modulate adhesion and subendothelial basement membrane assembly

doi: 10.7554/eLife.85165

Figure Lengend Snippet: ( A ) HUVECs plated on FN-coated coverslips for 20 min were stained for SAM68, F-actin, cortactin and phospho-tyrosine. Scale bars=10 μm. Dotted squares depict enlarged areas (10 μm wide) shown in the same panel. ( B ) Labelling of SAM68 and FAK was performed on HUVECs plated on FN-coated coverslips for the indicated times; dotted squares depict enlarged areas shown in the same panel.

Article Snippet: The SAM68 WT lentivirus expression vector was generated by insertion of a FLAG-tagged SAM68 coding sequence into pLenti-CMV-MCS-GFP-SV-puro plasmid (gift from Paul Odgren Addgene plasmid # 73582) between XbaI and MluI restriction sites.

Techniques: Staining

Journal: eLife

Article Title: Coalescent RNA-localizing and transcriptional activities of SAM68 modulate adhesion and subendothelial basement membrane assembly

doi: 10.7554/eLife.85165

Figure Lengend Snippet: ( A ) Scheme of the experimental procedure used to induce and image artificial adhesion sites in contact with FN-coated beads. ( B ) Labelling of SAM68 and vinculin was performed 20 min after seeding cells on FN-coated beads. Dotted squares in top panels depict enlarged z-projections shown in the middle panel. Orthogonal views are shown in bottom panels. Scale bars=5 μm. ( C ) Immunolabeling of α5β1 and activated FAK (pFAK-Y397) were performed 20 min after deposition of FN-coated beads onto siCTRL- or siSAM68-transfected cells and pFAK-Y397 foci were quantified (n=at least 8 beads per condition, N=3). ( D ) siSAM68-transfected cells were transduced with lentiviral constructions encoding SAM68 WT and mutants shown in the left panel of the figure. Immunolabeling of activated FAK (pFAK-Y397) was performed 20 min after deposition of FN-coated beads onto cells and pFAK-Y397 foci were quantified (n=at least 5 beads per condition, N=4). Statistics: p-values: *<0.05 **<0.01. Student’s t-test (paired CTRL-siSAM68) was used for ( C, D ). Figure 3—source data 1. Quantification of pFAK397 foci from . Figure 3—source data 2. Quantification of pFAK397 foci from .

Article Snippet: The SAM68 WT lentivirus expression vector was generated by insertion of a FLAG-tagged SAM68 coding sequence into pLenti-CMV-MCS-GFP-SV-puro plasmid (gift from Paul Odgren Addgene plasmid # 73582) between XbaI and MluI restriction sites.

Techniques: Immunolabeling, Transfection, Transduction

Journal: eLife

Article Title: Coalescent RNA-localizing and transcriptional activities of SAM68 modulate adhesion and subendothelial basement membrane assembly

doi: 10.7554/eLife.85165

Figure Lengend Snippet: ( A ) Immunofluorescence staining of α5β1 integrin was performed to identify and quantify fibrillar adhesion in siRNA transfected cells plated overnight on glass coverslips (n=at least 15; N=3). ( B ) Immunofluorescence staining of FN was performed on siRNA transfected cells plated on glass coverslips and quantification on whole-coverslip scans is expressed as the ratio of FN-stained area to the number of cells (N=8). Representative 40x field views. ( C ) Representative high magnification images of FN staining are shown with areas between the dotted lines selected for fluorescence intensity profiles. ( D ) Western blot analysis of cell-associated FN in siRNA transfected cells with densitometric quantification indicated below (N=3). ( E ) qPCR analysis of total FN (tFN) and Extra Domain-containing isoform expression in siRNA transfected cells using the indicated qPCR primer pairs (N=7). ( F ) Measurements of Luciferase activity driven by the FN1 promoter when SAM68 is overexpressed (N=5). ( G ) DNA fragments located in the FN1 promoter were quantified by qPCR in anti-SAM68 or IgG immunoprecipitated complexes (N=3). Statistics: p-values: *<0.05 **<0.01 ***<0.001 ****<0.0001. Student’s t-test (paired CTRL-siSAM68) was used for ( A, B, D, E, F ). Statistical analysis of fold enrichment in ( G ) was performed with R using pairwise t-test with p-values adjusted using ‘Bonferroni correction’. Figure 5—source data 1. Quantification of endothelial fibrillar adhesions. Figure 5—source data 2. Western blot uncropped membranes. Figure 5—source data 3. Quantification of FN1 mRNA levels. Figure 5—source data 4. Quantification of FN1 promotor reporter activity. Figure 5—source data 5. Quantification of SAM68 protein recruitment onto the FN1 promotor.

Article Snippet: The SAM68 WT lentivirus expression vector was generated by insertion of a FLAG-tagged SAM68 coding sequence into pLenti-CMV-MCS-GFP-SV-puro plasmid (gift from Paul Odgren Addgene plasmid # 73582) between XbaI and MluI restriction sites.

Techniques: Immunofluorescence, Staining, Transfection, Fluorescence, Western Blot, Expressing, Luciferase, Activity Assay, Immunoprecipitation

Journal: eLife

Article Title: Coalescent RNA-localizing and transcriptional activities of SAM68 modulate adhesion and subendothelial basement membrane assembly

doi: 10.7554/eLife.85165

Figure Lengend Snippet: Migration of individual siRNA-transfected cells was analyzed by time lapse microscopy. ( A ) Representation of individual tracks of the experiment presented in for the second siRNA directed against SAM68. ( B ) Additional quantification of average speed and total distance travelled are shown (N=3). Statistics: p-values: *<0.05. Student’s t-test (paired CTRL-siSAM68) was used.

Article Snippet: The SAM68 WT lentivirus expression vector was generated by insertion of a FLAG-tagged SAM68 coding sequence into pLenti-CMV-MCS-GFP-SV-puro plasmid (gift from Paul Odgren Addgene plasmid # 73582) between XbaI and MluI restriction sites.

Techniques: Migration, Transfection, Time-lapse Microscopy

Journal: eLife

Article Title: Coalescent RNA-localizing and transcriptional activities of SAM68 modulate adhesion and subendothelial basement membrane assembly

doi: 10.7554/eLife.85165

Figure Lengend Snippet: Distribution of average sprout length per bead observed in the 3D angiogenesis assay presented in . Results from two SAM68-targeting siRNAs in four different experiments are shown (N1 to N4).

Article Snippet: The SAM68 WT lentivirus expression vector was generated by insertion of a FLAG-tagged SAM68 coding sequence into pLenti-CMV-MCS-GFP-SV-puro plasmid (gift from Paul Odgren Addgene plasmid # 73582) between XbaI and MluI restriction sites.

Techniques: Angiogenesis Assay

Journal: eLife

Article Title: Coalescent RNA-localizing and transcriptional activities of SAM68 modulate adhesion and subendothelial basement membrane assembly

doi: 10.7554/eLife.85165

Figure Lengend Snippet: Upon integrin activation, a cytoplasmic fraction of SAM68 participates transiently in adhesion complex stabilization, through regulation of integrin signaling and localization of β-actin mRNA. In the nucleus, SAM68 concomitantly regulates the expression of key subendothelial matrix genes, thereby promoting basement membrane assembly and conditioning. These coalescent functions of SAM68 enhance endothelial cell adaptation to their microenvironment and point to an important role for SAM68 during angiogenesis.

Article Snippet: The SAM68 WT lentivirus expression vector was generated by insertion of a FLAG-tagged SAM68 coding sequence into pLenti-CMV-MCS-GFP-SV-puro plasmid (gift from Paul Odgren Addgene plasmid # 73582) between XbaI and MluI restriction sites.

Techniques: Activation Assay, Expressing

Journal: eLife

Article Title: Coalescent RNA-localizing and transcriptional activities of SAM68 modulate adhesion and subendothelial basement membrane assembly

doi: 10.7554/eLife.85165

Figure Lengend Snippet:

Article Snippet: The SAM68 WT lentivirus expression vector was generated by insertion of a FLAG-tagged SAM68 coding sequence into pLenti-CMV-MCS-GFP-SV-puro plasmid (gift from Paul Odgren Addgene plasmid # 73582) between XbaI and MluI restriction sites.

Techniques: Transduction, Sequencing, Blocking Assay