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pcdna3 0 ha sam68 wt dna plasmid (Addgene inc)

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Addgene inc pcdna3 0 ha sam68 wt dna plasmid
Pcdna3 0 Ha Sam68 Wt Dna Plasmid, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/pcdna3 0 ha sam68 wt dna plasmid/product/Addgene inc
Average 93 stars, based on 8 article reviews

pcdna3 0 ha sam68 wt dna plasmid - by Bioz Stars, 2026-03

93/100 stars

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Journal: eLife

Article Title: SAFB regulates hippocampal stem cell fate by targeting Drosha to destabilize Nfib mRNA

doi: 10.7554/eLife.74940

Figure Lengend Snippet:

Article Snippet: Recombinant DNA reagent , pcDNA3 HA Sam68 WT (plasmid) , , RRID: Addgene_17690 , pcDNA3 expression vector.

Techniques: Protease Inhibitor, Recombinant, Clone Assay, Bicinchoninic Acid Protein Assay, Mutagenesis, Transfection, Sequencing, esiRNA, Blocking Assay, Plasmid Preparation, Expressing, Software

Journal: eLife

Article Title: SAFB regulates hippocampal stem cell fate by targeting Drosha to destabilize Nfib mRNA

doi: 10.7554/eLife.74940

Figure Lengend Snippet:

Article Snippet: Recombinant DNA reagent , pcDNA3 HA Sam68 WT (plasmid) , , RRID: Addgene_17690 , pcDNA3 expression vector.

Techniques: Control, Protease Inhibitor, Recombinant, Cloning, Bicinchoninic Acid Protein Assay, Mutagenesis, Transfection, Sequencing, esiRNA, Blocking Assay, Plasmid Preparation, Expressing, Software

( 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.

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

( 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 .

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 .

Techniques: Immunolabeling, Transfection, Transduction

( 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.

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.

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

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.

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.

Techniques: Migration, Transfection, Time-lapse Microscopy

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).

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).

Techniques: Angiogenesis Assay

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

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.

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:

Techniques: Transduction, Sequencing, Blocking Assay

Figure 1. SAM68 regulates endothelial cell spreading, formation and maturation of adhesion sites. (A) siRNA-transfected cells plated overnight on uncoated glass coverslips were stained for F-actin for cell shape analysis (n=50 cells per condition; N=3). Scale bar=50 μm. Spreading Index is expressed as the ratio of occupied area (hatched area) to the cell surface (solid grey area) and elongation ratio is expressed as the ratio of the major to minor cell axis. (B) Magnification images of endothelial cells from the experimental setting described in (A). Sacle bars=20 μm and 10 μm (zoomed). Average fiber number was quantified using FIJI software (n=16 cells per condition; N=3). (C) Vinculin staining was performed on siRNA-transfected cells plated overnight on glass coverslips. Analysis of adhesion sites was performed using FIJI software by quantifying at least 15 cells per condition (N=3). Statistics: p-values: *<0.05 **<0.01; ****<0.0001. Student’s t-test (paired CTRL-siSAM68) was used for (A, B, C). Pearson’s chi-squared test was used for adhesion length distribution (C).

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: Figure 1. SAM68 regulates endothelial cell spreading, formation and maturation of adhesion sites. (A) siRNA-transfected cells plated overnight on uncoated glass coverslips were stained for F-actin for cell shape analysis (n=50 cells per condition; N=3). Scale bar=50 μm. Spreading Index is expressed as the ratio of occupied area (hatched area) to the cell surface (solid grey area) and elongation ratio is expressed as the ratio of the major to minor cell axis. (B) Magnification images of endothelial cells from the experimental setting described in (A). Sacle bars=20 μm and 10 μm (zoomed). Average fiber number was quantified using FIJI software (n=16 cells per condition; N=3). (C) Vinculin staining was performed on siRNA-transfected cells plated overnight on glass coverslips. Analysis of adhesion sites was performed using FIJI software by quantifying at least 15 cells per condition (N=3). Statistics: p-values: *<0.05 **<0.01; ****<0.0001. Student’s t-test (paired CTRL-siSAM68) was used for (A, B, C). Pearson’s chi-squared test was used for adhesion length distribution (C).

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: Transfection, Staining, Software

Figure 2. SAM68 localization in spreading cells. (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.

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: Figure 2. SAM68 localization in spreading cells. (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

Figure 3. SAM68 regulates integrin signaling and RNA composition at adhesion sites. (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

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: Figure 3. SAM68 regulates integrin signaling and RNA composition at adhesion sites. (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

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

Figure 4. SAM68 is involved local delivery of β-actin mRNA at adhesion sites. (A) smRNA FISH and SAM68 stainings were performed 20 min after deposition of FN-coated beads. Scale bars top image=10 μm, enlarged area 5 μm. Arrowheads point to overlapping signals between β-actin mRNA and SAM68 protein. (B) smRNA FISH of β-actin performed on cells 20 min after addition of FN-coated beads to cultures of siCTRL- or siSAM68-transfected cells (n=at least 12 beads per condition, N=3). Scale bars = left 10 μm, enlarged area 5 μm. (C) smiRNA FISH staining of β-actin performed on cells 20 min after deposition of FN-coated beads onto endothelial cells transfected with CTRL or blocking oligonucleotides (#SBE1 and #SBE2, as indicated) (n=at least 12 beads per condition, N=3). Statistics: p-values: *<0.05 **<0.01. Student’s t-test (paired CTRL-siSAM68) was used.

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: Figure 4. SAM68 is involved local delivery of β-actin mRNA at adhesion sites. (A) smRNA FISH and SAM68 stainings were performed 20 min after deposition of FN-coated beads. Scale bars top image=10 μm, enlarged area 5 μm. Arrowheads point to overlapping signals between β-actin mRNA and SAM68 protein. (B) smRNA FISH of β-actin performed on cells 20 min after addition of FN-coated beads to cultures of siCTRL- or siSAM68-transfected cells (n=at least 12 beads per condition, N=3). Scale bars = left 10 μm, enlarged area 5 μm. (C) smiRNA FISH staining of β-actin performed on cells 20 min after deposition of FN-coated beads onto endothelial cells transfected with CTRL or blocking oligonucleotides (#SBE1 and #SBE2, as indicated) (n=at least 12 beads per condition, N=3). Statistics: p-values: *<0.05 **<0.01. 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: Transfection, Staining, Blocking Assay

Figure 5. SAM68 is involved in FN assembly and expression. (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

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: Figure 5. SAM68 is involved in FN assembly and expression. (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

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: Expressing, Immunofluorescence, Staining, Transfection

Figure 6. SAM68 regulates ECM protein deposition and mRNA biogenesis of matrisome genes. (A) qPCR analysis of selected mRNA expression in siRNA transfected cells (N=5). (B) Representative images of collagen IV and perlecan staining of siRNA transfected cells 48 hr after plating on uncoated glass coverslips. Scale bars=50 μm. (C) Quantification of collagen IV and perlecan staining (N=5). (D) (left) Schematic of detachment assay used to determine release of siRNA-transfected cells from their self-assembled ECM support. (right) Quantification of adherent, trypsin-resistant cells (N=3). Statistics: p-values: *<0.05 **<0.01 ***<0.001 ****<0.0001. Student’s t-test (paired CTRL-siSAM68) was used for (A, C, D).

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: Figure 6. SAM68 regulates ECM protein deposition and mRNA biogenesis of matrisome genes. (A) qPCR analysis of selected mRNA expression in siRNA transfected cells (N=5). (B) Representative images of collagen IV and perlecan staining of siRNA transfected cells 48 hr after plating on uncoated glass coverslips. Scale bars=50 μm. (C) Quantification of collagen IV and perlecan staining (N=5). (D) (left) Schematic of detachment assay used to determine release of siRNA-transfected cells from their self-assembled ECM support. (right) Quantification of adherent, trypsin-resistant cells (N=3). Statistics: p-values: *<0.05 **<0.01 ***<0.001 ****<0.0001. Student’s t-test (paired CTRL-siSAM68) was used for (A, C, D).

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: Expressing, Transfection, Staining

Figure 7. SAM68 depletion regulates endothelial cell migration and angiogenic sprouting behavior. (A) Migration of individual siRNA-transfected cells was analyzed by time lapse microscopy. Individual tracks of one representative experiment are plotted and cell velocity as well total distance travelled are quantified (N=3). (B) Representative images from a wound assay experiment on siRNA transfected cells are shown together with quantification of

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: Figure 7. SAM68 depletion regulates endothelial cell migration and angiogenic sprouting behavior. (A) Migration of individual siRNA-transfected cells was analyzed by time lapse microscopy. Individual tracks of one representative experiment are plotted and cell velocity as well total distance travelled are quantified (N=3). (B) Representative images from a wound assay experiment on siRNA transfected cells are shown together with quantification of

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

$Figure 8. SAM68 regulates integrin adhesion maturation and matrix conditioning in endothelial cells. Upon integrin activation, a cytoplasmic fraction of SAM68 participates transiently in adhesion complex stabilization, through regulation of integrin signaling and localization of β-actin mRNA. In the nucleus, SAM68 concomitantly regulates the expression of key subendothelial matrix genes, thereby promoting basement membrane assembly and conditioning. These coalescent functions of SAM68 enhance endothelial cell adaptation to their microenvironment and point to an important role for SAM68 during angiogenesis.$

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: Figure 8. SAM68 regulates integrin adhesion maturation and matrix conditioning in endothelial cells. 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, Membrane

Journal: Nature Communications

Article Title: Fast-exchanging spirocyclic rhodamine probes for aptamer-based super-resolution RNA imaging

doi: 10.1038/s41467-023-39611-1

Figure Lengend Snippet: a Schematic illustration of the construct used for expressing RhoBAST-tagged GFP mRNA. b Confocal images of HEK293T cells expressing GFP-RhoBAST 16 or GFP (control) mRNA incubated with SpyRho (100 nM) for 1 h before imaging. At least three independent experiments were carried out with similar results. c Normalized fluorescence profile along the dashed line in panel b (lower left). d Schematic illustration of the constructs used for expressing CGG-containing FMR1-GFP mRNAs fused to 16 repeats of RhoBAST and HaloTag7-Sam68. e Confocal images of live Cos7 cells expressing CGG 99 -FMR1-GFP-RhoBAST 16 , CGG 99 -FMR1-GFP or GFP (control) mRNA and HaloTag7-Sam68 fusion protein (plasmid ratio 10:1) incubated with SpyRho (100 nM) and MaP700-Halo (200 nM, SiR channel) for 1 h before imaging. At least three independent experiments were carried out with similar results. f Normalized fluorescence profiles along the dashed lines shown in e . The fluorescence in the TMR channel was normalized to the highest value detected for CGG 99 -FMR1-GFP mRNA. Scale bars, 5 µm.

Article Snippet: The vector backbone and the HaloTag7 insert were amplified by PCR using the pcDNA3-HA-Sam68-WT vector (Addgene, plasmid #17690) and pcDNA5-FRT- Halo-SNAP-NLS plasmid (kindly provided by the Johnsson group, Heidelberg) as templates.

Techniques: Construct, Expressing, Incubation, Imaging, Fluorescence, Plasmid Preparation

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