Journal: eLife

Article Title: Secreted exosomes induce filopodia formation

doi: 10.7554/eLife.101673

Figure Lengend Snippet: ( A ) Purified LEVs and SEVs were run on a colloidal blue-stained gel. Four arrows denote SEV bands that were cut and submitted for proteomics, along with notable proteins identified (see for the full proteomics results). ( B ) B16F1 total cell lysate (TCL), LEVs, and density gradient purified SEVs were run on an SDS-PAGE gel and probed by western blot for endoglin, and EV positive (HSP70, TSG101, flotillin-1, and CD63) and negative (GM130) markers. ( C ) Total cell lysate (TCL) and small EVs (SEVs) from endoglin-KD (shEng) and control (shScr) B16F1 cells were run on an SDS-PAGE gel and probed by western blot for endoglin, EV marker TSG101, and EV-negative marker GM130. ( D ) Representative images from control (shScr) or endoglin-KD (shEng) B16F1 cell lines incubated for 18 hr with no EVs (left panels), or with SEVs purified from control (+shScr SEVs) or shEng cell lines (+shEng SEVs) (right panels). Arrowheads indicate example filopodia. Scale bar = 10 mm. ( E ) Quantification of filopodia in control (shScr) and endoglin knockdown (shEng) cells treated with the indicated number of LEVs or SEVs for 18 hr (≥20 cells per condition per biological replicate, from three biological replicates). ( F ) Filopodia number in B16F1 control (shLacZ) or exosome-depleted (shHrs) cells treated with indicated numbers of LEVs, control (shScr) SEVs, or endoglin-KD (shEng1) SEVs for 18 hr. ≥20 cells per condition per biological replicate, from three biological replicates. Representative images for this experiment are shown in . ( G, H ) B16F1 cells were transfected with tdTomato-F-Tractin and imaged live every 30 s for 15 min. Only filopodia that form and fully retract during the duration of each video were quantified. ( G ) De novo filopodia formation. ( H ) Filopodia lifetime, defined as the time from initial filopodia formation to full retraction. Bars represent mean and error bars are SEM. (³25 total cells per type per biological replicate, from three biological replicates) ns, not significant; * p<0.05; ** p<0.01; *** p<0.001. Figure 4—source data 1. PDF file containing original blot for , indicating the relevant bands. Figure 4—source data 2. Original file for Coomassie blue gel displayed in . Figure 4—source data 3. PDF file containing the original western blots from , indicating the relevant bands. Figure 4—source data 4. Original files for western blot analysis displayed in . Figure 4—source data 5. PDF file containing the original western blots from , indicating the relevant bands. Figure 4—source data 6. Original files for western blot analysis displayed in .

Article Snippet: Antibody , anti-human Endoglin (Rabbit monoclonal) , Cell Signaling , 4335 , WB 1:1000.

Techniques: Purification, Staining, SDS Page, Western Blot, Control, Marker, Incubation, Knockdown, Transfection

Journal: eLife

Article Title: Secreted exosomes induce filopodia formation

doi: 10.7554/eLife.101673

Figure Lengend Snippet: ( A ) Nanoparticle tracking analysis traces for B16F1 control (shScr) and endoglin-KD (shEng) SEVs. ( B ) SEV secretion rates from B16F1 shEng stable lines. N=5 biological replicates. ( C ) Representative western blot of Endoglin-KD in transient siRNA-transfected B16F1 cells. ( D ) Filopodia numbers in siRNA-transfected B16F1 cells (≥23 cells per condition per biological replicate, from three biological replicates). ( E ) Images from control and shHrs cells incubated with purified EV, corresponding to graph in . Scale bar in wide field and zoom insets = 10 mm. Error bars, SEM. ns, not significant; * p<0.05; ** p<0.01; *** p<0.001. Figure 4—figure supplement 1—source data 1. PDF file containing the original western blots from , indicating the relevant bands. Figure 4—figure supplement 1—source data 2. Original files for western blot analysis displayed in .

Article Snippet: Antibody , anti-human Endoglin (Rabbit monoclonal) , Cell Signaling , 4335 , WB 1:1000.

Techniques: Control, Western Blot, Transfection, Incubation, Purification

Journal: eLife

Article Title: Secreted exosomes induce filopodia formation

doi: 10.7554/eLife.101673

Figure Lengend Snippet: ( A ) Western blot of Endoglin KD in HT1080 cells. ( B ) Nanoparticle tracking analysis traces of SEVs purified from shScr and shEng HT1080 cells showing size distribution (diameter) of SEVs and particles/mL/cell (N=3 biological replicates). ( C ) SEV secretion rates of HT1080 shScr and shEng HT1080 cells. ( D ) Representative images of HT1080 shScr and shEng cells. Images have been edited with brightness and contrast for ease of visibility. Scale bar in wide field and zoom insets = 10 mm. ( E ) Quantitation of filopodia density for control and shEng HT1080 cells.≥20 cells per condition per biological replicate, from four biological replicates. Error bars, SEM. ns, not significant; * p<0.05; ** p<0.01; *** p<0.001. Figure 4—figure supplement 2—source data 1. PDF file containing the original western blots from , indicating the relevant bands. Figure 4—figure supplement 2—source data 2. Original files for western blot analysis displayed in .

Article Snippet: Antibody , anti-human Endoglin (Rabbit monoclonal) , Cell Signaling , 4335 , WB 1:1000.

Techniques: Western Blot, Purification, Quantitation Assay, Control

Journal: eLife

Article Title: Secreted exosomes induce filopodia formation

doi: 10.7554/eLife.101673

Figure Lengend Snippet: ( A ) Western blot showing b1-integrin, TGFb1, ALK1 levels in control (shScr) and endoglin-KD (shEng) B16F1 SEVs. ( B ) Filopodia density analysis of B16F1 shScr and shEng cells treated with BMP-9.≥20 cells per condition per biological replicate, from three biological replicates. ( C ) Filopodia density analysis of B16F1 shScr and shEng cells treated with TGFb1.≥20 cells per condition per biological replicate, from three biological replicates. ( D ) Filopodia density analysis of B16F1 shScr and shEng cells plated on 20 µg/ml fibronectin (FN) or 100 µg/mL poly-D-lysine (PDL). ≥20 cells per condition per biological replicate, from three biological replicates. ( E ) Filopodia density analysis of B16F1 shScr and shEng cells plated on PDL or 2 µg/mL rhTHSD7A for the indicated time points, then fixed and stained for filopodia. ≥20 cells per condition per biological replicate, from biological replicates. Figure 6—figure supplement 1—source data 1. PDF file containing the original western blots from , indicating the relevant bands. Figure 6—figure supplement 1—source data 2. Original files for western blot analysis displayed in .

Article Snippet: Antibody , anti-human Endoglin (Rabbit monoclonal) , Cell Signaling , 4335 , WB 1:1000.

Techniques: Western Blot, Control, Staining

Journal: eLife

Article Title: Secreted exosomes induce filopodia formation

doi: 10.7554/eLife.101673

Figure Lengend Snippet: ( A ) Western blot analysis of total cell lysates (TCL) and SEVs from HT1080 control and shEng cells +/-rescue with WT endoglin or control expression vectors. The figure was made from cropped images of membranes to remove irrelevant lanes. ( B ) Quantification of endoglin expression (normalized to flotillin-1 as a loading control, and relative to shScr control) from triplicate Western blots as in A. ( C ) Quantification of THSD7A expression (relative to flotillin-1 as a loading control, and relative to shScr control) from triplicate Western blots as in A. ( D ) Quantification of filopodia in HT1080 control cells and shEng cells rescued with WT endoglin expression. N=3, at least 30 total cells per condition. ( E ) Representative confocal images of THSD7A-mScarlet-expressing control and shEng HT1080 cells immunostained for CD63. Box 1 shows extracellular THSD7A and CD63 deposits. Box 2 shows intracellular CD63-positive MVEs. For both boxes, the zoomed images have been adjusted for brightness and contrast (to equivalent levels for control and shEng cells) for easy visualization. Note that the overlap of THSD7A (magenta) and CD63 (green) gives a white signal, pointed out with white arrowheads in the shEng merged image in Zoom 2. Scale bar is 10 mm in wider field view and 5 mm in zoom insets. ( F ) Quantification of colocalization of internal CD63 and mScarlet signals in HT1080 cells from nonadjusted images.≥20 cells per condition per biological replicate, from three biological replicates. Error bars, SEM. ns, not significant; * p<0.05; ** p<0.01; *** p<0.001. Figure 7—source data 1. PDF file containing the original western blots from , indicating the relevant bands. Figure 7—source data 2. Original files for western blot analysis displayed in .

Article Snippet: Antibody , anti-human Endoglin (Rabbit monoclonal) , Cell Signaling , 4335 , WB 1:1000.

Techniques: Western Blot, Control, Expressing

Journal: eLife

Article Title: Secreted exosomes induce filopodia formation

doi: 10.7554/eLife.101673

Figure Lengend Snippet: Control and endoglin-KD HT1080 cells were plated on coverslips coated with poly-D-lysine (PDL) or THSD7A. In some cases, cells were treated with the Cdc42 inhibitor ML141 (10 µM) or transfected with the dominant active Cdc42 mutant Q61L, as indicated.≥20 cells per condition per biological replicate, from three biological replicates. Error bars, SEM. ns, not significant; * p<0.05; ** p<0.01; *** p<0.001.

Article Snippet: Antibody , anti-human Endoglin (Rabbit monoclonal) , Cell Signaling , 4335 , WB 1:1000.

Techniques: Control, Transfection, Mutagenesis

Journal: eLife

Article Title: Secreted exosomes induce filopodia formation

doi: 10.7554/eLife.101673

Figure Lengend Snippet: ( A ) In tumor cells, endoglin and THSD7A are trafficked into intralumenal vesicles (ILV) in multivesicular endosomes (MVEs) for secretion. Inhibiting the exosome biogenesis pathway by blocking Hrs or inhibiting MVE docking by blocking Rab27a subsequently reduces exosome secretion and filopodia formation. SEVs carrying THSD7A can induce filopodia on target cells via Cdc42, leading to increased cell motility and metastasis. When endoglin levels are lowered (such as by KD), THSD7A is retained inside cells in CD63-positive endosomes, and its levels in SEVs are greatly decreased. The drop in THSD7A levels in endoglin-KD EVs could be due either to a lack of trafficking into ILVs or, alternatively, enhanced lysosomal degradation of THSD7A-containing MVEs. Given that THSD7A accumulates in CD63-positive endolysosomal compartments in endoglin-KD cells , the latter possibility seems more likely. The cartoon was created using BioRender.com . ( B ) In primary neurons, exosome biogenesis is controlled by the formation of ILVs by Hrs and MVE docking is controlled by Rab27b. Knockdown of either of these proteins results in reduced formation of filopodia, dendritic spines, and synapses in both cortical and hippocampal neurons. Similar to cancer cells, THSD7A is carried in neuronal SEVs and induces filopodia. The cartoon was created using BioRender.com .

Article Snippet: Antibody , anti-human Endoglin (Rabbit monoclonal) , Cell Signaling , 4335 , WB 1:1000.

Techniques: Blocking Assay, Knockdown