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b16 f1 mouse melanoma cells (ATCC)

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ATCC b16 f1 mouse melanoma cells
B16 F1 Mouse Melanoma Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 97/100, based on 1202 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/b16+f1+mouse+melanoma+cells/B16-F1/pm42032025-161-0-5
Average 97 stars, based on 1202 article reviews

b16 f1 mouse melanoma cells - by Bioz Stars, 2026-10

97/100 stars

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Knock-Out:

Article Title: The NHSL1-A complex interacts with the Arp2/3 complex and controls cell migration efficiency and chemotaxis
Article Snippet: .. HEK293FT cells (Thermo Fisher Scientific R70007), B16-F1 mouse melanoma cells (ATCC CRL-6323), and the B16-F1 NHSL1 knockout cell line were cultured in Dulbecco’s modified Eagle’s medium containing 1% penicillin/streptomycin, 2 mM L-glutamine and 10% fetal bovine serum (Gibco). ..

Cell Culture:

Article Title: NHSL3 interacts with Ena/VASP proteins and the Scar/WAVE complex and promotes cell migration
Article Snippet: .. HEK293FT cells (Thermo Fisher Scientific R70007) and B16-F1 mouse melanoma cells (ATCC CRL-6323) were cultured in Dulbeccos modified Eagle’s medium containing penicillin/streptomycin, L-glutamine and 10% fetal bovine serum. .. Transient transfections in HEK293FT cells were carried out using Lipofectamine 2000 (Invitrogen) according to manufacturer’s instructions or PEI (Sigma 913375 or 408727: add 2ug DNA in 100ul OptiMEM to 4ul 2mg/ml PEI in 100ul OptiMEM, mix and incubate for 20 min room temperature).

Article Title: A novel role of erythropoietin in skin pigmentation through melanin production.
Article Snippet: .. B16/F1 mouse melanoma cells (CRL-6323, ATCC, Manassas, VA, USA) and HaCaT human keratinocytes were cultured in Dulbecco’s Modified Eagle Medium (30-2002, ATCC, Manassas, VA, USA) containing 10% fetal bovine serum and 1% penicillin and streptomycin. ..

Modification:

Tissue:

Article Title: Profilin promotes lamellipodium protrusion by tuning the antagonistic activities of CP and VASP
Article Snippet: .. B16-F1 mouse melanoma cells (purchased from American Type Culture Collection, Manassas, VA, CRL-6323) were cultivated according to standard tissue culture conditions. .. In brief, cells were grown at 37°C in an atmosphere containing 7.5% CO2 in Dulbecco’s modified Eagle medium (DMEM) containing 4.5 g/l glucose (Life Technologies, Thermo Fisher Scientific, Germany) and 10% fetal calf serum (FCS; Gibco, Paisley, UK), 2 mM glutamine and 1% penicillin-streptomycin (Thermo Fisher Scientific).

Derivative Assay:

Article Title: Unleashed Actin Assembly in Capping Protein-Deficient B16-F1 Cells Enables Identification of Multiple Factors Contributing to Filopodium Formation.
Article Snippet: .. NIH 3T3 fibroblasts (ATCC CRL-1658), B16-F1 mouse melanoma cells (ATCC CRL6323) and derived mutants were cultivated at 37 ◦C and 5% CO2 in high-glucose DMEM culture medium (Lonza, Cologne, Germany) supplemented with 1% penicillin-streptomycin (Biowest, Nuaille’, France), 10% FBS (Biowest) and 2 mM UltraGlutamine (Lonza). .. A total of 3 h after seeding onto 35 mm diameter wells (Sarstedt, Nümbrecht, Germany), the cells were transfected with 1 μg (B16-F1) or 3 μg (NIH 3T3) plasmid DNA using JetPRIME transfection reagent (PolyPlus) at a ratio of 1 μg of DNA to 2 μL of the transfection reagent, according to the manufacturer’s protocol.

Article Title: Unleashed Actin Assembly in Capping Protein-Deficient B16-F1 Cells Enables Identification of Multiple Factors Contributing to Filopodium Formation
Article Snippet: .. NIH 3T3 fibroblasts (ATCC CRL-1658), B16-F1 mouse melanoma cells (ATCC CRL-6323) and derived mutants were cultivated at 37 °C and 5% CO 2 in high-glucose DMEM culture medium (Lonza, Cologne, Germany) supplemented with 1% penicillin-streptomycin (Biowest, Nuaille’, France), 10% FBS (Biowest) and 2 mM UltraGlutamine (Lonza). .. A total of 3 h after seeding onto 35 mm diameter wells (Sarstedt, Nümbrecht, Germany), the cells were transfected with 1 μg (B16-F1) or 3 μg (NIH 3T3) plasmid DNA using JetPRIME transfection reagent (PolyPlus) at a ratio of 1 μg of DNA to 2 μL of the transfection reagent, according to the manufacturer’s protocol.

Image Search Results

B16F1 cellular behaviors of A@DFX, AB@DFX and RGD AB@DFX NPs (A) Fluorescence microscopy images of cells treated with various FITC-labeled NPs for 6 h. Scale bars, 10 μm. ( n = 3 independent biological replicates). (B) Cell viability assessment after 24 h of incubation with cells by MTT assay. Data are expressed as means ± SD ( n = 3 independent biological replicates). Statistical analysis: one-way ANOVA followed by Tukey’s post hoc test. ∗ p < 0.05. (C) Western blot analysis of p -AKT and PD-L1 expression of cells treated with various NPs for 6 h ( n = 3 independent biological replicates).

Journal: iScience

Article Title: Targeted delivery and controlled release of deferasirox for melanoma therapy

doi: 10.1016/j.isci.2026.115303

Figure Lengend Snippet: B16F1 cellular behaviors of A@DFX, AB@DFX and RGD AB@DFX NPs (A) Fluorescence microscopy images of cells treated with various FITC-labeled NPs for 6 h. Scale bars, 10 μm. ( n = 3 independent biological replicates). (B) Cell viability assessment after 24 h of incubation with cells by MTT assay. Data are expressed as means ± SD ( n = 3 independent biological replicates). Statistical analysis: one-way ANOVA followed by Tukey’s post hoc test. ∗ p < 0.05. (C) Western blot analysis of p -AKT and PD-L1 expression of cells treated with various NPs for 6 h ( n = 3 independent biological replicates).

Article Snippet: Mouse Melanoma Cells (B16F1) , ATCC , CRL-6323.

Techniques: Fluorescence, Microscopy, Labeling, Incubation, MTT Assay, Western Blot, Expressing

Pharmacokinetics and biodistribution of various NPs in B16F1 tumor-bearing mice (A) Plasma concentration-time curve of DFX of mice intravenously injected with DFX, A@DFX NPs, AB@DFX NPs and RGD AB@DFX NPs during 24 h. Data are expressed as means ± SD ( n = 3 mice). (B) Ex vivo fluorescence images of tumor and major organs (heart, liver, spleen, lung, and kidney) of mice intravenously injected with DiR-labeled NPs ( RGD AB@DiR NPs, AB@DiR NPs and A@DiR NPs) and DiR; the fluorescence images were collected at 24 h post-injection ( n = 3 mice). (C) Quantitative fluorescence intensity analysis in (B). Data are expressed as means ± SD ( n = 3 mice).

Journal: iScience

Article Title: Targeted delivery and controlled release of deferasirox for melanoma therapy

doi: 10.1016/j.isci.2026.115303

Figure Lengend Snippet: Pharmacokinetics and biodistribution of various NPs in B16F1 tumor-bearing mice (A) Plasma concentration-time curve of DFX of mice intravenously injected with DFX, A@DFX NPs, AB@DFX NPs and RGD AB@DFX NPs during 24 h. Data are expressed as means ± SD ( n = 3 mice). (B) Ex vivo fluorescence images of tumor and major organs (heart, liver, spleen, lung, and kidney) of mice intravenously injected with DiR-labeled NPs ( RGD AB@DiR NPs, AB@DiR NPs and A@DiR NPs) and DiR; the fluorescence images were collected at 24 h post-injection ( n = 3 mice). (C) Quantitative fluorescence intensity analysis in (B). Data are expressed as means ± SD ( n = 3 mice).

Article Snippet: Mouse Melanoma Cells (B16F1) , ATCC , CRL-6323.

Techniques: Drug discovery, Clinical Proteomics, Concentration Assay, Injection, Ex Vivo, Fluorescence, Labeling

In vivo therapeutic effect of RGD AB@DFX NPs in B16F1 tumor-bearing mice (A) Tumor growth curves during 21 days of treatment with PBS, A NPs, DFX, A@DFX NPs, AB@DFX NPs and RGD AB@DFX NPs. Data are expressed as means ± SD ( n = 3 mice). Statistical analysis: one-way ANOVA followed by Tukey’s post hoc test. ∗ p < 0.05. (B) Photographs of tumors at the end of 21 days of various treatments ( n = 3 mice). (C) PD-L1 expression of tumor tissues at the end of 21 days of various treatments ( n = 3 independent biological replicates). (D) H&E staining of tumor tissue sections at the end of 21 days of various treatments. Scale bars, 100 μm.

Journal: iScience

Article Title: Targeted delivery and controlled release of deferasirox for melanoma therapy

doi: 10.1016/j.isci.2026.115303

Figure Lengend Snippet: In vivo therapeutic effect of RGD AB@DFX NPs in B16F1 tumor-bearing mice (A) Tumor growth curves during 21 days of treatment with PBS, A NPs, DFX, A@DFX NPs, AB@DFX NPs and RGD AB@DFX NPs. Data are expressed as means ± SD ( n = 3 mice). Statistical analysis: one-way ANOVA followed by Tukey’s post hoc test. ∗ p < 0.05. (B) Photographs of tumors at the end of 21 days of various treatments ( n = 3 mice). (C) PD-L1 expression of tumor tissues at the end of 21 days of various treatments ( n = 3 independent biological replicates). (D) H&E staining of tumor tissue sections at the end of 21 days of various treatments. Scale bars, 100 μm.

Article Snippet: Mouse Melanoma Cells (B16F1) , ATCC , CRL-6323.

Techniques: In Vivo, Expressing, Staining

Biocompatibility of RGD AB@DFX NPs in B16F1 tumor-bearing mice (A) H&E staining of major organs (heart, liver, spleen, lung and kidney) at the end of 21 days of treatments with PBS, A NPs, DFX, A@DFX NPs, AB@DFX NPs and RGD AB@DFX NPs. Scale bars, 100 μm. (B) Blood routine indices of mice at the end of 21 days of various treatments. Data are expressed as means ± SD ( n = 3 mice).

Journal: iScience

Article Title: Targeted delivery and controlled release of deferasirox for melanoma therapy

doi: 10.1016/j.isci.2026.115303

Figure Lengend Snippet: Biocompatibility of RGD AB@DFX NPs in B16F1 tumor-bearing mice (A) H&E staining of major organs (heart, liver, spleen, lung and kidney) at the end of 21 days of treatments with PBS, A NPs, DFX, A@DFX NPs, AB@DFX NPs and RGD AB@DFX NPs. Scale bars, 100 μm. (B) Blood routine indices of mice at the end of 21 days of various treatments. Data are expressed as means ± SD ( n = 3 mice).

Article Snippet: Mouse Melanoma Cells (B16F1) , ATCC , CRL-6323.

Techniques: Staining

B16F1 cells stably expressing shHrs or control plasmid (shLacZ) were transiently transfected with tdTomato-fTractin and seeded onto PDL-coated glass bottom MatTek Plates 24 hr post-transfection. Live cells were imaged every 30 s for 15 min. Movies were inverted, de-noised, and brightness/contrast was adjusted to facilitate visualization of filopodia.

Journal: eLife

Article Title: Secreted exosomes induce filopodia formation

doi: 10.7554/eLife.101673

Figure Lengend Snippet: B16F1 cells stably expressing shHrs or control plasmid (shLacZ) were transiently transfected with tdTomato-fTractin and seeded onto PDL-coated glass bottom MatTek Plates 24 hr post-transfection. Live cells were imaged every 30 s for 15 min. Movies were inverted, de-noised, and brightness/contrast was adjusted to facilitate visualization of filopodia.

Article Snippet: B16F1 mouse melanoma cells (ATCC CRL-6323) were maintained in DMEM supplemented with 10% FBS.

Techniques:

B16F1 cells stably expressing shEng or control plasmid (shScr) were transiently transfected with tdTomato-fTractin and seeded onto PDL-coated glass bottom MatTek plates 24 hr post-transfection. Live cells were imaged every 30 s for 15 min. Movies were inverted and brightness/contrast was adjusted to facilitate visualization of filopodia.

Journal: eLife

Article Title: Secreted exosomes induce filopodia formation

doi: 10.7554/eLife.101673

Figure Lengend Snippet: B16F1 cells stably expressing shEng or control plasmid (shScr) were transiently transfected with tdTomato-fTractin and seeded onto PDL-coated glass bottom MatTek plates 24 hr post-transfection. Live cells were imaged every 30 s for 15 min. Movies were inverted and brightness/contrast was adjusted to facilitate visualization of filopodia.

Article Snippet: B16F1 mouse melanoma cells (ATCC CRL-6323) were maintained in DMEM supplemented with 10% FBS.

Techniques:

$( A ) Western blot of Hrs-KD in B16F1 cells. ( B ) Western blot of Rab27a-KD in B16F1 cells. ( C ) Nanoparticle tracking analysis representative traces showing particle size distributions that correspond to the data for control SEVs and LEVs in . ( D ) Western blot of gradient fractions for SEVs prepared by the cushion density gradient (DG) method. TCL, total cell lysate. LEVs also run on gel. ( E ) TEM of negatively stained LEVs and SEVs prepared by the cushion-DG method. Scale bar = 200 nm in each image. Figure 2—figure supplement 1—source data 1. PDF file containing the original western blots from , indicating the relevant bands. Figure 2—figure supplement 1—source data 2. Original files for western blot analysis displayed in . Figure 2—figure supplement 1—source data 3. PDF file containing the original western blots from , indicating the relevant bands. Figure 2—figure supplement 1—source data 4. Original files for western blot analysis displayed in . Figure 2—figure supplement 1—source data 5. PDF file containing the original western blots from , indicating the relevant bands. Figure 2—figure supplement 1—source data 6. Original files for western blot analysis displayed in .$

Journal: eLife

Article Title: Secreted exosomes induce filopodia formation

doi: 10.7554/eLife.101673

Figure Lengend Snippet: ( A ) Western blot of Hrs-KD in B16F1 cells. ( B ) Western blot of Rab27a-KD in B16F1 cells. ( C ) Nanoparticle tracking analysis representative traces showing particle size distributions that correspond to the data for control SEVs and LEVs in . ( D ) Western blot of gradient fractions for SEVs prepared by the cushion density gradient (DG) method. TCL, total cell lysate. LEVs also run on gel. ( E ) TEM of negatively stained LEVs and SEVs prepared by the cushion-DG method. Scale bar = 200 nm in each image. Figure 2—figure supplement 1—source data 1. PDF file containing the original western blots from , indicating the relevant bands. Figure 2—figure supplement 1—source data 2. Original files for western blot analysis displayed in . Figure 2—figure supplement 1—source data 3. PDF file containing the original western blots from , indicating the relevant bands. Figure 2—figure supplement 1—source data 4. Original files for western blot analysis displayed in . Figure 2—figure supplement 1—source data 5. PDF file containing the original western blots from , indicating the relevant bands. Figure 2—figure supplement 1—source data 6. Original files for western blot analysis displayed in .

Article Snippet: B16F1 mouse melanoma cells (ATCC CRL-6323) were maintained in DMEM supplemented with 10% FBS.

Techniques: Western Blot, Control, Staining

( A ) EVs secreted from equal numbers of control (shLacZ), Rab27a-KD, and Hrs-KD B16F1 cells over 48 hr were quantified using NanoSight particle tracking analysis (N=3 biological replicates). ( B ) Representative images of control (shLacZ), Hrs-KD, and Rab27a-KD B16F1 cells stained with rhodamine-phalloidin. Arrowheads show examples of filopodia. Images have been edited with brightness and contrast for ease of visibility. ( C ) Quantification of filopodia from images as in B (≥27 total cells per condition from three biological replicates). Filopodia number per 500 mm 2 cell area. ( D ) Representative images of filopodia in B16F1 control (shLacZ) and exosome-depleted (shHrs) cells treated for 18 hr with LEVs or SEVs isolated from control cells. Arrowheads show examples of filopodia. Images have been edited with brightness and contrast for ease of visibility. ( E ) Quantification of filopodia from images as in D (≥20 total cells per condition from three biological replicates). ( F ) Filopodia number in B16F1 shScr cells treated with indicated numbers of purified LEVs or SEVs, for 18 hr (≥25 total cells per condition from three biological replicates). ( G ) Control (shLacZ) and exosome-depleted (shHrs) B16F1 cells were transiently transfected with tdTomato-F-tractin to visualize filopodia formation. Live images were taken every 30 s for 15 min and newly formed filopodia were counted at each time point. Only filopodia that form and fully retract during the duration of the video were quantified. (≥20 total cells per type per biological replicate, from three biological replicates). ( H ) Lifetime of newly formed filopodia from G. Lifetime is defined as the time from first formation of the filopodia to full retraction. Bars represent mean and error bars are SEM. Scale bars in wide field and zoom insets = 10 mm. Error bars, SEM. ns, not significant; * p<0.05; ** p<0.01; *** p<0.001.

Journal: eLife

Article Title: Secreted exosomes induce filopodia formation

doi: 10.7554/eLife.101673

Figure Lengend Snippet: ( A ) EVs secreted from equal numbers of control (shLacZ), Rab27a-KD, and Hrs-KD B16F1 cells over 48 hr were quantified using NanoSight particle tracking analysis (N=3 biological replicates). ( B ) Representative images of control (shLacZ), Hrs-KD, and Rab27a-KD B16F1 cells stained with rhodamine-phalloidin. Arrowheads show examples of filopodia. Images have been edited with brightness and contrast for ease of visibility. ( C ) Quantification of filopodia from images as in B (≥27 total cells per condition from three biological replicates). Filopodia number per 500 mm 2 cell area. ( D ) Representative images of filopodia in B16F1 control (shLacZ) and exosome-depleted (shHrs) cells treated for 18 hr with LEVs or SEVs isolated from control cells. Arrowheads show examples of filopodia. Images have been edited with brightness and contrast for ease of visibility. ( E ) Quantification of filopodia from images as in D (≥20 total cells per condition from three biological replicates). ( F ) Filopodia number in B16F1 shScr cells treated with indicated numbers of purified LEVs or SEVs, for 18 hr (≥25 total cells per condition from three biological replicates). ( G ) Control (shLacZ) and exosome-depleted (shHrs) B16F1 cells were transiently transfected with tdTomato-F-tractin to visualize filopodia formation. Live images were taken every 30 s for 15 min and newly formed filopodia were counted at each time point. Only filopodia that form and fully retract during the duration of the video were quantified. (≥20 total cells per type per biological replicate, from three biological replicates). ( H ) Lifetime of newly formed filopodia from G. Lifetime is defined as the time from first formation of the filopodia to full retraction. Bars represent mean and error bars are SEM. Scale bars in wide field and zoom insets = 10 mm. Error bars, SEM. ns, not significant; * p<0.05; ** p<0.01; *** p<0.001.

Article Snippet: B16F1 mouse melanoma cells (ATCC CRL-6323) were maintained in DMEM supplemented with 10% FBS.

Techniques: Control, Staining, Isolation, Purification, Transfection

( 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 .

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: B16F1 mouse melanoma cells (ATCC CRL-6323) were maintained in DMEM supplemented with 10% FBS.

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

( 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 .

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: B16F1 mouse melanoma cells (ATCC CRL-6323) were maintained in DMEM supplemented with 10% FBS.

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

( 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 .

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: B16F1 mouse melanoma cells (ATCC CRL-6323) were maintained in DMEM supplemented with 10% FBS.

Techniques: Western Blot, Control, Staining

( A ) Native gel Western blot of B16F1 SEVs. ( B ) Standard western blot of HT1080 SEVs. ( C ) Western blot of cortical neuron total cell lysate (TCL) and SEVs. ( D ) Representative images and quantitation of filopodia number in control (lipofectamine) and THSD7A-mScarlet-transfected HT1080 cells. Arrowheads indicate THSD7A at the ends of filopodia (white arrowheads) or in extracellular deposits (red arrowheads). Scale bars in wide field and zoom insets = 10 mm. ( E ) (Left) Western blot of control shRNA (NTC) and shTHSD7A (C-04, C05, C-06) - expressing HT1080 cell lines. Vinculin is used as a loading control and numbers below the blot indicate normalized THSD7A levels. (Right) Filopodia counts in control and shTHSD7A HT1080 cells. ≥20 cells per condition per biological replicate, from three biological replicates. ( F ) THSD7A coated coverslips rescue filopodia defect in shEng B16F1 and HT1080 cells.≥20 cells per condition per biological replicate, from three biological replicates. ( G, H ) Cortical neurons were transfected with a FLAG-THSD7A expression vector or vector control, fixed, and stained with an antibody against THSD7A, and imaged by confocal microscopy. ( G ) Representative images. Arrows indicate THSD7A localization to the tips of filopodia. Scale bar = 5 mm. ( H ) Quantification of filopodia in neurons expressing FLAG-THSD7A or control vector. n=42 neurons from three separate experiments (biological replicates). ( I ) Rescue of filopodia numbers in shHrs neurons plated on dishes coated with various concentrations of recombinant human THSD7A, as indicated. Error bars, SEM. ns, not significant; * p<0.05; ** p<0.01; *** p<0.001. Figure 6—source data 1. PDF file containing the original western blots and Ponceau stain from , indicating the relevant bands. Figure 6—source data 2. Original files for western blot and Ponceau analysis displayed in . Figure 6—source data 3. PDF file containing the original western blots from , indicating the relevant bands. Figure 6—source data 4. Original files for western blot analysis displayed in . Figure 6—source data 5. PDF file containing the original western blots from , indicating the relevant bands. Figure 6—source data 6. Original files for western blot analysis displayed in . Figure 6—source data 7. PDF file containing the original western blots from , indicating the relevant bands. Figure 6—source data 8. Original files for western blot analysis displayed in .

Journal: eLife

Article Title: Secreted exosomes induce filopodia formation

doi: 10.7554/eLife.101673

Figure Lengend Snippet: ( A ) Native gel Western blot of B16F1 SEVs. ( B ) Standard western blot of HT1080 SEVs. ( C ) Western blot of cortical neuron total cell lysate (TCL) and SEVs. ( D ) Representative images and quantitation of filopodia number in control (lipofectamine) and THSD7A-mScarlet-transfected HT1080 cells. Arrowheads indicate THSD7A at the ends of filopodia (white arrowheads) or in extracellular deposits (red arrowheads). Scale bars in wide field and zoom insets = 10 mm. ( E ) (Left) Western blot of control shRNA (NTC) and shTHSD7A (C-04, C05, C-06) - expressing HT1080 cell lines. Vinculin is used as a loading control and numbers below the blot indicate normalized THSD7A levels. (Right) Filopodia counts in control and shTHSD7A HT1080 cells. ≥20 cells per condition per biological replicate, from three biological replicates. ( F ) THSD7A coated coverslips rescue filopodia defect in shEng B16F1 and HT1080 cells.≥20 cells per condition per biological replicate, from three biological replicates. ( G, H ) Cortical neurons were transfected with a FLAG-THSD7A expression vector or vector control, fixed, and stained with an antibody against THSD7A, and imaged by confocal microscopy. ( G ) Representative images. Arrows indicate THSD7A localization to the tips of filopodia. Scale bar = 5 mm. ( H ) Quantification of filopodia in neurons expressing FLAG-THSD7A or control vector. n=42 neurons from three separate experiments (biological replicates). ( I ) Rescue of filopodia numbers in shHrs neurons plated on dishes coated with various concentrations of recombinant human THSD7A, as indicated. Error bars, SEM. ns, not significant; * p<0.05; ** p<0.01; *** p<0.001. Figure 6—source data 1. PDF file containing the original western blots and Ponceau stain from , indicating the relevant bands. Figure 6—source data 2. Original files for western blot and Ponceau analysis displayed in . Figure 6—source data 3. PDF file containing the original western blots from , indicating the relevant bands. Figure 6—source data 4. Original files for western blot analysis displayed in . Figure 6—source data 5. PDF file containing the original western blots from , indicating the relevant bands. Figure 6—source data 6. Original files for western blot analysis displayed in . Figure 6—source data 7. PDF file containing the original western blots from , indicating the relevant bands. Figure 6—source data 8. Original files for western blot analysis displayed in .

Article Snippet: B16F1 mouse melanoma cells (ATCC CRL-6323) were maintained in DMEM supplemented with 10% FBS.

Techniques: Western Blot, Quantitation Assay, Control, Transfection, shRNA, Expressing, Plasmid Preparation, Staining, Confocal Microscopy, Recombinant

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