kat2a primary cell based screening assays cell culture ht1080 human fibrosarcoma cell line ht1080  (ATCC)

Journal: bioRxiv

Article Title: Nuclear blebs are composed of variable chromatin states but consistently enrich transcription initiation relative to elongation

doi: 10.64898/2026.03.10.710873

Figure Lengend Snippet: Example images of a nuclear bleb in HT1080 cells stably expressing NLS-GFP (green) and H2B-mCherry (gray). (A, B) Super plot nuclear bleb to body ratio (bleb/body) of NLS-GFP and H2B-mCherry (A) in cells in between nuclear ruptures, biological triplicates n = 22, and (B) in cells no nuclear rupture taken every hour, n = 5, 5, 3. Example images and super plots of nuclear bleb to body ratio for Hoechst 33342 DNA stain (cyan) and H3 (magenta) for cell lines and drug treatments (C) MEF WT n = 8, 15, 7, (D) MEF VPA n = 13, 11, 10, (E) HT1080 WT n = 10, (F) HT1080 VPA n = 8, 9, 13. White arrows denote the nuclear bleb. Mean ± s.e.m. is graphed. Statistical significance is denoted by *P<0.05, **P<0.01, ***P<0.001 or ns (not significant) via two-tailed paired Student’s t-test. Scale bars: 10 µm.

Article Snippet: Human fibrosarcoma HT1080 cells obtained from the American Tissue Culture Collection (ATCC) were cultured and passaged similarly.

Techniques: Stable Transfection, Expressing, Staining, Two Tailed Test

Journal: bioRxiv

Article Title: Nuclear blebs are composed of variable chromatin states but consistently enrich transcription initiation relative to elongation

doi: 10.64898/2026.03.10.710873

Figure Lengend Snippet: Examples of nuclear blebs in (A) MEF cells, (B) human HT1080 cells, and (C) human prostate cancer cell lines imaged via Hoechst (DNA) and euchromatin markers H3K27ac and H3K9ac. Super plots of nuclear bleb to body ratios biological triplicates in (A) MEF and (B) HT100 wild type (WT), increased euchromatin (VPA), decreased heterochromatin (DZNep), and lamin A knockdown (LA KD, MEF only). (C) Super plots of nuclear bleb to body ratios in human prostate cancer cell lines LNCaP n = 33, PC3 n = 30, and DU145 n = 29. Mean ± s.e.m. is graphed in A, B, and C. Statistical significance is denoted by * P <0.05, ** P <0.01, *** P <0.001 or ns (not significant) via One way Anova with post-hoc Turkey test. Scale bars: 10 µm.

Article Snippet: Human fibrosarcoma HT1080 cells obtained from the American Tissue Culture Collection (ATCC) were cultured and passaged similarly.

Techniques: Knockdown

Journal: bioRxiv

Article Title: Nuclear blebs are composed of variable chromatin states but consistently enrich transcription initiation relative to elongation

doi: 10.64898/2026.03.10.710873

Figure Lengend Snippet: Examples of nuclear blebs in (A) MEF cells, (B) human HT1080 cells, and (C) human prostate cancer cell lines imaged via Hoechst (DNA) and heterochromatin markers H3K27me3 and H3K9me2,3. Super plots of nuclear bleb to body ratios biological triplicates in (A) MEF and (B) HT100 wild type (WT), increased euchromatin (VPA), decreased heterochromatin (DZNep), and lamin A knockdown (LA KD, MEF only). (C) Super plots of nuclear bleb to body ratios in human prostate cancer cell lines LNCaP n = 10, 10, 9, PC3 n = 11, 11, 11, and DU145 n = 24. Mean ± s.e.m. is graphed in A, B, and C. Statistical significance is denoted by * P <0.05, ** P <0.01, *** P <0.001 or ns (not significant) via One way Anova with post-hoc Turkey test. Scale bars: 10 µm.

Article Snippet: Human fibrosarcoma HT1080 cells obtained from the American Tissue Culture Collection (ATCC) were cultured and passaged similarly.

Techniques: Knockdown

Journal: bioRxiv

Article Title: Nuclear blebs are composed of variable chromatin states but consistently enrich transcription initiation relative to elongation

doi: 10.64898/2026.03.10.710873

Figure Lengend Snippet: Example images and super plots of nuclear bleb to body ratio for DNA (cyan) and RNA Pol II pSer 2 (gray, elongation) and pSer5 (magenta, initiation) for cell lines (A) MEF n = 18, (B) HT1080 n = 10, (C) LNCaP n = 6, 10, 10, (D), PC3 n = 9, 7, 13, (E ) DU145 n = 12, 11, 11. (F) Graph summarizing all cell lines show a significant enrichment of RNA Pol II pSer5 relative to pSer2 in the nuclear bleb to body ratio. Mean ± s.e.m. is graphed. Statistical significance between pSer2 and pSer5 is denoted by * P <0.05, ** P <0.01, *** P <0.001 or ns (not significant) via two-tailed paired Student’s t -test. Scale bars: 10 µm.

Article Snippet: Human fibrosarcoma HT1080 cells obtained from the American Tissue Culture Collection (ATCC) were cultured and passaged similarly.

Techniques: Two Tailed Test

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Structure-guided engineering of prototype foamy virus Env identifies key residues for heparan sulfate binding and enhances transduction efficiency

doi: 10.3389/fbioe.2026.1716928

Figure Lengend Snippet: Schematic overview of structure-based engineering and functional validation of PFV Env mutants for enhanced heparan sulfate binding. (A) Structure-based strategy to identify key residues in the prototype foamy virus (PFV) Env protein responsible for heparan sulfate (HS) binding. The workflow begins with PFV Env amino acid sequence-based structure prediction, followed by docking simulations with HS to identify the interacting residues. Key amino acids predicted to mediate HS binding were selected and substituted to generate Env point mutants, which were subsequently cloned into expression vectors for functional testing. (B) Experimental workflow for PFV vector production and transduction assays. Transfer plasmid encoding EGFP reporter and Gag/Pol, together with the helper plasmid expressing either wild type or mutant Env were co-transfected into HEK293FT cells. The resulting viral particles were harvested and used to transduce the HT1080 cells. After 3 days of incubation, transduction efficiency and Env functionality were assessed using fluorescence microscopy, Western blotting, and flow cytometry.

Article Snippet: The human fibrosarcoma cell line HT1080 was obtained from the Korean Cell Line Bank, and the human embryonic kidney cell line HEK293FT was obtained from Thermo Fisher Scientific (USA).

Techniques: Functional Assay, Biomarker Discovery, Binding Assay, Virus, Sequencing, Clone Assay, Expressing, Plasmid Preparation, Transduction, Mutagenesis, Transfection, Incubation, Fluorescence, Microscopy, Western Blot, Flow Cytometry

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Structure-guided engineering of prototype foamy virus Env identifies key residues for heparan sulfate binding and enhances transduction efficiency

doi: 10.3389/fbioe.2026.1716928

Figure Lengend Snippet: Functional characterization of PFV Env upper domain point mutants affecting HS binding and viral entry efficiency. (A) The structure shown represents the receptor-binding domain (RBD) of the PFV Env surface subunit (SU), modeled using AlphaFold2. The molecular docking pose of heparan sulfate (HS) is displayed on the positively charged pocket of the RBD upper domain (UD), with key interacting residues (R298, R440, and E446) highlighted. The binding free energy which calculated from Autodock-vina, −7.0 kcal/mol is labeled. The molecular docking simulation revealed three key residues, Arg(R)298, Arg(R)440, and Glu(E)446, located in UD. These residues form hydrogen bonding with negatively charged sulfate group of HS and are highlighted in red and cyan for negatively charged and positively charged residues, respectively. Based on this structural analysis, R298A, R440A, and E446A substitutions were designed to disrupt the HS binding. The distance between key residues and HS are labeled with black arrow. (B) Western blot analysis was performed to confirm the protein expression of wild type and Env variants (R298A, R440A, and E446A) in HEK293FT cells. The cells were co-transfected with the PFV transfer vector (v3) and Env plasmids at a 30:1 ratio. All three mutants showed Env protein levels comparable to those of the wild type, indicating that the point mutations did not affect protein expression or stability. β -actin was used as a loading control. Mock, HEK293FT cell line served as a negative control; PC, HEK293FT cells transfected pCMV-Env were used as a positive control. (C) To assess the effect of each Env variant on viral infectivity, supernatants from transfected HEK293FT cells were used to transduce HT1080 cells. Transduction efficiency was monitored by EGFP expression using fluorescence microscopy after 3 days. Phase-contrast images confirmed the equivalent cell density across conditions. Scale bars = 100 μm. (D,E) Flow cytometry was used to quantitatively measure the proportion of EGFP-positive HT1080 cells transduced with each viral construct. Representative histograms and bar graph quantifications ( N ≥ 3) are shown in (D) and (E) , respectively. The average number from at least three independent experiments is shown at the top of each bar. Error bars represent the standard deviation of biological triplicates.

Article Snippet: The human fibrosarcoma cell line HT1080 was obtained from the Korean Cell Line Bank, and the human embryonic kidney cell line HEK293FT was obtained from Thermo Fisher Scientific (USA).

Techniques: Functional Assay, Binding Assay, Labeling, Western Blot, Expressing, Transfection, Plasmid Preparation, Control, Negative Control, Positive Control, Variant Assay, Infection, Transduction, Fluorescence, Microscopy, Flow Cytometry, Construct, Standard Deviation

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Structure-guided engineering of prototype foamy virus Env identifies key residues for heparan sulfate binding and enhances transduction efficiency

doi: 10.3389/fbioe.2026.1716928

Figure Lengend Snippet: Structure-guided refinement of PFV Env mutants to dissect the HS-binding determinants in the upper domain. (A) Two-dimensional interaction diagram showing the predicted hydrophobic and electrostatic interactions between selected residues and HS sulfate groups. (B) Schematic representation of the domain organization of PFV Env (1–988 aa), highlighting the receptor-binding domain (RBD; aa 217–570), including upper domains (UD; aa 244-312, 375-494) based on structural predictions. (C) Western blot validation of Env protein expression in HEK293FT cells transfected with the PFV transfer vector and one of the six-point mutant Env constructs. The co-transfection ratio (30:1) was consistent with that of previous experiments. β -actin served as a loading control. Mock, HEK293FT cell line served as a negative control. (D) Fluorescence microscopy showing the differential infectivity of PFV vectors with each Env variant in HT1080 cells, as measured by EGFP expression. Phase-contrast images confirmed similar cell confluency across all conditions. Scale bars = 100 μm. (E,F) Flow cytometry was used to quantitatively measure the proportion of EGFP-positive HT1080 cells transduced with each viral construct. Representative histograms and bar graph quantifications ( N ≥ 3) are shown in (E,F) , respectively. The average number from at least three independent experiments is shown at the top of each bar. Data are presented as mean ± SEM of biological triplicates.

Article Snippet: The human fibrosarcoma cell line HT1080 was obtained from the Korean Cell Line Bank, and the human embryonic kidney cell line HEK293FT was obtained from Thermo Fisher Scientific (USA).

Techniques: Binding Assay, Western Blot, Biomarker Discovery, Expressing, Transfection, Plasmid Preparation, Mutagenesis, Construct, Cotransfection, Control, Negative Control, Fluorescence, Microscopy, Infection, Variant Assay, Flow Cytometry, Transduction

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Structure-guided engineering of prototype foamy virus Env identifies key residues for heparan sulfate binding and enhances transduction efficiency

doi: 10.3389/fbioe.2026.1716928

Figure Lengend Snippet: Functional screening of lower-domain Env variants. (A) Molecular docking simulation of the PFV Env RBD, highlighting candidate HS-binding residues in the lower domain (circled green). (B) Two-dimensional interaction diagram illustrating predicted hydrophobic and electrostatic interactions between selected lower domain residues and HS sulfate groups. (C) Schematic representation of PFV Env organization (1-988 aa), showing the localization of lower domain (LD) subregions (aa 217-243, 313-374, and 495-570), as determined by structural modeling. Guided by structure-based docking, seven candidate residues within the LD were selected for mutagenesis based on side-chain polarity and the potential for π-stacking interactions to enhance HS binding. The specific amino acid substitutions and their rationales are listed. (D) Western blot analysis of Env expression in HEK293FT cells co-transfected with vectors encoding each LD point mutant and the PFV packaging system. Mock, HEK293FT cell line served as a negative control. (E,F) EGFP fluorescence (E) and corresponding flow cytometry histograms (F) of HT1080 cells transduced with PFV particles carrying individual LD variants and representative flow cytometry histograms corresponding to these samples. (H,I) EGFP fluorescence imaging (H) and flow cytometry histograms (I) of HT1080 cells transduced with PFV particles containing double (LD var1,7; LD var5,6) or triple (LD var1,5,6; UD var5 and LD var5,6) variants. (G,J) Quantification ( N ≥ 3) of GFP-positive HT1080 cells by flow cytometry, summarizing means ± SEM from biological triplicates.

Article Snippet: The human fibrosarcoma cell line HT1080 was obtained from the Korean Cell Line Bank, and the human embryonic kidney cell line HEK293FT was obtained from Thermo Fisher Scientific (USA).

Techniques: Functional Assay, Binding Assay, Mutagenesis, Western Blot, Expressing, Transfection, Negative Control, Fluorescence, Flow Cytometry, Transduction, Imaging

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Structure-guided engineering of prototype foamy virus Env identifies key residues for heparan sulfate binding and enhances transduction efficiency

doi: 10.3389/fbioe.2026.1716928

Figure Lengend Snippet: Structure-based functional screening of chimeric PFV-SFV Env variants to identify critical residues in HS-mediated viral entry. (A) Structural alignment of the SFV RBD crystal structure (PDB: 8AEZ) with the PFV-SFV gorⅠⅠ chimera RBD predicted by Swiss-Model and AlphaFold2 (AF2). (B) Molecular docking simulation of the PFV-SFV chimera RBD, highlighting the potential HS-binding residues in the lower domain (circled in green). (C) Zoomed view of the interaction interfaces and key residues (upper panel) and two-dimensional interaction diagram (bottom panel) showing predicted hydrophobic and electrostatic interactions between selected residues and HS sulfate groups. (D) Schematic representation of the PFV Env domain organization, with the SFV gorⅠⅠ RBD (aa 208-557) replacing the PFV RBD (aa 217-570). Specific amino acid substitutions (var1-6) and their rationales are listed. (E) Western blot analysis confirmed the expression of wild type chimeric Env (PFV backbone with SFV RBD) and six chimeric Env point mutants (variants 1–6) in the HEK293FT cells. β-actin was used as a loading control. PFV wild type Env (PFV WT Env)-transfected HEK293FT cells were used as a positive control. Mock, HEK293FT cell line served as a negative control. (F) Infectivity of each construct was assessed by EGFP fluorescence in HT1080 cells transduced with the PFV vectors. (G) Flow cytometry analysis quantified the proportion of GFP-positive cells for PFV WT Env, chimeric WT Env, and chimeric Env.

Article Snippet: The human fibrosarcoma cell line HT1080 was obtained from the Korean Cell Line Bank, and the human embryonic kidney cell line HEK293FT was obtained from Thermo Fisher Scientific (USA).

Techniques: Functional Assay, Binding Assay, Western Blot, Expressing, Control, Transfection, Positive Control, Negative Control, Infection, Construct, Fluorescence, Transduction, Flow Cytometry

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Structure-guided engineering of prototype foamy virus Env identifies key residues for heparan sulfate binding and enhances transduction efficiency

doi: 10.3389/fbioe.2026.1716928

Figure Lengend Snippet: Establishment and functional validation of Tet-On–inducible PFV Env-expressing HEK293T cell lines. (A) Workflow for generating stable cell lines. HEK203FT virus producer cells were co-transfected with three plasmids (pCMV-Gag/Pol, pMD-VSV-G, and pLVX-Transgene). Lentiviral particles carrying the transgene were harvested and used to transduce the HEK293T cells. Stable cell populations were established through puromycin selection. (B) Schematic representation of the Tet-On doxycycline (Dox)-inducible system used to drive PFV Env expression in HEK293T cells. The Env cassette was stably integrated into the lentiviral vectors. (C) Western blot analysis confirmed the establishment of HEK293T cells harboring Tet-Env cassettes. Cell lysates were analyzed by Western blotting using anti-Env and anti-β-actin antibodies. The letters above the lanes indicate Env type and Dox treatment. NC, HEK293FT cell line served as a negative control; PC, PFV wild type Env (PFV WT Env)-transfected HEK293FT cells were used as a positive control. (D) Functional validation using an infection assay. HT1080 cells were infected with viral particles produced from Tet-Env-293T clones following Dox induction. Successful production of infectious viruses was confirmed by GFP expression observed under fluorescence microscopy. (E) Flow cytometry analysis of HT1080 cells infected with supernatants from individual TetEnv-293T clones with or without Dox treatment. (F) Quantification ( N ≥ 3) of GFP-positive HT1080 cells by flow cytometry, summarizing means ± SEM from biological triplicates.

Article Snippet: The human fibrosarcoma cell line HT1080 was obtained from the Korean Cell Line Bank, and the human embryonic kidney cell line HEK293FT was obtained from Thermo Fisher Scientific (USA).

Techniques: Functional Assay, Biomarker Discovery, Expressing, Stable Transfection, Virus, Transfection, Transduction, Selection, Western Blot, Negative Control, Positive Control, Infection, Produced, Clone Assay, Fluorescence, Microscopy, Flow Cytometry

Journal: eLife

Article Title: Secreted exosomes induce filopodia formation

doi: 10.7554/eLife.101673

Figure Lengend Snippet: HT1080 cells stably expressing pHLuorin_M153R-CD63-mScarlet were seeded onto glass bottom MatTek plates and imaged live. Images were taken every 10 s.

Article Snippet: HT1080 human fibrosarcoma cells (ATCC CCL-121) were maintained in DMEM supplemented with 10% bovine growth serum (BGS).

Techniques:

Journal: eLife

Article Title: Secreted exosomes induce filopodia formation

doi: 10.7554/eLife.101673

Figure Lengend Snippet: The indicated HT1080 cell types were induced to form spheroids and then mixed into 3D type I collagen. Spheroids were imaged every 30 min for 8 hr. Scale bar = 100 mm.

Article Snippet: HT1080 human fibrosarcoma cells (ATCC CCL-121) were maintained in DMEM supplemented with 10% bovine growth serum (BGS).

Techniques:

Journal: eLife

Article Title: Secreted exosomes induce filopodia formation

doi: 10.7554/eLife.101673

Figure Lengend Snippet: ( A ) Representative confocal image of HT1080 cells stained with phalloidin-Alexa fluor 488 and CD63 shown in red. The red channel has been edited using brightness and contrast tools for ease of visibility. Note the localization of the exosome marker CD63 in extracellular deposits and at or near the tips of filopodia (arrowheads). Representative of 20 images. Scale bar is 10 mm in each panel. ( B ) Time series of pHluorin-M153R-CD63-mScarlet movie in HT1080 cells. Yellow arrowheads indicate fusion sites and yellow arrows indicate filopodia. Note a filopodium forming shortly after MVE fusion. ( C ) Representative kymographs showing MVE docking (red), fusion (yellow), and filopodia formation in HT1080 cells. Yellow arrowheads denote MVE fusion events, and black arrowheads denote the formation of a filopodium. Each pixel is 10 s x 0.2857 mm. ( D ) Quantification of the time elapsed between MVE fusion and filopodia formation. n=420 kymographs from 46 cells from three independent experiments (biological replicates). ( E ) Primary cortical neurons were co-transfected with GFP-Rab27b (green) and mCherry as a filler to visualize filopodia (red) on DIV 5 and fixed for imaging on DIV 6. SV2 negative staining (no signal) identifies these structures as filopodia instead of dendritic spines. Arrows in merged images indicate localization of GFP-Rab27b to tips and bases of filopodia. Scale bars = 5 µm. ( F ) Percent GFP-Rab27b localization to tips and bases of filopodia in 70 individual cortical neurons from three independent experiments (biological replicates). Red line indicates the median. Error bars, SEM. ns, not significant; * p<0.05; ** p<0.01; *** p<0.001.

Article Snippet: HT1080 human fibrosarcoma cells (ATCC CCL-121) were maintained in DMEM supplemented with 10% bovine growth serum (BGS).

Techniques: Staining, Marker, Transfection, Imaging, Negative Staining

Journal: eLife

Article Title: Secreted exosomes induce filopodia formation

doi: 10.7554/eLife.101673

Figure Lengend Snippet: ( A ) WB of Rab27a KD in HT1080 cell lysates. ( B ) TEM of SEVs (purified by cushion DG) and LEVs from HT1080 cells. Scale bar = 200 nm in each image. ( C ) Secretion rates of SEVs from HT1080 cell lines (N=3). Nanoparticle tracking analysis traces of SEVs from shScr and shRab27a HT1080 cells showing size (diameter) distribution of SEVs and particles/mL/cell. ( D ) Representative images showing filopodia in control and Rab27a-KD H1080 cells. Images have been edited with brightness and contrast tools for ease of visibility. Scale bars in wide field and zoom insets = 10 mm. ( E ) Quantification of filopodia in control and Rab27a-KD HT1080 cell lines. ≥20 cells per condition per biological replicate, from three biological replicates. ( F ) Data from graph in E displayed as filopodia per cell. ( G ) Data in displayed as filopodia per cell. ( H ). Data from displayed as filopodia per cell. ( I ) Data from displayed as filopodia per cell. ( J ) Data from displayed as filopodia per cell. ( K ) Cell areas of cells used for quantification in . Error bars, SEM. ns, not significant; * p<0.05; ** p<0.01; *** p<0.001. Figure 2—figure supplement 2—source data 1. PDF file containing the original western blots from , indicating the relevant bands. Figure 2—figure supplement 2—source data 2. Original files for western blot analysis displayed in .

Article Snippet: HT1080 human fibrosarcoma cells (ATCC CCL-121) were maintained in DMEM supplemented with 10% bovine growth serum (BGS).

Techniques: Purification, Control, Western Blot

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: HT1080 human fibrosarcoma cells (ATCC CCL-121) were maintained in DMEM supplemented with 10% bovine growth serum (BGS).

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 ) Cartoon diagram of metastatic colony assay in avian embryos. On day 0, fluorescent HT1080 cells were injected (100,000 cells per egg) into the vein of the chicken embryo. On day 4, the egg was opened, the embryo was sacrificed, and a circular tool was used to punch holes through the shell. The chorioallantoic membrane (CAM) was peeled away from the shell, placed on a glass slide with a coverslip, and immediately imaged. The cartoon was created using BioRender.com . ( B ) Representative low power wide field images of colony formation in the CAM. Scale bar = 200 mm. ( C ) Representative high-power wide field images of colony formation in the CAM. Scale bar = 100 mm. ( D, E ) Quantification of CAM colony number ( D ) and size ( E ) from high-power images as in C. 4–7 eggs were harvested per replicate for each condition for three biological replicates. ( D ) Colony number is graphed per field of view using 25–30 fields of view per egg. ( E ) Quantification of the percent of large (≥5000 mm 2 ) colonies formed by control and shEng HT1080 cells. ( F ) 3D invasion in collagen. HT1080 cell spheroids were seeded in collagen gels and imaged for 8 hr. Invasion is quantified as fold area increase in the size of each spheroid over 8 hr. Scale bar = 100 mm. Error bars, SEM. ns, not significant; *p<0.05; ** p<0.01; *** p<0.001.

Article Snippet: HT1080 human fibrosarcoma cells (ATCC CCL-121) were maintained in DMEM supplemented with 10% bovine growth serum (BGS).

Techniques: Colony Assay, Injection, Membrane, Control

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: HT1080 human fibrosarcoma cells (ATCC CCL-121) were maintained in DMEM supplemented with 10% bovine growth serum (BGS).

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

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: HT1080 human fibrosarcoma cells (ATCC CCL-121) were maintained in DMEM supplemented with 10% bovine growth serum (BGS).

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: HT1080 human fibrosarcoma cells (ATCC CCL-121) were maintained in DMEM supplemented with 10% bovine growth serum (BGS).

Techniques: Control, Transfection, Mutagenesis