Journal: EMBO Reports

Article Title: Hepsin regulates TGFβ signaling via fibronectin proteolysis

doi: 10.15252/embr.202152532

Figure Lengend Snippet: A, B GSEA enrichment maps from tumors overexpressing hepsin (DOX + ) and control tumors (DOX − ). C Graph depicting quantification of Tgfβ1 mRNA levels (mean ± SD mRNA read counts), normalized to the housekeeping gene Pum1 , in mammary tissue of 6‐week‐old virgin female Wt and Hpn −/− mice. Student’s t ‐test was used for statistical analyses (n.s. = not significant, N = 3 indicates the number of biological replicates). D Immunoblot analysis of PAI‐1, hepsin, and β‐tubulin (loading control) in MCF10A‐pIND20‐HPN cells treated with doxycycline, galunisertib, and latent‐TGFβ (small latent complex) as indicated. Numbers below the PAI‐1 blot indicate PAI‐1 levels, normalized to β‐tubulin. E Immunoblot analysis of TGFβ‐V5 (antibody detecting V5), pSmad2/3, PAI‐1, and GAPDH (loading control) in cell lysates from MCF10A‐pIND20‐HPN cells with or without TGFβ‐V5 overexpression. F–I Silver‐stained protein gels with samples from in vitro protease activity assays with recombinant hepsin and fragments of fibronectin. The respective fragments (30 kDa (F), 45 kDa (G), 40 kDa (H), and 120 kDa (I); marked by arrowheads) were incubated (1 μg/reaction) with increasing concentrations of recombinant hepsin (nM). J Schematic mapping of the fibronectin fragments used in (G–J) onto full‐length fibronectin. RGD indicates the RGD‐binding domain in fibronectin. K Graph depicting quantification of Fn1 mRNA levels (mean ± SD mRNA read counts), normalized to the housekeeping gene Pum1 , in mammary tissue of 6‐week‐old virgin female Wt and Hpn −/− mice. Student’s t ‐test was used for statistical analyses (n.s. = not significant, N = 3 indicates the number of biological replicates).

Article Snippet: Fibronectin proteolytic fragment from human plasma, 45 kDa , Merck , F0162.

Techniques: Western Blot, Over Expression, Staining, In Vitro, Activity Assay, Recombinant, Incubation, Binding Assay

Journal: EMBO Reports

Article Title: Hepsin regulates TGFβ signaling via fibronectin proteolysis

doi: 10.15252/embr.202152532

Figure Lengend Snippet: Schematic illustration of proteins involved in TGFβ storage in the extracellular matrix. The small latent complex (SLC) is a non‐covalently linked tetramer of 2 LAP (latency‐associated peptide) (40 kDa) and 2 TGFβ1 (12 kDa). LTBP1 + SLC together form the large latent complex (LLC) through covalent bonds between LAP and LTBP1. LLC interacts with the ECM through non‐covalent interactions of LTBP with fibrillins and fibronectin. Immunoblot analysis of LAP in mammary whole tissue lysates from Wt and Hpn −/− mouse. The 90 kDa band represents unprocessed pro‐TGFβ, and the 40 kDa band corresponds to mature TGFβ LAP peptide. Schematic representation of the mouse experiment, showing orthotopic transplantation of Wt Wap‐Myc tumor cells into syngeneic Wt and Hpn −/− recipients. Immunoblot analysis of phospho‐Smad2/3, total Smad2/3, and GAPDH (loading control) expression in Wt tumors transplanted into either Wt or Hpn −/− recipients ( N = 10 tumors each). Western blot analysis of phospho‐Smad2/3, total Smad2/3, hepsin, and β‐tubulin (loading control) in MCF10A‐pIND20‐HPN cells treated with 1 µg/ml doxycycline for 48 hours (DOX; overexpression of hepsin) and the TGFβR1 inhibitor galunisertib (TGFβR1i, 10 µM) as indicated. Immunoblot analysis of conditioned medium (LAP peptide) and cell extracts (hepsin and vinculin) from control (DOX) or hepsin overexpressing (DOX + ) MCF10A‐pIND20‐HPN supplemented with 100 ng/ml latent‐TGFβ (small latent complex). Ponceau was used as a loading control for the cell culture medium. TGFβ ELISA assay to detect active and total TGFβ levels in conditioned medium from MCF10A‐pIND20‐HPN pL6‐TGFβ1 cells with (DOX + ) or without (DOX − ) hepsin overexpressing. The TGFβ ELISA assay detects active TGFβ levels (first 2 columns), but heat treatment of the conditioned medium activates all TGFβ, thus effectively measuring total TGFβ (right two columns). Data are presented as scatter plot (mean denoted by the black line), and paired Student’s t ‐test was used for statistical analyses. Silver‐stained protein gel with samples from an in vitro protease activity assay with recombinant SLC and hepsin. SLC was incubated with increasing concentrations of recombinant hepsin (numbers indicate the concentration of hepsin in nM). Western blots in (B, E, F) are representative of at least three repeats.

Article Snippet: Fibronectin proteolytic fragment from human plasma, 45 kDa , Merck , F0162.

Techniques: Western Blot, Transplantation Assay, Expressing, Over Expression, Cell Culture, Enzyme-linked Immunosorbent Assay, Staining, In Vitro, Activity Assay, Recombinant, Incubation, Concentration Assay

Journal: EMBO Reports

Article Title: Hepsin regulates TGFβ signaling via fibronectin proteolysis

doi: 10.15252/embr.202152532

Figure Lengend Snippet: Coomassie‐stained protein gels with cell lysates and concentrated culture supernatants of MCF10A‐pIND20‐HPN cells, with (DOX + ) or without (DOX − ) hepsin overexpression. M indicates the media only control. R and NR above the gels indicate reducing and non‐reducing conditions, respectively. Numbered arrowheads indicate areas of the gel that were analyzed by mass spectrometry; corresponding proteins differently expressed in hepsin overexpressing cells are listed on the left. The image with two lanes on the right is a copy of the indicated area with supernatant under reducing conditions, highlighting the part from which fibronectin was identified. Red boxes indicate the lanes that were analyzed by mass spectrometry. Immunoblot analyses of fibronectin expression in concentrated culture supernatant from MCF10A‐pIND20‐HPN cells with (DOX + ) or without (DOX − ) hepsin overexpression. Ponceau staining of the Western blot is shown as the loading control. Immunoblot analysis of fibronectin, hepsin, and β‐tubulin (loading control) in cell lysates from MCF10A‐pIND20‐HPN cells with (DOX + ) or without (DOX − ) hepsin overexpression. The graph shows the quantification of fibronectin levels. Student’s t ‐test was used for statistical analyses. Data are represented as mean ± SD. Silver‐stained protein gel with samples from an in vitro protease activity assay with recombinant hepsin and either full length purified plasma fibronectin (upper panel) or the 70 kDa most N‐terminal fragment of fibronectin (lower panel). Full‐length fibronectin (1 μg) or the 70 kDa fibronectin fragment (1 μg) was incubated with increasing concentrations of recombinant hepsin. Arrowheads indicate full‐length fibronectin, the 70 kDa fragment, the 50 kDa cleavage fragment generated by hepsin, and hepsin. The schematic figure shows the full‐length fibronectin protein and the 70 KDa N‐terminal fragment. The red arrow indicates the location of the putative hepsin cleavage site. RGD indicates the RGD‐binding domain in fibronectin. Experiments in (B, C, D) are representative of at least three repeats. Source data are available online for this figure.

Article Snippet: Fibronectin proteolytic fragment from human plasma, 45 kDa , Merck , F0162.

Techniques: Staining, Over Expression, Mass Spectrometry, Western Blot, Expressing, In Vitro, Activity Assay, Recombinant, Purification, Incubation, Generated, Binding Assay

Journal: EMBO Reports

Article Title: Hepsin regulates TGFβ signaling via fibronectin proteolysis

doi: 10.15252/embr.202152532

Figure Lengend Snippet: Immunoblot analysis of fibronectin in indicated tissues isolated from Wt, Hpn +/− and Hpn −/− mouse (representative of at least three repeats). *As the same tissue lysates were used as in Fig , the same GAPDH blot is used here as the loading control. Immunoblot analysis of fibronectin and β‐tubulin (loading control) in lysates from Wt and Hpn −/− mouse mammary tissue ( N = 5 animals each) from another hepsin knockout mouse model (Wu et al , ). Immunoblot analysis of fibronectin, hepsin, and β‐tubulin (loading control) in prostate lysates of control mice or mice with prostate‐specific hepsin overexpression (Klezovitch et al , ). N = 2 mice each, the two prostate lobes were run separately (A and B).

Article Snippet: Fibronectin proteolytic fragment from human plasma, 45 kDa , Merck , F0162.

Techniques: Western Blot, Isolation, Knock-Out, Over Expression

Journal: EMBO Reports

Article Title: Hepsin regulates TGFβ signaling via fibronectin proteolysis

doi: 10.15252/embr.202152532

Figure Lengend Snippet: Immunoblot analysis of fibronectin, pSmad2/3, total Smad2/3, and GAPDH (loading control) in cell lysates from MCF10A‐pIND20‐HPN cells without hepsin overexpression. The graph depicts quantification of pSmad2/3 normalized to total Smad2/3 levels, compared to siCtrl ( N = 3 biological repeats). Immunoblot analysis of cell lysates (pSmad2/3, total Smad2/3, and vinculin as loading control) and concentrated cell culture supernatant (anti‐V5 for TGFβ) from MCF10A‐pIND20‐HPN pL6‐TGFβ1‐V5 cells without hepsin overexpression. Ponceau staining is shown as a loading control for the Western blot with concentrated culture supernatant. The graph depicts quantification of pSmad2/3 normalized to total Smad2/3 levels, compared to siCtrl ( N = 3 biological repeats). TGFβ luciferase reporter assay of control or fibronectin silenced MCF10A‐pIND20‐HPN pL6‐TGFβ1 (SRE) TGFβ1 reporter cells with (DOX + ) or without (DOX − ) hepsin overexpression ( N = 7 biological repeats; Y‐axis shows fold change in relative light units (RLU)). Immunoblot analysis of cell lysates (fibronectin and β‐tubulin (loading control)) and concentrated cell culture supernatant (TGFβ) from Hs578T cells with knockdown of fibronectin. Ponceau staining is shown as the loading control for the Western blot with concentrated culture supernatant. A representative blot from three biological repeats is shown. Immunoblot analysis of fibronectin, pSmad2/3, total Smad2/3, hepsin, and GAPDH (loading control) in cell lysates from MCF10A‐pIND20‐HPN cells with (DOX + ) and without (DOX − ) hepsin overexpression, and with or without fibronectin silencing (siFN1). The graph depicts quantification of pSmad2/3 normalized to total Smad2/3 levels ( N = 3 biological repeats). TGFβ luciferase reporter assay of control or fibronectin silenced MCF10A‐pIND20‐HPN pL6‐TGFβ1 (SRE) TGFβ1 reporter cells, with (DOX + ) and without (DOX − ) hepsin overexpression. ( N = 6 biological repeats; Y‐axis shows fold change in relative light units (RLU)). Model figure depicting how hepsin regulates TGFβ signaling. Under Wt conditions, hepsin promotes degradation of fibronectin, which releases latent‐TGFβ from ECM stores, thus resulting in the induction of TGFβ signaling. In hepsin knockout mammary glands, TGFβ signaling is compromised as latent‐TGFβ cannot be released from ECM stores (Created with BioRender.com). Statistical analyses in (A, B, C, E, F) were done using Student’s t ‐test. Data in (A, B, C, E, F) are presented as dot plots where black lines represent the mean. Source data are available online for this figure.

Article Snippet: Fibronectin proteolytic fragment from human plasma, 45 kDa , Merck , F0162.

Techniques: Western Blot, Over Expression, Cell Culture, Staining, Luciferase, Reporter Assay, Knock-Out

Journal: EMBO Reports

Article Title: Hepsin regulates TGFβ signaling via fibronectin proteolysis

doi: 10.15252/embr.202152532

Figure Lengend Snippet: Uncropped Western blot corresponding to Fig . Uncropped Western blot corresponding to Fig . Red boxes indicate areas represented in the main Fig . Immunoblot analysis of fibronectin and the 12 kDa form of TGFβ in a panel of normal breast and breast cancer cell lines. β‐Tubulin was used as the loading control. TGFβ luciferase reporter assay of MCF10A‐pIND20‐HPN pL6‐TGFβ1 reporter cells harboring a Smad‐response element (SRE), with (DOX + ) and without (DOX − ) hepsin overexpression, and with or without galunisertib (TGFβR/ALK5 inhibitor). ( N = 4 biological repeats; Y‐axis shows fold change in relative light units (RLU)). The black line denotes the average. Paired Student’s t ‐test was used for statistical analyses. Titration of the TGFβ1 concentration in a luciferase assay with the MCF10A‐pIND20‐HPN pL6‐TGFβ1 (SRE) reporter cell line. Figure shows one biological repeat as a representative of three biological repeats; Y‐axis shows fold change in relative light units (RLU). Immunoblot for pSmad2/3, total Smad2/3, and vinculin as loading control from 0.01 ng/ml TGFβ1 and control treated MCF10A‐pIND20‐HPN pL6‐TGFβ1 reporter cells. The graph depicts quantification of pSmad2/3 normalized to total Smad2/3 levels ( N = 3 biological repeats) Source data are available online for this figure.

Article Snippet: Fibronectin proteolytic fragment from human plasma, 45 kDa , Merck , F0162.

Techniques: Western Blot, Luciferase, Reporter Assay, Over Expression, Titration, Concentration Assay

Journal: EMBO Reports

Article Title: Hepsin regulates TGFβ signaling via fibronectin proteolysis

doi: 10.15252/embr.202152532

Figure Lengend Snippet: Purified proteins used in the study.

Article Snippet: Fibronectin proteolytic fragment from human plasma, 45 kDa , Merck , F0162.

Techniques: Purification, Cell Attachment Assay, Recombinant

Journal: APL Bioengineering

Article Title: Extracellular matrix alignment dictates the organization of focal adhesions and directs uniaxial cell migration

doi: 10.1063/1.5052239

Figure Lengend Snippet: Microcontact-printed ECM screening arrays. (a) Photomask containing 252 individual ECM conditions, each containing a unique randomly generated array of line elements with varying alignments (angular dispersion), line densities, and line widths. (b) Overview of the negative microcontact-printing procedure. Briefly, AlexaFluor555-conjugated fibronectin (Fn555, red) was uniformly coated on flat PDMS stamps and a UV-ozone activated stamp containing the negative of the intended patterns was applied to remove background areas before transferring the remaining areas to the eventual cell culture substrate. (c) Tile-scan fluorescence image of a subsection of the Fn-printed (red) ECM screening array seeded with phalloidin-AlexaFluor488 stained 3T3s (green) (scale bar: 1 mm). (d) Fn555 microcontact-printed patterns demonstrating control over line alignment (left), line density (middle), and line width (right) (scale bars: 10 μ m).

Article Snippet: To enable pattern visualization, fibronectin from human plasma (Fn, Corning, Corning, NY) was fluorescently tagged with AlexaFluor555 succidinyl ester following the manufacturer's protocol (Invitrogen, Carlsbad, CA).

Techniques: Generated, Dispersion, Transferring, Cell Culture, Fluorescence, Staining, Control