EMBO/soda-nel huggingface dataset:
Hepsin regulates TGFβ signaling via fibronectin proteolysis (2021)
Figure 2

Figure 2. Overexpression of Hpn in Wap-Myc driven mammary tumors induces TGFβ signaling. (A) Schematic representation of the mouse experiment. (B) Immunoblot analysis of Wap-Myc mammary tumor lysates for the indicated TGFβ signaling markers and hepsin (T# denotes individual tumors). GAPDH was used as the loading control. Lysates were derived from Wap-Myc mammary tumors from 6 mice with and 6 mice without DOX-induced hepsin overexpression (see (A)). (C) Gene set enrichment analysis (GSEA) enrichment map of pathways upregulated in hepsin overexpressing Wap-Myc tumors (DOX+) compared with control tumors (DOX-) (N = 5 tumors each). Node size correlates with the number of genes in the signature; node color red correlates with enrichment in hepsin overexpressing Wap-Myc tumors. (D) GSEA graph comparing the expression of the HALLMARK_TGF_BETA_SIGNALING gene set in hepsin overexpressing (DOX+) to control (DOX-) Wap-Myc tumors (FDRp - p-value; FWERq -false discovery rate; NES-Normalized Enrichment Score). (E) Heatmap showing changes in expression of all genes in the HALLMARK_TGF_BETA_SIGNALING gene set in hepsin overexpressing (DOX+) compared to control (DOX-) Wap-Myc tumors. Red color indicates upregulation, and blue color indicates the dowregulation of the indicated genes.
Hepsin regulates TGFβ signaling via fibronectin proteolysis (2021)
Figure 3

Figure 3. Hepsin is a regulator of TGFβ storage. (A) 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. (B) 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. (C) Schematic representation of the mouse experiment, showing orthotopic transplantation of Wt Wap-Myc tumor cells into syngeneic Wt and Hpn-/- recipients. (D) 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). (E) Western blot analysis of phospho-Smad2/3, total Smad2/3, hepsin, and β-tubulin (loading control) in MCF10A-pIND20-HPN cells treated with doxycycline (DOX; overexpression of hepsin) and the TGFβR1 inhibitor Galunisertib (TGFβR1i, 10µM) as indicated. (F) 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 100ng/ml latent-TGFβ (Small Latent Complex). Ponceau was used as a loading control for the cell culture medium. (G) 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), paired student's t-test was used for statistical analyses. (H) 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.
Hepsin regulates TGFβ signaling via fibronectin proteolysis (2021)
Figure 4

Figure 4. Hepsin promotes the proteolytic processing of fibronectin. (A) 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 specrtometry (B) 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. (C) 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. A student's t-test was used for statistical analyses. Data are represented as mean±SD. (D) 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 (1μg) or the 70 kDa fibronectin fragment (1μg) were incubated with increasing concentrations of recombinant hepsin. Arrowheads indicate full-length fibronectin, the 70kDa fragment, the 50 kDa cleavage fragment generated by hepsin, and hepsin. The schematic figure shows the full-length fibronectin protein and the 70KDa N-terminal fragment. The red arrow indicates the location of the putative hepsin cleavage site. RGD indicated the RGD-binding domain in fibronectin. Experiments in (B, C, D) are representative of at least three repeats.
Hepsin regulates TGFβ signaling via fibronectin proteolysis (2021)
Figure 5

Figure 5. Hepsin reduces fibronectin levels in vivo. (A) 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 Figure 1F, the same GAPDH blot is used here as the loading control. (B) 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 knock out mouse model (Wu et al., 1998). (C) 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., 2004). N=2 mice each, the two prostate lobes were run separately (a and b).
Hepsin regulates TGFβ signaling via fibronectin proteolysis (2021)
Figure 6

Figure 6. Hepsin-mediated TGFβ1 signaling through the downregulation of fibronectin. (A) 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). (B) 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). (C) 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)). (D) 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 3 biological repeats is shown. (E) 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). (F) 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)). (G) 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 a student's t-test. Data in (A, B, C, E, F) are presented as dot plots where black lines represent the mean.

EXPANDED VIEW FIGURE LEGENDS
Comprehensive establishment and characterization of orthoxenograft mouse models of malignant peripheral nerve sheath tumors for personalized medicine. (2015)
Figure 2

Orthoxenograft mouse MPNST models closely resemble primary tumors

Orthotopic MPNST xenografts at passages 1 (OT P1) and 4 (OT P4) were histopathologically similar to their corresponding primary MPNST (PT) in hematoxylin–eosin staining of paraffin-embedded tumor sections from patients MPNST-NF1-001 and MPNST-SP-002. Main panels show a general view of the tumors at low magnification (40×); inset pictures were taken at higher magnification (400×).

Orthotopic xenograft and primary MPNSTs exhibited similar immunohistochemical features. A representative immunostained section of vimentin, CD34, S100, and Ki-67 is shown for primary tumors (PT) and orthotopic tumors (OT) from patients MPNST-NF1-001 and MPNST-SP-002. Positive antibody signals are shown in brown, and the hematoxylin counterstain in blue. Main panels show pictures at high magnification (400×); inset pictures show mitotic cells present in these tumors.
Comprehensive establishment and characterization of orthoxenograft mouse models of malignant peripheral nerve sheath tumors for personalized medicine. (2015)
Figure 3

Human stroma is lost after engraftment and replaced by murine cells

Stromal elements of the primary tumors were labeled with anti-human CD34 but not anti-mouse CD34; patient-derived xenografts are labeled with anti-mouse CD34 only and no anti-human marker. Representative sections (at 40× and 400× magnification) of the primary tumors (PT) and the orthoxenograft tumors at passages 1 (OT P1) and 4 (OT P4) were labeled with anti-human CD34 (H) and anti-mouse CD34 (M).

Sanger sequencing of the germline NF1 mutation c.350T>A present in the patient that developed two different tumors (MPNST-NF1-001 and MPNST-NF1-002). Sequencing results from normal tissue, primary tumor (PT), and orthotopic xenograft tumors (OT) are shown. The sequence of the NF1 region revealed WT NF1 alleles in primary tumor samples, and not in the corresponding derived orthoxenografts, indicating probable loss of the human stromal cells.

SNP array of primary tumor (PT) and orthotopic xenograft passages 1 (OT P1) and 4 (OT P4) for MPNST-NF1-001 and MPNST-NF1-002 tumors. Results correspond to chromosome 17. Images show the B-allele frequency (BAF) for the different samples as scatter plots and the copy number callings below them represented by thick horizontal lines: 2n regions are shown in gray, gained regions in orange, lost regions in green, and LOH regions are represented in blue. The vertical red line indicates the location of the NF1 locus. In addition, the percentage of tumor versus stromal cells for each sample is represented in a pie chart (blue, stromal cells; red, tumor cells).
Activation of the medial preoptic area (MPOA) ameliorates loss of maternal behavior in a Shank2 mouse model for autism (2021)
Figure 1

Figure 1: Shank2-/- mice ignore and abandon their pups after delivery

(A) In contrast to the immediate care of Shank2+/+ dams shortly after delivery Shank2-/- dams neglect the offspring. No proper nest building is observed, and pups are lying randomly scattered within the bedding. Right diagram: Average percentage of pups surviving until weaning per pregnancy. None of the litters of Shank2-/- mice survived after delivery, Mann-Whitney test, ***P<0.001, Shank2+/+ n = 12, Shank2-/- n = 9.

(B-F) (B, a-h) Series of pictures displaying the neglected appearance of pups delivered by Shank2-/- dams: (B,a, C), the percentage of pups gathered in the nest location is significantly reduced in Shank2-/- mice. Mann-Whitney test, ***P<0.001. (B,d, D) Shank2-/- mothers fail to remove extra-embryonical tissue after delivery, Mann-Whitney test, *P=0.036. (B,e-f, E) Shank2-/- dams showed impaired placentophagia (arrowheads), Mann-Whitney test, ***P<0.001. (B,g-h, F) Shank2-/- dams attack their pups inducing injury in the head and body region (arrowheads), Mann-Whitney test, *P=0.013, Shank2+/+, n = 12, Shank2-/- n = 9.

Data information: All data are presented as mean ± SEM.
Activation of the medial preoptic area (MPOA) ameliorates loss of maternal behavior in a Shank2 mouse model for autism (2021)
Figure 4

Figure 4: Shank2-/- mice display no gross abnormalities in hormonal gene and receptor expression.

(A-G) qRT-PCR analysis of mRNA expression levels of hormonal genes and hormonal receptors normalized to house-keeping gene HMBS of 9-10 week old naive and postnatal day 1 Shank2+/+ and Shank2-/- mice: (A) Oxytocin gene expression did not differ between Shank2+/+ and Shank2-/- mice, one-way ANOVA, P=0.335. (B) Vasopressin gene expression did not differ between Shank2+/+ and Shank2-/- mice, one-way ANOVA, P=0.945. (C) Prolactin gene expression did not differ between Shank2+/+ and Shank2-/- mice, one-way ANOVA, P=0.202. Additionally, no significant difference was evident in (D) oxytocin receptor, one-way ANOVA, P=0.675, (E) prolactin receptor, one-way ANOVA, P=0.912, (F) vasopressin receptor, one-way ANOVA, P=0.418 and (G) estrogen receptor alpha expression, one-way ANOVA, P=0.890 in comparison to Shank2+/+ mice. All data are presented as mean ± SEM, NS: not significant. Pituitary gland, (n=3 pituitary glands pooled per value, n=3 hypothalamus (Oxytocin, Vasopressin, Oxytocin receptor, Vasopressin receptor, estrogen alpha) n=2-3 hypothalamus (Prolactin receptor)). HYP, hypothalamus; PIT, pituitary gland.
Activation of the medial preoptic area (MPOA) ameliorates loss of maternal behavior in a Shank2 mouse model for autism (2021)
Figure 6

Figure 6: DREADD-based activation of MPOA neurons ameliorates social bonding in Shank2-/- mice

(A) Shank2+/+ and Shank2-/- mice were bilaterally injected with a Cre-dependent DREADD virus (pAAV-hSyn-DIO-hM3D (Gq)-mCherry) and (pENN.AAV.hSyn.HI.eGFP-Cre)) into the MPOA region. After four weeks, a pup-exposure assay was performed once a day for five days after intraperitoneal injection of vehicle or 5 mg/kg CNO (30 min before pup exposure). Maternal behavior was analyzed at the final 20 min of pup exposure on day 5 (test day).

(B) Confocal images showing the location and expression of the stereotaxically injected viral constructs in the MPOA region. (a) Merge of GFP(CRE) and mCherry (DREAAD) (scale bar: 500 µm) (b-d) Magnified views of the MPOA region (scale bar: 20 µm), (b) GFP & mCherry merge, (c) GFP, (d) mCherry. The dotted lines outline the MPOA and the third ventricle (middle line).

(C) A significant genotype x treatment interaction was detected for pup retrieval, two-way ANOVA, effect of genotype: P=0.187, effect of treatment: P=0.759, genotype x treatment interaction: *P=0.031. Shank2+/+ vehicle injected n = 6, Shank2-/- vehicle injected n = 6, Shank2+/+ CNO injected n = 6, Shank2-/- CNO injected n = 7.

(D-G) DREADD activation by CNO injection selectively rescues major components of social attachment behavior in Shank2-/- mice: (D) grooming, two-way ANOVA, effect of genotype: *P=0.018, effect of treatment: **P=0.002, genotype x treatment interaction: ***P=0.001. (E) Crouching, two-way ANOVA, effect of genotype: P=0.091, effect of treatment: P=0.869, genotype x treatment interaction: P=0.663, (F) nest building, two-way ANOVA, effect of genotype: P=0.415, effect of treatment: P=0.341 genotype x treatment interaction: P=0.093, (G) Maternal interaction, two-way ANOVA, effect of genotype: *P=0.034, effect of treatment: P=0.149, genotype x treatment interaction: *P=0.045. Shank2+/+ vehicle injected n = 6, Shank2-/- vehicle injected n = 6, Shank2+/+ CNO injected n = 6, Shank2-/- CNO injected n = 7.

Data information: All data are presented as mean ± SEM, NS: not significant. AC, anterior commissure and MPOA, medial preoptic area.
Prdx4 limits caspase-1 activation and restricts inflammasome-mediated signaling by extracellular vesicles (2019)
Figure 1

Figure 1. Prdx4 protects from LPS-induced septic shock.

A Percent body weight of male Prdx4 WT and KO mice over the 72 h course of LPS (4.5 mg/kg BW) or PBS injection (i.p.). Each circle represents a mean of n=7 mice, vertical lines indicate SEM *, p < 0.05; **, p < 0.01; ***, p < 0.001; (two-way-ANOVA, Bonferroni-posttest).

B-D Cytokine concentration in Prdx4 WT and KO mice in response to LPS injection. (B) Cxcl1 levels in serum (left) or peritoneal lavage (right) at indicated time points after LPS or PBS injection. (C) TNF-α levels in serum (left) or peritoneal lavage (right) at indicated time points after LPS or PBS injection. (D) IL-1β levels in serum (left) or peritoneal lavage (right) at indicated time points after LPS or PBS injection. Each dot (B-D) represents an individual mouse. Horizontal lines indicate mean. **, p < 0.01; ***, p < 0.001; n.s. not significant (two-tailed t-test). Data are representative of two independent experiments.
Prdx4 limits caspase-1 activation and restricts inflammasome-mediated signaling by extracellular vesicles (2019)
Figure 2

Figure 2. Role of IL-1 receptor blockade and myeloid-specific ablation of Prdx4 in the endotoxin shock model.

A Percent body weight of male Prdx4 WT and KO mice over the 48 h course of LPS (4.5 mg/kg BW) injection (i.p.) and treatment with IL-1 receptor antagonist (IL-1RA) Anakinra (200 µg/mouse) or control. Arrows indicate time point of Anakinra injection. Each circle represents a mean of n=5 mice, vertical lines indicate SEM. ***, p < 0.001; (two-way-ANOVA, Bonferroni-posttest).

B Serum concentration of Cxcl1, TNF-α and IL-1β in Prdx4 WT and KO mice injected with LPS, LPS and IL-1RA or control. Horizontal lines indicate mean. *, p < 0.05; **, p < 0.01; ***, p < 0.001; n.s. not significant (two-tailed t-test).

C Percent body weight of male Prdx4-flox and Prdx4-∆LysMCre mice over the 48 h course of 4.5 mg/kg BW LPS ( i.p.). Each circle represents a mean of n=7 mice, vertical lines indicate SEM. *, p < 0.05; ***, p < 0.001; (two-way-ANOVA, Bonferroni-posttest).

D Serum concentration of Cxcl1, TNF-α and IL-1β in Prdx4-flox and Prdx4-∆LysMCre mice injected with LPS. Each dot (B, D) represents an individual mouse. Horizontal lines indicate mean. *, p < 0.05; ***, p < 0.001; n.s. not significant (two-tailed t-test). Data are representative of two independent experiments.
Prdx4 limits caspase-1 activation and restricts inflammasome-mediated signaling by extracellular vesicles (2019)
Figure 3

Figure 3: Prdx4-deficient macrophages display elevated cytokine responses and inflammasome activation.

A Concentration of Cxcl1, TNF-α and IL-1β in the supernatants of Prdx4 WT and KO BMDMs in response to a time course of LPS stimulation (100 ng/ml LPS, time points indicated).

B IL-1β release of Prdx4 WT and KO BMDMs, untreated or primed for 6 h with LPS (100 ng/ml) and then pulsed for indicated time points with ATP (5 mM).

C Western blot analysis of IL-1β in cell lysates and supernatants of Prdx4 WT and KO BMDMs, primed with LPS (100 ng/ml) and pulsed with ATP (5 mM) for 4 h or left untreated. Dashed line indicates vertical slice.

D Immunofluorescence microscopy of ASC speck formation in Prdx4 WT and KO BMDMs in response to Nigericin (10 µg/ml) stimulation for 45 min of LPS-primed cells. Cells were stained with an antibody to ASC and nuclei were counterstained using DAPI. Scale bar indicates 20 µm. ASC speck-positive cells were counted and expressed as percentage of total cells. Bars represent a mean of n=4 mice, vertical lines indicate SD. n.s. not significant (two-tailed t-test).

E Western blot analysis of caspase-1 cleavage in the supernatant of Prdx4 WT and KO BMDMs in response to Nigericin (10 µg/ml) stimulation for 1 h after priming with LPS (100 ng/ml), LPS-priming alone or without stimulation. Whole cell lysates were analyzed for pro-caspase-1 and Gapdh levels.

F IL-1β release in Prdx4 WT and KO BMDMs, untreated or primed for 6 h with LPS (100 ng/ml) and then pulsed for 3 h with ATP (5 mM) or Nigericin (10 µg/ml) or transfected for 3 h with poly (dA:dT) or Flagellin (1 µg/ml each) or treated with transfection agent only.

G Quantification of cell death by LDH-release in Prdx4 WT and KO BMDMs, untreated or primed for 6 h with LPS (100 ng/ml) and then pulsed for 3 h with ATP (5 mM) or Nigericin (10 µg/ml) or transfected for 3 h with poly (dA:dT) or flagellin (1 µg/ml each) or treated with transfection agent only.

Data information: (A, B) Each dot represents a mean of n=3 mice, vertical lines indicate SD. **p < 0.01; ***, p < 0.001; (two-way-ANOVA, Bonferroni-posttest). (F, G) Bars represent a mean of n=3 mice, vertical lines indicate SD. *, p < 0.05; **, p < 0.01; ***, p < 0.001; n.s. not significant (two-tailed t-test). All data are representative of two independent experiments.