Journal: Neoplasia (New York, N.Y.)

Article Title: Inhibition of fibronectin accumulation suppresses TUMOR growth

doi: 10.1016/j.neo.2021.06.012

Figure Lengend Snippet: Changes in fibronectin affect blood vessels and proliferation. (A) Tumor lesions exhibit less fibronectin staining in sections from knockdown (Kd) tumors compared to controls (CT). Bars represent 100 μm. (B) Kd tumors contain less fibronectin as determined by ELISA. Pieces obtained from tumor samples were weighed and lysed in protein lysis buffer to evaluate by ELISA. The amount of fibronectin was corrected to total protein measured by the BCA method. N=3/6 replicates. (C) Expression of fibronectin mRNA originating from the cancer cells (human) and the host (murine) was diminished. Using primers specific for human and murine fibronectin it was possible to differentiate between both in qPCR analyses. Human fibronectin was corrected to human HPRT and murine fibronectin to murine β-actin. N=5/3 and 7/5 for the two graphs (left to right). (D) The area of CD31 + blood vessels diminishes as does the number of vessels stained with both CD31 (in red) and the pericyte markers α-smooth muscle actin (α-SMA) or desmin (in green). Examples are shown. Bars represent 100 μm. N=11/10 for CD31 + area and stainings with αSMA and 7/7 for stainings with desmin. (E) Endothelial cells from knockdown tumors produce less fibronectin. Isolated endothelial cells were cultured in the presence of fibronectin-depleted FCS and fibronectin evaluated in the conditioned media by ELISA. N=11/5. (F) Proliferation in tumor sections was diminished in Kd tumors as evidenced by the decrease in the percentage of Ki67 + cells. N=9/9. Examples are shown. Bars represent 100 μm. (G) Tumor sections were stained using TUNEL to mark apoptotic cells. No difference was seen between CT and Kd tumors. N=3/3. Examples are shown. Bars represent 100 μm. All pairs were evaluated using t-test. * P <0.05, ** P <0.005, *** P <0.0001.

Article Snippet: We also used rat anti-mouse CD31 (BIO-RAD; MCA2388GA; 1:100), mouse anti-alpha-smooth muscle actin (α-SMA)-Cy3 (Merck; #A2547; 1:200), rabbit anti-desmin (Dianova; #DLN-13732; 1:100), rabbit monoclonal antibody against YAP (Cell signaling #14074, 1:200), goat anti-rabbit-Alexa 647 (Abcam #ab150079; 1:1000), goat anti-mouse-Cy2 (Dianova; #115-225-166; 1:1000), goat anti-rat-Alexa 594 (Dianova; #112-585-062; 1:1000).

Techniques: Staining, Enzyme-linked Immunosorbent Assay, Lysis, Expressing, Isolation, Cell Culture, TUNEL Assay

Journal: Neoplasia (New York, N.Y.)

Article Title: Inhibition of fibronectin accumulation suppresses TUMOR growth

doi: 10.1016/j.neo.2021.06.012

Figure Lengend Snippet: Histology of pUR4-treated tumors confirms changes in matrix and reveals a decrease in proliferation. (A) Stained tumor sections show a decrease in fibronectin and collagen in pUR4-treated animals and no changes in R1R2-treated animals. Bars represent 100 μm. (B) Western blots from the tumors confirm a decrease in fibronectin and collagen with pUR4 treatment, but collagen I did not decrease with R1R2 therapy. N=4/4/4/4 replicates/treatment. Relevant pairs were evaluated by t-tests. * P <0.05. (C) Proliferation was diminished after treatment with pUR4. Sections were stained for Ki67. Bars represent 100 μm. N=9/9/9/9. Pairs were evaluated by t-tests. ** P <0.01. (D) Apoptosis was not affected in vivo by the various treatments. Sections were stained for TUNEL. Examples are shown. Bars represent 100 μm. N=9/9/9/9. Pairs were evaluated by t-tests. (E) Evaluation of blood vessels shows a decrease in area of CD31 + vessels with pUR4 treatment as well as the number of CD31 + αSMA + or CD31 + desmin + double positive vessels (adjusted to the area). Sections from representative tumors with bars representing 100μm. These are intratibial tumors from mice treated for 10 days with the peptides. N=12/12/11/12 for CD31 alone or with α-smooth muscle actin (αSMA) and 11/10/7/7 for staining with desmin. Pairs were compared by t-test. * P <0.05, **** P <0.0001.

Article Snippet: We also used rat anti-mouse CD31 (BIO-RAD; MCA2388GA; 1:100), mouse anti-alpha-smooth muscle actin (α-SMA)-Cy3 (Merck; #A2547; 1:200), rabbit anti-desmin (Dianova; #DLN-13732; 1:100), rabbit monoclonal antibody against YAP (Cell signaling #14074, 1:200), goat anti-rabbit-Alexa 647 (Abcam #ab150079; 1:1000), goat anti-mouse-Cy2 (Dianova; #115-225-166; 1:1000), goat anti-rat-Alexa 594 (Dianova; #112-585-062; 1:1000).

Techniques: Staining, Western Blot, In Vivo, TUNEL Assay

Journal: Cell Reports

Article Title: The onset of circulation triggers a metabolic switch required for endothelial to hematopoietic transition

doi: 10.1016/j.celrep.2021.110103

Figure Lengend Snippet: Ncx1 −/− embryos display an aberrant peri-aortic microenvironment (A) Confocal WM-IF analysis of E9.5 (22-25sp) +/+ and Ncx1 −/− embryos. Left panels show maximum intensity projections. Boxed area is magnified in the middle and right panels (single 2.5 μm-thick slices). Arrowheads indicate α-SMA + peri-aortic SMCs, absent from Ncx1 −/− embryos (asterisks). Yellow dashed arrow: distance between dorsal aorta (da) and vitelline artery (va). N = 3 ( +/+ ), N = 3 ( Ncx1 −/− ) embryos analyzed. Scale bars: 300 μm (3D), 50 μm (slice). (B) Distance between dorsal aorta and vitelline artery as a measurement of the sub-aortic mesenchyme thickness. Measurements done on images from N = 7 ( +/+ ), N = 5 ( Ncx1 −/− ) different embryos (1-4 images/embryo; 5 measurements / image; 16 ( +/+ ), 17 ( Ncx1 −/− ) different images used. Data are mean ± SD. (C) Flow cytometric analysis of macrophages (Ter119 - CD45 + F4/80 + CD11b + ) in E9.5 (21-25sp) +/+ and Ncx1 −/− caudal part (CP). N = 3 ( +/+ ), N = 3 ( Ncx1 −/− ) embryos were analyzed individually in 2 independent experiments. (D) Quantification of flow cytometric analysis in (C). Data are mean ± SD. (E) Confocal WM-IF of E9.5 (21-24sp) +/+ and Ncx1 −/− embryos (single 2.5 μm-thick slice representative of N = 4 ( +/+ ), N = 4 ( Ncx1 −/− ) embryos). Arrowheads: peri-aortic F4/80 + macrophages. Scale bars: 30 μm. (F) Uniform Manifold Approximation and Projection (UMAP; ( Becht et al., 2018 )) of the E9.5 (20-23sp) +/+ and Ncx1 −/− PAS scRNA-Seq dataset. Cells were isolated from 4 embryos for each genotype. (G) Percentage of cells in each PAS scRNA-Seq cluster. (H) Bubble plot showing marker genes for each PAS scRNA-Seq cluster. Dot size indicates the percentage of expressing cells; color intensity indicates expression level. (I) Bubble plot showing expression of genes encoding for hematopoietic niche signals in niche cell subsets. Expression is shown separately for +/+ and Ncx1 −/− cells.

Article Snippet: Mouse anti-mouse/rat/human α-SMA (Cy3 conjugate), clone 1A4 , Merck , Cat# C6198; RRID: AB_476856.

Techniques: Isolation, Marker, Expressing

Journal: Cell Reports

Article Title: The onset of circulation triggers a metabolic switch required for endothelial to hematopoietic transition

doi: 10.1016/j.celrep.2021.110103

Figure Lengend Snippet:

Article Snippet: Mouse anti-mouse/rat/human α-SMA (Cy3 conjugate), clone 1A4 , Merck , Cat# C6198; RRID: AB_476856.

Techniques: Blocking Assay, Recombinant, Modification, TaqMan Assay, Expressing, RNA Sequencing Assay, Mutagenesis, Knock-Out, Software

Journal: Physiological Reports

Article Title: The development of hepatic stellate cells in normal and abnormal human fetuses – an immunohistochemical study

doi: 10.14814/phy2.12504

Figure Lengend Snippet: Hepatic Mesenchymal Cells in Control Fetuses. (A) cRBP-1-positive cell numbers at different gestational ages in control fetuses. The mean number of stellate cells/hpf from 10 hpf was calculated for each control fetus. Although there is wide variation in numbers of stellate cells at each gestational age, there is a statistically significant reduction in stellate cell density with increasing gestational age ( r = −0.3576, P = 0.0186). Hepatic mesenchymal cells stained for cRBP-1 (B), GFAP (C, D), and α SMA (E) in a 19 week gestational control fetus. (B) Numerous hepatic stellate cells are present, showing oval nuclei and long cell processes expressing cRBP-1 antigen (arrow and inset). (C) Numerous stellate-shaped perisinusoidal cells expressing GFAP. Rounded GFAP+ve cells in the sinusoidal spaces (arrows) show similar nuclear features and appear to be transitional forms between intravascular and perisinusoidal cells. (D) GFAP stain showing some submesothelial cells expressing this antigen. There are relatively abundant stellate cells beneath the liver capsule. (E) α SMA shows scant stellate cells in the liver lobules. (score 0.5 for α SMA); Original magnification ×400.

Article Snippet: Sections were incubated with mouse anti- α SMA primary antibody (Biocare Medical), followed by a secondary MACH antimouse HRP (Biocare Medical) antibody and TSATM-FITC signal amplification (Life Technologies).

Techniques: Control, Staining, Expressing

Journal: Physiological Reports

Article Title: The development of hepatic stellate cells in normal and abnormal human fetuses – an immunohistochemical study

doi: 10.14814/phy2.12504

Figure Lengend Snippet: Clinical details and expression of cRBP-1, GFAP, α -SMA, and WT1 in Renal Agenesis Cases versus Controls (modified from Loo et al. 2012b )

Article Snippet: Sections were incubated with mouse anti- α SMA primary antibody (Biocare Medical), followed by a secondary MACH antimouse HRP (Biocare Medical) antibody and TSATM-FITC signal amplification (Life Technologies).

Techniques: Expressing, Modification, Control

Journal: Physiological Reports

Article Title: The development of hepatic stellate cells in normal and abnormal human fetuses – an immunohistochemical study

doi: 10.14814/phy2.12504

Figure Lengend Snippet: Hepatic Mesenchymal Cells in Renal Agenesis Fetuses. (A) cRBP-1-positive cell numbers at different gestational ages in Renal Agenesis Fetuses. Numbers of hepatic stellate cells/hpf expressing cRBP-1 in fetuses with bilateral renal agenesis, with or without cardiac defects compared to matched control fetal liver. Mean of 50 hpf is provided for each case. There were significantly fewer hepatic stellate cells in cases of bilateral renal agenesis fetuses with cardiac defects (BRA + CHD; gestational ages 17–41 weeks) versus both controls (gestational ages 15–29 weeks) and bilateral renal agenesis fetuses without cardiac defects (BRA; gestational ages 18–21 weeks) (ANOVA, P = 0.0023), independent of gestational age. Results for individual fetuses are presented in Table . Hepatic mesenchymal cells stained for cRBP-1 (B), α SMA (C) and GFAP (D–G) in renal agenesis fetuses. (B) cRBP-1 shows fewer stellate cells and these have shorter cell processes (small arrow) compared with control (see Fig. ). Circulating mesenchymal cells are present in the blood vessel (large arrow). (C) There are many more perisinusoidal cells expressing α SMA (score 3 for α SMA) versus the control fetus (see Fig. ); Original Magnification, ×400. (D) Many intravascular and perisinusoidal cells, some with stellate morphology, expressing GFAP. (E) High power view of (D) showing GFAP+ve intravascular cells (small arrow) and stellate shaped perisinusoidal cells with stellate morphology (large arrow) and cells of the transitional forms between intravascular and perisinusoidal cells (arrowhead). (F) In a fetus with bilateral renal agenesis, cardiac and WT1 defects, there are numerous round intravascular cells expressing GFAP antigen (arrows), while stellate cells in the perisinusoidal space with characteristic long processes are scant. These are not concentrated beneath the mesothelium as in control cases (as seen in Fig. ). (G) GFAP immunohistochemistry shows abundant stellate cells beneath the capsule in a renal agenesis fetus without cardiac or WT1 defects.

Article Snippet: Sections were incubated with mouse anti- α SMA primary antibody (Biocare Medical), followed by a secondary MACH antimouse HRP (Biocare Medical) antibody and TSATM-FITC signal amplification (Life Technologies).

Techniques: Expressing, Control, Staining, Immunohistochemistry

Journal: Physiological Reports

Article Title: The development of hepatic stellate cells in normal and abnormal human fetuses – an immunohistochemical study

doi: 10.14814/phy2.12504

Figure Lengend Snippet: Colocalization of α SMA and cRBP-1. Dual immunofluorescence for (A) α SMA (green), (B) cRBP-1 (red) and (C) DAPI (blue) in renal agenesis fetus, showing a large round cell (mesenchymal stem cell, arrowhead) and an activated stellate cell (arrow) expressing α SMA (A). There is another similar round cell coexpressing both antigens (large arrow), presumably a stem cell in early transition to a stellate cell (D, merge). Stellate cells expressing cRBP-1 antigen alone are also seen (B). (Original magnification, ×630).

Article Snippet: Sections were incubated with mouse anti- α SMA primary antibody (Biocare Medical), followed by a secondary MACH antimouse HRP (Biocare Medical) antibody and TSATM-FITC signal amplification (Life Technologies).

Techniques: Immunofluorescence, Expressing

Journal: Physiological Reports

Article Title: The development of hepatic stellate cells in normal and abnormal human fetuses – an immunohistochemical study

doi: 10.14814/phy2.12504

Figure Lengend Snippet: Colocalization of α SMA and GFAP. Dual immunofluorescence for (A) α SMA (green), (B) GFAP (red) and (C) DAPI (blue) in renal agenesis fetus showing a hepatic stellate cell (arrow) and mesenchymal stem cell (arrowhead) expressing α SMA only (A), and a mesenchymal stem cell coexpressing both α SMA and GFAP (B) possibly in transition to a hepatic stellate cell (large arrow) and (D, merge). (Original magnification, ×630).

Article Snippet: Sections were incubated with mouse anti- α SMA primary antibody (Biocare Medical), followed by a secondary MACH antimouse HRP (Biocare Medical) antibody and TSATM-FITC signal amplification (Life Technologies).

Techniques: Immunofluorescence, Expressing

Journal: Physiological Reports

Article Title: The development of hepatic stellate cells in normal and abnormal human fetuses – an immunohistochemical study

doi: 10.14814/phy2.12504

Figure Lengend Snippet: Hepatic mesenchymal cells in main liver of 27 week diaphragm agenesis fetus showing (A) GFAP, (B) cRBP-1, and (C) α SMA expressing mesenchymal cells. (A) GFAP is expressed in large, rounded intravascular cells (small arrow), and perisinusoidal stellate cells (large arrow). (B) cRBP-1 is weakly expressed in ductal plate cells but there are very scant perisinusoidal stellate cells expressing this antigen. (C) Increased numbers of stellate-shaped perisinusoidal cells express α SMA antigen (large arrow) as well as a few large, rounded intravascular cells, including one in a central vein in the lower right of the photomicrograph (small arrow).

Article Snippet: Sections were incubated with mouse anti- α SMA primary antibody (Biocare Medical), followed by a secondary MACH antimouse HRP (Biocare Medical) antibody and TSATM-FITC signal amplification (Life Technologies).

Techniques: Expressing

Journal: Cells

Article Title: α2-Adrenergic Receptor in Liver Fibrosis: Implications for the Adrenoblocker Mesedin

doi: 10.3390/cells9020456

Figure Lengend Snippet: TaqMan gene expression assays used for the mRNA analysis.

Article Snippet: α-SMA (IHC) (mouse monoclonal) , 61001 , Progen, Heidelberg, Germany.

Techniques: Gene Expression

Journal: Cells

Article Title: α2-Adrenergic Receptor in Liver Fibrosis: Implications for the Adrenoblocker Mesedin

doi: 10.3390/cells9020456

Figure Lengend Snippet: Antibodies used for Western blot and immunofluorescence analyses.

Article Snippet: α-SMA (IHC) (mouse monoclonal) , 61001 , Progen, Heidelberg, Germany.

Techniques: Western Blot, Immunofluorescence

Journal: Cells

Article Title: α2-Adrenergic Receptor in Liver Fibrosis: Implications for the Adrenoblocker Mesedin

doi: 10.3390/cells9020456

Figure Lengend Snippet: Influence of mesedin on the expression of α-adrenergic receptors in murine hepatic stellate cells (M1-4HSCs). ( A – D ) Immunofluorescence analysis of α1/α-ARs (red) and α-SMA (green) in M1-4HSCs treated with 10 µM mesedin vs untreated controls (ctrl.). The scale bar corresponds to 100 µm, and the cell nuclei were stained with DAPI. ( E , F ) Quantification of α1-AR + /α-SMA + and α2-AR/α-SMA + M1-4HSCs upon incubation with mesedin vs the control (ctrl). ( G ) Western blot and densitometric analysis of α2-AR and β2-AR in M1-4HSCs with and without mesedin. Student’s t -test, n = 4, p < 0.05 (*), p < 0.01 (**).

Article Snippet: α-SMA (IHC) (mouse monoclonal) , 61001 , Progen, Heidelberg, Germany.

Techniques: Expressing, Immunofluorescence, Staining, Incubation, Control, Western Blot

Journal: Cells

Article Title: α2-Adrenergic Receptor in Liver Fibrosis: Implications for the Adrenoblocker Mesedin

doi: 10.3390/cells9020456

Figure Lengend Snippet: Influence of mesedin on α-smooth muscle actin (α-SMA) expression in M1-4HSCs. (A) Quantification of α-SMA mRNA in mesedin-treated M1-4HSCs vs controls (ctrl.), n = 6. ( B ) Quantification of α-SMA + M1-4HSCs with and without mesedin, n = 5. ( C ) Western blot and densitometric analysis of α-SMA in M1-4HSCs treated with mesedin, n = 4. Student’s t -test; data are shown as the mean ± SEM, p < 0.05 (*), p < 0.01 (**), p < 0.0001 (****).

Article Snippet: α-SMA (IHC) (mouse monoclonal) , 61001 , Progen, Heidelberg, Germany.

Techniques: Expressing, Western Blot

Journal: Cells

Article Title: α2-Adrenergic Receptor in Liver Fibrosis: Implications for the Adrenoblocker Mesedin

doi: 10.3390/cells9020456

Figure Lengend Snippet: Transforming growth factor-β (TGF-β) and platelet-derived growth factor (PDGF) expression in mesedin-treated M1-4HSCs. ( A ) TGF-β (red) and α-SMA (green) staining of M1-4HSCs under control conditions (ctrl.) and incubation with mesedin. Cell nuclei were stained with DAPI; scale bar 100 µm. Quantification of α-SMA/TGF-β-positive M1-4HSCs ( n = 9, p < 0.001 (***), Student’s t -test; data shown as the mean ± SEM). ( B ) TGF-β expression as analyzed by Western blot and a corresponding densitometric analysis of M1-4HSCs treated with mesedin vs the control (ctrl.) ( n = 4, p < 0.05 (*), Student’s t -test, data shown as the mean ± SEM). ( C ) PDGF Western blot of M1-4HSCs with and without mesedin (ctrl.), n = 4.

Article Snippet: α-SMA (IHC) (mouse monoclonal) , 61001 , Progen, Heidelberg, Germany.

Techniques: Derivative Assay, Expressing, Staining, Control, Incubation, Western Blot

Journal: Cells

Article Title: Depletion of Bone Marrow-Derived Fibrocytes Attenuates TAA-Induced Liver Fibrosis in Mice

doi: 10.3390/cells8101210

Figure Lengend Snippet: The antifibrotic effect was not accompanied by a reduction of myofibroblasts. ( a ) Western blot analysis and optical densitometry thereof revealed that the hepatic α-SMA levels were increased following TAA-treatment but unchanged by fibrocyte ablation. Two individual western blots were included in the analysis, a representative blot is shown. Arbitrary unit. SC n = 2; Ctrl, FC-Abl. n = 6. Mean + SEM is depicted. ( b ) Immunohistochemical staining of α-SMA (brown) demonstrated the periportal accumulation of myofibroblasts in TAA-treated animals and an unchanged expression pattern in result of the fibrocyte ablation. Representative stainings are shown. Magnification 40× and 200×, bars 400 and 50 µm. ( c ) Hepatic gene expression levels of Acta2 , Tgfb, and Pdgfb were comparable at the end of the experiment (full data in ).

Article Snippet: Western blot experiments were performed as described previously [ ] using 1:1.000 diluted antibodies against α-SMA (Mouse anti α-SMA monoclonal antibody, 61001, Progen, Heidelberg, Germany), Bax (Rabbit anti Bax polyclonal antibody, #2772), and Bcl-2 (Rabbit anti Bcl-2 polyclonal antibody, #2876).

Techniques: Western Blot, Immunohistochemical staining, Staining, Expressing, Gene Expression