Journal: Scientific Reports

Article Title: TGFβ3-mediated induction of Periostin facilitates head and neck cancer growth and is associated with metastasis

doi: 10.1038/srep20587

Figure Lengend Snippet: ( A,B ) The transcriptional and translational statuses of POSTN were determined in CAFs and NFs, 6 representative HNC cell lines and normal oral epithelial cells (titled normal) using western blotting ( A ) and real-time PCR ( B ). ( C ) Representative images of immunohistochemical staining showed the distribution of POSTN expression in adjacent normal tissues and HNC tissues. ( D,E ) POSTN expressions in NFs and CAFs were detected using immunohistochemical staining ( D ) and confocal microscopy ( E ). ( F ) The protein levels of POSTN, FAP and α-SMA were determined by a western blot analysis. ( G ) POSTN mRNA levels in 5 pairs of NFs and CAFs were measured by real-time PCR. ( H ) POSTN protein levels in the media of NFs and CAFs were determined by ELISA analyses. (**** p < 0.0001).

Article Snippet: The blots were probed with a mouse monoclonal POSTN antibody (Sigma, USA, 1:1 000), a rabbit polyclonal POSTN antibody [polyclonal] (Sigma, USA; 1:1 000), a mouse monoclonal α-SMA antibody [1A4] (Abcam, USA; 1:300) and a rabbit monoclonal FAP antibody [S9B] (Abcam, USA; 1:1 000).

Techniques: Western Blot, Real-time Polymerase Chain Reaction, Immunohistochemical staining, Staining, Expressing, Confocal Microscopy, Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: Gene silencing of TACE enhances plaque stability and improves vascular remodeling in a rabbit model of atherosclerosis

doi: 10.1038/srep17939

Figure Lengend Snippet: ( A ) Representative immnofluorescence images showing TACE positive staining cells in green; ( B ) RAM-11 positive staining cells in red; ( C ) DAPI positive staining cells in blue; ( D ) TACE, RAM-11 and DAPI positive staining cells in merged image; ( E ) TACE positive staining cells in green; ( F ) α-actin positive staining cells in red; ( G ) DAPI positive staining cells in blue; ( H ) TACE, α-actin and DAPI positive staining cells in merged image; ( I ) correlation analysis between TACE and macrophage positive staining areas; ( J ) correlation between TACE positive staining area and neovessel number in plaques. Bar = 100 μm

Article Snippet: Serial sections were subjected to immunostaining for RAM11 monoclonal antibody (1:200, Lab Vision Neomarkers, USA), mouse anti-human smooth muscle cell α-actin 1A4 monoclonal antibody (1:100, Boster, China), goat anti-human PECAM-1 (CD31) polyclonal antibody (1:100, Santa Cruz Biotechology, USA), goat anti-human TACE polyclonal antibody (1:100, Santa Cruz Biotecholog, USA), mouse anti-human P65 monoclonal antibody (1:200, Santa Cruz Biotecholog, USA), goat anti-human iNOS polyclonal antibody (1:200, Santa Cruz Biotecholog, USA), goat anti-human ICAM-1 polyclonal antibody (1:200, Santa Cruz Biotecholog, USA), and goat anti-human TGFβ1 polyclonal antibody (1:200, Santa Cruz Biotecholog, USA).

Techniques: Staining

Journal: Scientific Reports

Article Title: Gene silencing of TACE enhances plaque stability and improves vascular remodeling in a rabbit model of atherosclerosis

doi: 10.1038/srep17939

Figure Lengend Snippet: ( A–C ) Representative RAM-11 immunostaining showing the macrophage content in the Control group ( A ), Mock group ( B ), and TACE shRNA group ( C ) at week 16; ( D – F ) Representative Oil red O staining showing the lipid content in the Control group ( D ), Mock group ( E ), and TACE shRNA group ( F ) at week 16; ( G – I ) Representative α-actin immunostaining showing the content of smooth muscle cells in the Control group ( G ), Mock group ( H ), and TACE shRNA group ( I ) at week 16; ( J – L ) Representative Sirius red staining showing the collagen content in the Control group ( J ), Mock group ( K ), and TACE shRNA group ( L ) at week 16; ( M ) Quantitative analysis of ( A – C ); (N) Quantitative analysis of ( D – F ); ( O ) Quantitative analysis of ( G – I ); ( P ) Quantitative analysis of ( J – L ). + P < 0.05 vs . Control group, + + P < 0.01 vs . Control group, # P < 0.05 vs . Mock group, * P < 0.01 vs . Mock group. Bar = 250 μm.

Article Snippet: Serial sections were subjected to immunostaining for RAM11 monoclonal antibody (1:200, Lab Vision Neomarkers, USA), mouse anti-human smooth muscle cell α-actin 1A4 monoclonal antibody (1:100, Boster, China), goat anti-human PECAM-1 (CD31) polyclonal antibody (1:100, Santa Cruz Biotechology, USA), goat anti-human TACE polyclonal antibody (1:100, Santa Cruz Biotecholog, USA), mouse anti-human P65 monoclonal antibody (1:200, Santa Cruz Biotecholog, USA), goat anti-human iNOS polyclonal antibody (1:200, Santa Cruz Biotecholog, USA), goat anti-human ICAM-1 polyclonal antibody (1:200, Santa Cruz Biotecholog, USA), and goat anti-human TGFβ1 polyclonal antibody (1:200, Santa Cruz Biotecholog, USA).

Techniques: Immunostaining, Control, shRNA, Staining

Journal: Scientific Reports

Article Title: Long-term safety and stability of angiogenesis induced by balanced single-vector co-expression of PDGF-BB and VEGF 164 in skeletal muscle

doi: 10.1038/srep21546

Figure Lengend Snippet: V, VIP and P myoblast clones, expressing low, medium or high levels of one or both factors, as well as control cells (Ctrl), were implanted into calf muscles of SCID mice. Frozen sections were obtained 4 weeks after implantation and immunostained for CD31 (endothelium, in red), NG2 (pericytes, in green) and α-SMA (smooth muscle cells, in cyan); n = 3–8 samples/group. In each panel, the right image is a higher-magnification view of the area marked by the white dotted box in the left image. Size bars = 20 μm; asterisks indicate angioma-like aberrant vascular structures.

Article Snippet: The following primary antibodies and dilutions were used: rat monoclonal anti-mouse PECAM-1 (clone MEC13.3, BD Biosciences, Basel, Switzerland) at 1:100; mouse monoclonal anti-mouse α-SMA (clone 1A4, MP Biomedicals, Basel, Switzerland) at 1:400; rabbit polyclonal anti-NG2 (Chemicon International, Hampshire, UK) at 1:200.

Techniques: Clone Assay, Expressing

Journal: Scientific Reports

Article Title: Long-term safety and stability of angiogenesis induced by balanced single-vector co-expression of PDGF-BB and VEGF 164 in skeletal muscle

doi: 10.1038/srep21546

Figure Lengend Snippet: V and VIP myoblasts clones, expressing low, medium or high levels, as well as control cells (Ctrl), were implanted into calf muscles of SCID mice. Frozen sections were obtained 4 months after implantation and immunostained for CD31 (endothelium, in red), NG2 (pericytes, in green) and α-SMA (smooth muscle cells, in cyan); n = 3–7 samples/group. In each panel, the right image is a higher-magnification view of the area marked by the white dotted box in the left image. High VEGF levels caused the progressive growth of macroscopic angiomas ( f ) and required early termination of the experiment after 9 weeks (n = 4). Size bars = 20 μm, except in ( f ): size bar = 100 μm; asterisks in ( f ) indicate large angioma-like aberrant vascular structures.

Article Snippet: The following primary antibodies and dilutions were used: rat monoclonal anti-mouse PECAM-1 (clone MEC13.3, BD Biosciences, Basel, Switzerland) at 1:100; mouse monoclonal anti-mouse α-SMA (clone 1A4, MP Biomedicals, Basel, Switzerland) at 1:400; rabbit polyclonal anti-NG2 (Chemicon International, Hampshire, UK) at 1:200.

Techniques: Clone Assay, Expressing

Journal: Scientific Reports

Article Title: Long-term safety and stability of angiogenesis induced by balanced single-vector co-expression of PDGF-BB and VEGF 164 in skeletal muscle

doi: 10.1038/srep21546

Figure Lengend Snippet: Capillaries and arterioles were identified by immunostaining on frozen sections of the same muscles analyzed in and , 4 months after implantation of control cells (Ctrl) or V and VIP myoblasts clones, expressing low, medium or high levels. ( a–f ) Endothelium is stained in red (CD31) and smooth muscle cells in cyan (α-SMA). In each panel, the right image is a higher-magnification view of the area marked by the white dotted box in the left image. Size bars = 50 μm in all panels. ( g ) Quantification of the number of arterioles adjacent to the areas of increased angiogenesis, which were strictly localized to the sites of myoblast engraftment. Data are shown as mean ± SEM (n = 14 measurements/group in 2 tissues/group); ****p < 0.0001 vs control and for indicated comparisons; *p < 0.05 for indicated comparisons (ANOVA with Sidak multiple comparisons test).

Article Snippet: The following primary antibodies and dilutions were used: rat monoclonal anti-mouse PECAM-1 (clone MEC13.3, BD Biosciences, Basel, Switzerland) at 1:100; mouse monoclonal anti-mouse α-SMA (clone 1A4, MP Biomedicals, Basel, Switzerland) at 1:400; rabbit polyclonal anti-NG2 (Chemicon International, Hampshire, UK) at 1:200.

Techniques: Immunostaining, Clone Assay, Expressing, Staining

Journal: Oncotarget

Article Title: Breast cancer cell cyclooxygenase-2 expression alters extracellular matrix structure and function and numbers of cancer associated fibroblasts

doi: 10.18632/oncotarget.14912

Figure Lengend Snippet: Representative images of α-SMA immunostained sections obtained from A . MDA-MB-231 and D . Clone 13 tumors. Magnified FOVs showing immunostained CAFs and the accuracy of the algorithm in identifying CAFs are presented in B . and C . for the MDA-MB-231 tumor section and in E . and F . for the Clone 13 tumor section. G . Quantification of immunostaining identified the presence of higher CAFs in MDA-MB-231 tumors (N=5) compared to Clone 13 tumors (N=6). Values represent Mean ± SEM. #p ≤ 0.084. H . Representative α-SMA immunoblot obtained from an MDA-MB-231 and a Clone 13 tumor. GAPDH was used as a loading control.

Article Snippet: Expression levels of COX-2, α-SMA, Col1A1, and VEGF were determined by immunoblotting after blocking with 5% nonfat milk, with goat anti-COX-2 antibody (1:500, Cayman Chemical, Ann Arbor, Michigan), a monoclonal antibody against α-SMA (Clone 1A4, 1:1000), a rabbit polyclonal antibody against Col1A1 (ORIGENE, Rockville, MD), or an anti-VEGF polyclonal antibody (1:2000, Millipore Temecula, CA), and visualized with HRP (horseradish peroxidase)-conjugated secondary antibodies using the SuperSignal West Pico Chemiluminescent substrate kit (Thermo Scientific, Rockford, IL).

Techniques: Immunostaining, Western Blot

Journal: Oncotarget

Article Title: Breast cancer cell cyclooxygenase-2 expression alters extracellular matrix structure and function and numbers of cancer associated fibroblasts

doi: 10.18632/oncotarget.14912

Figure Lengend Snippet: Representative 5 μm-thick H&E and corresponding α-SMA immunostained sections obtained from lungs of mice injected with A, B . MDA-MB-231 (N=5) and C, D . Clone 13 (N=3) cells. E . Spearman correlation between sum of metastatic nodule pixels (reflecting total nodule area) and sum of strongly positive pixels (reflecting number of CAFs) in lungs obtained from each mouse. A significant correlation was observed supporting the role of CAFs in the formation of metastasis.

Article Snippet: Expression levels of COX-2, α-SMA, Col1A1, and VEGF were determined by immunoblotting after blocking with 5% nonfat milk, with goat anti-COX-2 antibody (1:500, Cayman Chemical, Ann Arbor, Michigan), a monoclonal antibody against α-SMA (Clone 1A4, 1:1000), a rabbit polyclonal antibody against Col1A1 (ORIGENE, Rockville, MD), or an anti-VEGF polyclonal antibody (1:2000, Millipore Temecula, CA), and visualized with HRP (horseradish peroxidase)-conjugated secondary antibodies using the SuperSignal West Pico Chemiluminescent substrate kit (Thermo Scientific, Rockford, IL).

Techniques: Injection

Journal: Oncotarget

Article Title: Breast cancer cell cyclooxygenase-2 expression alters extracellular matrix structure and function and numbers of cancer associated fibroblasts

doi: 10.18632/oncotarget.14912

Figure Lengend Snippet: A . PGE 2 expression in COX-2 overexpressing cells; cells were exposed to 50nM TPA for 24hrs to induce COX-2 expression. Values represents Mean ± SEM from four independent experiments; * p≤ 0.005. B . 3D visualization of Col1 fibers in empty vector expressing and COX-2 overexpressing SUM-149 tumors. The FOV image size was 423.5×423.5×12μm 3 . C . Quantification of Col1 fiber volume and fiber density. COX-2 overexpressing tumors (N=5) had significantly lower inter-fiber distance and significantly higher percent fiber volume compared to SUM-149-EV tumors (N=5). Values represent Mean ± SEM. * p≤ 0.05, ** p≤ 0.005. D . Representative images of α-SMA immunostained sections obtained from SUM-149-EV (top) and SUM-149-COX-2FL (bottom) tumors. Magnified FOVs at 20X show immunostained CAFs for SUM-149-EV (top) and SUM-149-COX-2FL (bottom) tumors. E . Quantification of immunostained sections identified a higher number of CAFs in SUM-149-COX-2FL tumors (N=5) compared to SUM-149-EV tumors (N=5). Values represent Mean ± SEM. # p=0.06. F . Representative high resolution 20X H&E images of lung section showing emboli formation following intravenous injection of SUM-149-EV cells (left) and SUM-149-COX-2FL cells (right). G . Representative high-resolution 20X images of lung showing pulmonary metastatic foci following intravenous injection of SUM-149-EV cells (left) and SUM-149-COX-2FL (right).

Article Snippet: Expression levels of COX-2, α-SMA, Col1A1, and VEGF were determined by immunoblotting after blocking with 5% nonfat milk, with goat anti-COX-2 antibody (1:500, Cayman Chemical, Ann Arbor, Michigan), a monoclonal antibody against α-SMA (Clone 1A4, 1:1000), a rabbit polyclonal antibody against Col1A1 (ORIGENE, Rockville, MD), or an anti-VEGF polyclonal antibody (1:2000, Millipore Temecula, CA), and visualized with HRP (horseradish peroxidase)-conjugated secondary antibodies using the SuperSignal West Pico Chemiluminescent substrate kit (Thermo Scientific, Rockford, IL).

Techniques: Expressing, Plasmid Preparation, Injection

Journal: PLoS ONE

Article Title: The SULFs, Extracellular Sulfatases for Heparan Sulfate, Promote the Migration of Corneal Epithelial Cells during Wound Repair

doi: 10.1371/journal.pone.0069642

Figure Lengend Snippet: A and B: Immunofluorescent staining with anti SULF1 antibody (R1.1) (right panels, red) and nuclear staining with DAPI (left panels, blue). Corneal wounding of wild-type mice was performed with an Algerbrush. Eyeballs were fixed at 8 and 72 hrs (A) or 4 and 8 hrs (B) post wounding, corneas were prepared for whole mount staining, and imaged at 4× by immunofluorescent microscopy (composite images are prepared digitally). A contralateral eye served as a non-wounded control (NW). The pair in each row shows the same field. Scale bar: 1 mm. C: Imaging of the 4-hr post-injury corneal whole mount at 40× by spinning disc confocal microscopy. The field shown is centered on the wound edge. SULF1 (yellow) is only found in the superficial epithelial layer (X-Z and Y-Z panels). Scale bar: 70 µm. D: Whole mounts of 18 hr post-wounded corneas stained with either anti SULF1 antibody (R1.1) or rabbit anti-DNP (IgG control) and imaged by laser scanning microscopy at 20×. The wound margin is visible in the upper right-hand side corner and outlined by the dotted white line. E: Whole mount of cornea 24 hrs post-wounding stained with R1.1 and imaged by laser scanning microscopy at 20×. The dotted white line approximates the center of the original wound. Scale bars: 100 µm.

Article Snippet: Immunoblotting was performed with 1 μg/ml mouse SULF1 mAb 1A4 which recognizes the C-terminal 50-kDa subunit (Novus Biologicals, Littleton, CO), 3 μg/ml G1.6 for SULF1 (see ) and 1 μg/ml R2.3 and R2.1 for SULF2, both of which detect the N-terminal 75-kDa subunit .

Techniques: Staining, Microscopy, Control, Imaging, Confocal Microscopy, Laser-Scanning Microscopy

Journal: PLoS ONE

Article Title: The SULFs, Extracellular Sulfatases for Heparan Sulfate, Promote the Migration of Corneal Epithelial Cells during Wound Repair

doi: 10.1371/journal.pone.0069642

Figure Lengend Snippet: A: Epithelial defects (∼1 mm diameter) created at the center of wild-type (WT), Sulf1 −/− , Sulf2 −/− , and Sulf1 −/− / Sulf2 −/− corneas (left panel) were visualized by fluorescent staining (arrows) immediately post-wounding (left panel) and 24 hrs after wounding (right panel) with individual mice used for each condition. Representative results are shown for each genotype at each time point. Scale bar: 1 mm. B: The two-dimensional projection of the wound area was quantified using ImageJ software, and the percent remaining epithelial defect was determined (Area t24 /Area t0 ×100) (+ SEM, 11–14 eyes/genotype; *p<0.05, **p<0.01 compared to WT). Statistical significance was evaluated by one-way analysis of variance (ANOVA) with a Tukey's range post-test. P values of <0.05 were considered significant.

Article Snippet: Immunoblotting was performed with 1 μg/ml mouse SULF1 mAb 1A4 which recognizes the C-terminal 50-kDa subunit (Novus Biologicals, Littleton, CO), 3 μg/ml G1.6 for SULF1 (see ) and 1 μg/ml R2.3 and R2.1 for SULF2, both of which detect the N-terminal 75-kDa subunit .

Techniques: Staining, Software

Journal: PLoS ONE

Article Title: The SULFs, Extracellular Sulfatases for Heparan Sulfate, Promote the Migration of Corneal Epithelial Cells during Wound Repair

doi: 10.1371/journal.pone.0069642

Figure Lengend Snippet: Confluent monolayers of MCE cells of the indicated genotype were wounded by a pipette tip and monitored by time-lapse microscopy for 24 hrs. Wound healing was determined as the average linear speed of the wound edge over 8 hrs. Sulf1 −/− MCE migrated at a reduced rate compared to wild-type (WT) and Sulf2 −/− cells. Data are expressed as means+SEM, n = 3 per group. Statistical significance was evaluated with a Student t-test. *p = 0.023, **p = 0.009.

Article Snippet: Immunoblotting was performed with 1 μg/ml mouse SULF1 mAb 1A4 which recognizes the C-terminal 50-kDa subunit (Novus Biologicals, Littleton, CO), 3 μg/ml G1.6 for SULF1 (see ) and 1 μg/ml R2.3 and R2.1 for SULF2, both of which detect the N-terminal 75-kDa subunit .

Techniques: Transferring, Time-lapse Microscopy

Journal: PLoS ONE

Article Title: The SULFs, Extracellular Sulfatases for Heparan Sulfate, Promote the Migration of Corneal Epithelial Cells during Wound Repair

doi: 10.1371/journal.pone.0069642

Figure Lengend Snippet: A: RT-PCR analysis for SULF expression was performed on cDNA prepared from THCE, MCE and 293T cells transfected with pcDNA Sulf1 (S1) or pcDNA Sulf2 (S2) as positive controls. First-strand cDNA synthesis was also performed with reverse transcriptase omitted as a control for genomic DNA contamination (RT-). B: Detergent lysates of human primary corneal epithelial cells (PCE), THCE, MCE and pcDNA SULF1 (S1) or pcDNA SULF2 -transfected 293T (S2) cells were subjected to Western blot analysis with 1A4 (SULF1) and R2.1/2.3 (SULF2). SULF1 was detected in MCE cells by the presence of its C-terminal subunit (∼50 kDa), while SULF2 was absent. SULF2 was detected in THCE and PCE cells (as the 125 kDa proprotein). C: SULF2 in the indicated THCE cells. Western blotting for SULF2 (R2.1/R2.3) was performed on conditioned media (CM) and detergent lysates from these cells. Density of each band was measured with ImageJ, values were normalized to β-actin of the Cnt shRNA-treated cells, and the percent SULF2 expression (shown at the bottom of the respective lanes) was calculated relative to the expression of the protein in Cnt shRNA cells. shRNA1 and shRNA2 reduced SULF2 expression by ∼83% and ∼98% respectively. D: and E: Effects of SULF2 knockdown on the cell-surface sulfation was determined by staining with RB4CD12. D: Immunofluorescent staining of RB4CD12 or control MPB49 antibody in THCE control- and SULF2-knockdown cells. E: Flow cytometry analysis of RB4CD12 or control MPB49 staining in THCE control- and SULF2-knockdown cells. SULF2 knockdown increased the level of RB4CD12 epitope on the cell surface by 72±2% (mean ± SEM, n = 3 experiments). A representative result is shown.

Article Snippet: Immunoblotting was performed with 1 μg/ml mouse SULF1 mAb 1A4 which recognizes the C-terminal 50-kDa subunit (Novus Biologicals, Littleton, CO), 3 μg/ml G1.6 for SULF1 (see ) and 1 μg/ml R2.3 and R2.1 for SULF2, both of which detect the N-terminal 75-kDa subunit .

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Transfection, cDNA Synthesis, Reverse Transcription, Control, Western Blot, shRNA, Knockdown, Staining, Flow Cytometry

Journal: PLoS ONE

Article Title: The SULFs, Extracellular Sulfatases for Heparan Sulfate, Promote the Migration of Corneal Epithelial Cells during Wound Repair

doi: 10.1371/journal.pone.0069642

Figure Lengend Snippet: A: and D: Representative phase-contrast images of the gap at 0 and 12 hr post-scratch in mock-transfected (Cnt shRNA) and SULF2-knockdown cells ( HSULF2 shRNA2) transfected with pcDNA or pc MSulf2 (A) or with pcDNA or pc HSulf1 (C). B: and E: Percent gap remaining at 6 and 12 hrs was determined for the indicated conditions. Data are expressed as mean+SEM, n = 4 replicate wells per treatment. **p<0.01, *p<0.05, Student t-test. C and F: Overexpression of SULF in SULF2 knockdown THCE cells by plasmid transfection. SULF was detected in lysates of the indicated THCE cells by Western blotting with R2.1/2.3 for SULF2, 75 kDa subunit (C) and G1.6 for SULF1, 75 kDa subunit (F). β-actin was used as a loading control. A high level of endogenous SULF2 was observed in Cnt shRNA-treated cells (C, Lane 1), while HSULF2 shRNA almost completely eliminated protein expression (Lane 2). Transient expression with pcMSULF2 in SULF2 knockdown cells partially restored SULF2 expression but below the endogenous level in Cnt shRNA (Lane 3). SULF1 expression was detected only transient transfection with pcHSULF1 (F, Lane 3).

Article Snippet: Immunoblotting was performed with 1 μg/ml mouse SULF1 mAb 1A4 which recognizes the C-terminal 50-kDa subunit (Novus Biologicals, Littleton, CO), 3 μg/ml G1.6 for SULF1 (see ) and 1 μg/ml R2.3 and R2.1 for SULF2, both of which detect the N-terminal 75-kDa subunit .

Techniques: Transfection, shRNA, Knockdown, Over Expression, Plasmid Preparation, Western Blot, Control, Expressing