Journal: Journal of biomedical materials research. Part A

Article Title: Development of hybrid scaffolds with natural extracellular matrix deposited within synthetic polymeric fibers 3

doi: 10.1002/jbm.a.36078

Figure Lengend Snippet: PDTEC:PEG polymer fiber mat were washed in dH2O to remove PEG. Mouse NIH 3T3 fibroblasts were grown for 7 days within the PDTEC fiber mat. Cells were fixed and stained with R457 anti-fibronectin antiserum (green) and DAPI to stain the nuclei (blue). Confocal images shown are (A) maximum intensity projection (scale bar = 50 μm), (B) maximum intensity projection of the view orthogonal to (A) showing the depth of the matrix within the scaffold, and (C) an alpha-blended volume view (scale bar = 25 μm).

Article Snippet: After 24 h, cultures were fixed, permeabilized for 15 min in 0.5% Triton X (Sigma) in PBS, and stained to visualize the decellularized matrix with R457 polyclonal antiserum diluted 1:100, actin cytoskeleton with either rhodamine-phalloidin or Texas Red-X phalloidin (ThermoFisher) 1:50, nuclei with 1 μg/mL DAPI, or newly deposited human fibronectin matrix with HFN7.1 monoclonal anti-fibronectin antibody (DSHB, University of Iowa) diluted at 1:100.

Techniques: Polymer, Staining

Journal: Journal of biomedical materials research. Part A

Article Title: Development of hybrid scaffolds with natural extracellular matrix deposited within synthetic polymeric fibers 3

doi: 10.1002/jbm.a.36078

Figure Lengend Snippet: After PEG removal, NIH3T3 cells were cultured within the scaffolds for 7 d and then fixed and stained with anti-fibronectin antibodies (green) and DAPI (blue). A and C are the same microscope field; (A) shows the bright field image of the synthetic fibers and (C) shows the fluorescence signal of matrix and nuclei. In parallel, a similar culture was decellularized and then fixed and stained as above. B and D show the same field by bright field and fluorescence imaging. The fiber mat organization is not affected by the decellularization procedure (compare A and B). The absence of DAPI staining in D shows that cells are removed by decellularization while matrix fibrils remain in place. Scale bars = 50 μm.

Article Snippet: After 24 h, cultures were fixed, permeabilized for 15 min in 0.5% Triton X (Sigma) in PBS, and stained to visualize the decellularized matrix with R457 polyclonal antiserum diluted 1:100, actin cytoskeleton with either rhodamine-phalloidin or Texas Red-X phalloidin (ThermoFisher) 1:50, nuclei with 1 μg/mL DAPI, or newly deposited human fibronectin matrix with HFN7.1 monoclonal anti-fibronectin antibody (DSHB, University of Iowa) diluted at 1:100.

Techniques: Cell Culture, Staining, Microscopy, Fluorescence, Imaging

Journal: Journal of biomedical materials research. Part A

Article Title: Development of hybrid scaffolds with natural extracellular matrix deposited within synthetic polymeric fibers 3

doi: 10.1002/jbm.a.36078

Figure Lengend Snippet: Human MSCs were plated on a hybrid ECM – polymer scaffold or on a polymer scaffold without ECM. Cells were allowed to spread for 24 h, then cell shapes were visualized by staining actin filaments with rhodamine-phalloidin (red) and DAPI (blue). Samples were also stained with anti-fibronectin antiserum to show the NIH 3T3 matrix within the scaffold. B, C, E, F are higher magnification images of the samples in A and D. Scale bars = 50 μm.

Article Snippet: After 24 h, cultures were fixed, permeabilized for 15 min in 0.5% Triton X (Sigma) in PBS, and stained to visualize the decellularized matrix with R457 polyclonal antiserum diluted 1:100, actin cytoskeleton with either rhodamine-phalloidin or Texas Red-X phalloidin (ThermoFisher) 1:50, nuclei with 1 μg/mL DAPI, or newly deposited human fibronectin matrix with HFN7.1 monoclonal anti-fibronectin antibody (DSHB, University of Iowa) diluted at 1:100.

Techniques: Polymer, Staining

Journal: Journal of biomedical materials research. Part A

Article Title: Development of hybrid scaffolds with natural extracellular matrix deposited within synthetic polymeric fibers 3

doi: 10.1002/jbm.a.36078

Figure Lengend Snippet: Cells were plated as in Figure 5 but then grown for 7 d before staining with rhodamine-phalloidin and DAPI. Human MSC-produced and assembled fibronectin was detected with a human fibronectin-specific monoclonal antibody. Considerably more fibronectin matrix was deposited by cells attached to the hybrid ECM scaffold than to the scaffold alone (compare A and B with D and E). Note that the fibers remain intact throughout the 7 d culture (C, F). Scale bar = 50 μm.

Article Snippet: After 24 h, cultures were fixed, permeabilized for 15 min in 0.5% Triton X (Sigma) in PBS, and stained to visualize the decellularized matrix with R457 polyclonal antiserum diluted 1:100, actin cytoskeleton with either rhodamine-phalloidin or Texas Red-X phalloidin (ThermoFisher) 1:50, nuclei with 1 μg/mL DAPI, or newly deposited human fibronectin matrix with HFN7.1 monoclonal anti-fibronectin antibody (DSHB, University of Iowa) diluted at 1:100.

Techniques: Staining, Produced

Journal: Investigative Ophthalmology & Visual Science

Article Title: Protective Effects of Estradiol on Disease Progression in a Murine Model of Fuchs Endothelial Corneal Dystrophy

doi: 10.1167/iovs.66.15.64

Figure Lengend Snippet: In vivo estradiol (E2) treatment ameliorates the corneal endothelial phenotype in an FECD mouse model. ( A ) Effects of E2 on the corneal endothelial phenotype were investigated in Col8a2 Q455K/Q455K mice, a mouse model of FECD, by supplying E2-supplemented drinking water ad libitum. Representative specular microscopy images of the corneal endothelium from the wild-type, FECD, and FECD + E2 groups. FECD mice exhibited extensive guttae formation and enlarged endothelial cells compared to wild-type mice. E2 treatment reduced guttae formation and maintained smaller endothelial cells with a higher cell density compared with untreated FECD mice. ( B ) Quantification of guttae formation in the wild-type ( n = 9), FECD ( n = 11), and FECD + E2 ( n = 12) groups. The percentage of guttae area was significantly lower in the FECD + E2 group (0.55 ± 0.23%) compared with the FECD group (0.97 ± 0.22%, P < 0.001). ( C ) Quantification of endothelial cell density (ECD). The FECD + E2 group maintained a significantly higher endothelial cell density (2,263 ± 177 cells/mm²) compared with the FECD group (2058 ± 118 cells/mm²; P = 0.004). ( D, E ) A sex-stratified analysis shows that E2 administration significantly reduced the percentage of guttae area in both male mice ( D ; FECD, n = 6; FECD + E2, n = 6; P = 0.002) and female mice ( E ; FECD, n = 5; FECD + E2, n = 6; P = 0.018) compared with their respective untreated controls. ( F, G ) Similarly, the analysis of endothelial cell density, stratified by sex, shows that E2 treatment resulted in a significantly higher density in both male mice ( F ; FECD, n = 6; FECD + E2, n = 6; P = 0.049) and female mice ( G ; FECD, n = 5; FECD + E2, n = 6; P = 0.013). ( H, I ) Quantitative PCR analysis shows that the expression of the ECM-related genes FN1 ( H ) and COL1A1 ( I ), which was elevated in the FECD group, was significantly suppressed by E2 administration. ( J ) Immunofluorescence for fibronectin ( green ) revealed minimal deposition in wild-type corneas, whereas its accumulation was evident in the corneas of FECD model mice. This pathological deposition was markedly suppressed following E2 treatment. Nuclei were counterstained with DAPI ( blue ). Images are representative of four independent experiments. Scale bar = 100 µm.

Article Snippet: The TaqMan primers used were GAPDH , Hs00266705_mL; FN1 , Hs00365052_mL; BGN , Hs00959143_mL; and COL1A1 , Hs00164004_mL (Applied Biosystems).

Techniques: In Vivo, Microscopy, Real-time Polymerase Chain Reaction, Expressing, Immunofluorescence

Journal: Stem Cell Research & Therapy

Article Title: Administration of cytokine-induced myeloid-derived suppressor cells ameliorates renal fibrosis in diabetic mice

doi: 10.1186/s13287-018-0915-0

Figure Lengend Snippet: Renal ECM expression and MDSC distribution in STZ-treated diabetic mice. a Diabetes induced in mice using one dose of streptozotocin (STZ, 180 mg/kg) intraperitoneally, and blood glucose levels maintained over 350 mg/dl. Four weeks later, mice were sacrificed and ECM expression in kidney examined. Kidney cryostat sections histochemically stained with anti-fibronectin mAb, anti-collagen type IV mAb, or anti-alpha smooth muscle actin mAb (left panel, brown, 400× magnification). Bar graph shows quantification of differences in ECM expression of kidney between STZ-treated diabetic mice and untreated mice (right panel, * P < .05). b Blood and urine collected for serum creatinine and protein analyses when mice sacrificed. Level of differences in serum creatinine and proteinuria between STZ-treated diabetic mice and untreated mice (* P < .05). c MDSC ratios (CD11b + /Gr-1 + ) in BM, blood, spleen, and kidneys of STZ-treated diabetic mice and untreated mice compared. Isolated cells were two-color stained with specific mAbs against CD11b and Gr-1 for flow analyses. Double-positive CD11b and Gr-1 cells represent MDSCs (* P < .05). d Cryostat sections of spleen and kidney from STZ-treated diabetic mice and untreated mice double-stained with anti-CD11b (green) and anti-Gr-1 (red) mAbs and evaluated under fluorescent microscope (400× magnification; upper panel). Double-positive cells counted. In total, 10 high-power fields randomly selected in each section. Data expressed as mean CD11b + /Gr-1 + cells ± 1 SD (lower panel, * P < .05). Data representative of three separate experiments. α-SMA alpha-smooth muscle actin, ECM extracellular matrix, MDSC myeloid-derived suppressor cell

Article Snippet: Protein extracts were separated by SDS-PAGE and transferred onto PVDF membranes before being probed with antibodies against fibronectin (15613-1-AP; Proteintech, Chicago, IL, USA) or GAPDH (sc-32,233; Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Expressing, Staining, Isolation, Microscopy, Derivative Assay

Journal: Stem Cell Research & Therapy

Article Title: Administration of cytokine-induced myeloid-derived suppressor cells ameliorates renal fibrosis in diabetic mice

doi: 10.1186/s13287-018-0915-0

Figure Lengend Snippet: MMCs produced more fibronectin and inflammatory cytokines in hyperglycemic environment. a Fibronectin protein expression, assessed through western blotting, in MMCs exposed to 5 mM, 25 mM, and 35 mM of glucose as well as TGF-β (2 ng/ml) with 5 mM of glucose for 24 h. Quantitative ratios of fibronectin expression compared with that of GAPDH (* P < .05). b Fibronectin expression pattern of MMCs compared through immunofluorescence staining (red for fibronectin, blue for nuclear DAPI stain; 400× magnification). c Expression of chemokines, growth factors, and immunomodulators in conditioned medium from MMCs cultured in normal (5 mM) or high glucose (25 mM) levels for 72 h using a cytokine array kit. Bar graph constructed for each cytokine using median value in arbitrary units of three independent assays (* P < .05). d Expression of fibronectin mRNA ratios from MMCs cocultured with or without cMDSCs at ratio of 1:4 in different glucose concentrations or stimulated with TGF-β for 24 h determined using qPCR (* P < .05). MMCs cocultured with cMDSCs assayed using Transwell method (top layer, cMDSC; bottom layer, MMC). GAPDH glyceraldehyde 3-phosphate dehydrogenase, IP10 interferon gamma-induced protein 10, JE CCL2, KC CXCL1, M-CSF macrophage colony-stimulating factor, MDSC myeloid-derived suppressor cell, MIP-2 macrophage inflammatory protein 2, MMC mouse mesangial cell, RANTES regulated on activation, normal T cells expressed and secreted, SDF-1 stromal-cell-derived factor-1, TGF-β transforming growth factor beta, TIMP-1 tissue inhibitors of metalloproteinases

Article Snippet: Protein extracts were separated by SDS-PAGE and transferred onto PVDF membranes before being probed with antibodies against fibronectin (15613-1-AP; Proteintech, Chicago, IL, USA) or GAPDH (sc-32,233; Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Produced, Expressing, Western Blot, Immunofluorescence, Staining, Cell Culture, Construct, Derivative Assay, Activation Assay

Journal: Stem Cell Research & Therapy

Article Title: Administration of cytokine-induced myeloid-derived suppressor cells ameliorates renal fibrosis in diabetic mice

doi: 10.1186/s13287-018-0915-0

Figure Lengend Snippet: Adoptive transfer of cMDSCs into STZ-treated diabetic mice. a Cytokine-induced MDSCs (1 × 10 7 cells) adoptively transferred into STZ mice through pudendal vein once a week (on days 5, 13, and 20). Mice in each group then sacrificed on day 27 (upper panel). Blood sugar levels and body weight (lower panel) measured twice a week until mice sacrificed in subgroups of untreated mice (untreated, n = 5), STZ-treated diabetic mice (STZ, n = 5), and cMDSC-treated STZ mice (STZ + cMDSC, n = 5). b Kidneys harvested and weights measured on day 27. Ratios of kidney weight to body weight of the three groups compared (left panel). GFRs of the three groups examined and compared (right panel) (* P < .05). c Kidney cryostat sections histochemically stained with anti-fibronectin mAb, anti-collagen type IV mAb, or anti-alpha-smooth muscle actin mAb (left panel, brown; 400× magnification) and examined using a microscope. Bar graph presents quantification of ECM expression of renal cortex of untreated mice, STZ mice, and cMDSC-treated STZ mice (right upper panel) (* P < .05). Areas of fibronectin expression within the glomeruli of the three groups quantified (right lower panel) (* P < .05). d Presence of MDSCs in kidneys analyzed through histochemical staining with anti-Gr-1 mAb (left panel, brown; 400× magnification) and examined using a microscope. Bar graph illustrates positive MDSCs counted in a total of 10 high-power fields randomly selected in each section (right panel) (* P < .05). α-SMA alpha-smooth muscle actin, cMDSC cytokine-induced myeloid-derived suppressor cell, ECM extracellular matrix, GFR glomerular filtration rate, iv intravenous, STZ streptozotocin

Article Snippet: Protein extracts were separated by SDS-PAGE and transferred onto PVDF membranes before being probed with antibodies against fibronectin (15613-1-AP; Proteintech, Chicago, IL, USA) or GAPDH (sc-32,233; Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Adoptive Transfer Assay, Staining, Microscopy, Expressing, Derivative Assay, Filtration

Journal: Neural Regeneration Research

Article Title: Mechanisms responsible for the inhibitory effects of epothilone B on scar formation after spinal cord injury

doi: 10.4103/1673-5374.202921

Figure Lengend Snippet: Effects of EpoB on responsive scar formation at the injury site. (A–D) Masson's trichrome staining of the SCI site at different time points. At both time points, the collagenous staining (blue, arrows) was weaker in the EpoB group (SCI + EpoB) than in the (normal saline) vehicle group at (A, B) 3 days post-SCI and (C, D) 21 days post-SCI. Blue staining indicates collagenous material, and red or dark red staining indicates nervous tissue. (E–H) The FN immunofluorescent staining (red, arrows) was reduced in the EpoB group at both (E, F) 3 days post-SCI and (G, H) 21 days post-SCI. (I–L) GFAP staining (red) did not show an apparent difference. (M–P) The β-tubulin staining (red) was stronger in the EpoB group than in the vehicle group. Nuclei are stained with DAPI (blue). Scale bars: (A–D) 500 μm, (E–H) 100 μm, (I–L) 100 μm, and (M–P) 50 μm. (Q, R) Western blot analysis and quantification (gray value: marker/β-actin) confirmed the histochemical staining results. * P < 0.05, ** P < 0.01, *** P < 0.001. Data are expressed as the mean ± SD ( n = 3; one-way analysis of variance followed by Dunnett's test). EpoB: Epothilone B; SCI: spinal cord injury; GFAP: glial fibrillary acidic protein; DAPI: 4′,6-diamidino-2-phenylindole; FN: fibronectin.

Article Snippet: The following primary antibodies were used: mouse anti–neuron-glial antigen 2 (anti-NG2) (1:200, ab50009; Abcam, Cambridge, UK), rabbit anti-platelet-derived growth factor receptor β (anti-PDGFRβ) (1:100, ab32570; Abcam), rabbit polyclonal anti-glial fibrillary acidic protein (anti-GFAP, 1:100, ZA-0117; Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China), rabbit anti-β-tubulin (9F3) (1:200, #2128; Cell Signaling Technology, Inc., Danvers, MA, USA), and rabbit polyclonal anti-fibronectin (1:100, ab2413; Abcam).

Techniques: Staining, Western Blot, Marker

Journal: Neural Regeneration Research

Article Title: Mechanisms responsible for the inhibitory effects of epothilone B on scar formation after spinal cord injury

doi: 10.4103/1673-5374.202921

Figure Lengend Snippet: Expression of NG2, PDGFRβ in injured spinal cord after treatment with EpoB. (A–H) Immunofluorescence staining of the SCI site at different time points and examination under a confocal microscope. (A–D) Number of NG2 (green)-immunoreactive cells was higher in the vehicle group than in the EpoB group. (E–H) Number of PDGFRβ (red)-immunoreactive cells was higher in the vehicle group (normal saline) than in the EpoB group (SCI + EpoB). Nuclei are stained with DAPI (blue). Scale bars: (A–H) 50 μm. (I, J) The quantification (gray value ratio of marker/β-actin) confirmed the histochemical staining results: the NG2 and PDGFRβ expression levels were decreased in the EpoB group. * P < 0.05, ** P < 0.01. Data are expressed as the mean ± SD ( n = 3; one-way analysis of variance followed by Dunnett's test). FN: Fibronectin; NG2: neuron-glial antigen 2; PDGFRβ: platelet-derived growth factor receptor β; EpoB: epothilone B; DAPI: 4′,6-diamidino-2-phenylindole.

Article Snippet: The following primary antibodies were used: mouse anti–neuron-glial antigen 2 (anti-NG2) (1:200, ab50009; Abcam, Cambridge, UK), rabbit anti-platelet-derived growth factor receptor β (anti-PDGFRβ) (1:100, ab32570; Abcam), rabbit polyclonal anti-glial fibrillary acidic protein (anti-GFAP, 1:100, ZA-0117; Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China), rabbit anti-β-tubulin (9F3) (1:200, #2128; Cell Signaling Technology, Inc., Danvers, MA, USA), and rabbit polyclonal anti-fibronectin (1:100, ab2413; Abcam).

Techniques: Expressing, Immunofluorescence, Staining, Microscopy, Marker, Derivative Assay

Journal: Neural Regeneration Research

Article Title: Mechanisms responsible for the inhibitory effects of epothilone B on scar formation after spinal cord injury

doi: 10.4103/1673-5374.202921

Figure Lengend Snippet: EpoB treatment improved the post-SCI behavior recovery and regulated gene expression. (A) EpoB enhanced the expression of β-tubulin III, suppressed the expression of FN, and minimally affected GFAP (* P < 0.05, ** P < 0.01). (B) EpoB suppressed NG2 and PDGFRβ mRNA expression in pericytes (* P < 0.05, ** P < 0.01, *** P < 0.001). (C) Behavioral test with the BBB score system. EpoB treatment (SCI + EpoB) significantly improved hindlimb performance at 2 weeks post-SCI compared with the vehicle group (normal saline) (* P < 0.05, ** P < 0.01). Data are expressed as the mean ± SD ( n = 3; one-way analysis of variance followed by Dunnett's test). BBB: Basso, Beattie, and Bresnahan locomotor scale; EpoB: epothilone B; SCI: spinal cord injury; GFAP: glial fibrillary acidic protein; FN: fibronectin; NG2: neuron-glial antigen 2; PDGFRβ: platelet-derived growth factor receptor β.

Article Snippet: The following primary antibodies were used: mouse anti–neuron-glial antigen 2 (anti-NG2) (1:200, ab50009; Abcam, Cambridge, UK), rabbit anti-platelet-derived growth factor receptor β (anti-PDGFRβ) (1:100, ab32570; Abcam), rabbit polyclonal anti-glial fibrillary acidic protein (anti-GFAP, 1:100, ZA-0117; Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China), rabbit anti-β-tubulin (9F3) (1:200, #2128; Cell Signaling Technology, Inc., Danvers, MA, USA), and rabbit polyclonal anti-fibronectin (1:100, ab2413; Abcam).

Techniques: Expressing, Derivative Assay