Journal: Neuro-Oncology

Article Title: CRN2 enhances the invasiveness of glioblastoma cells

doi: 10.1093/neuonc/nos388

Figure Lengend Snippet: Involvement of CRN2 in cell proliferation, adhesion, matrix degradation, and invasion. (A) To determine the proliferation rate, 7 × 10 4 cells per cell line were seeded into 60 mm plates at day 0 and counted at day 1 and day 2. For each cell line, the mean proliferation rate between day 1 and day 2 and the standard errors were calculated from 12 measurements derived from 4 independent experiments. (B) Adhesion of the U373 tumor cell lines (1 × 10 5 cells/well of a 96-well plate) to a monolayer of primary human aortic endothelial cells was quantitated using a fluorescence-based protocol. Mean adhesion values and standard errors were calculated from measurements of 16 wells. (C) Matrix degradation in conjunction with the formation of invadopodia was determined using fluorescently labeled gelatin. Invadopodia at the ventral surface of the cells are defined by presence of an F-actin core, matrix degradation by the loss of the TRITC-gelatin signal. The total area of matrix degradation per cell was normalized to the cell area. Mean values per cell line and standard errors were calculated from 30 different cells derived from 3 independent experiments. (D) 0.2 × 10 5 U373 cells per well were seeded on the upper surface of a collagen I–coated semi-porous membrane in a 96-well format. The number of cells that had invaded the collagen matrix and migrated to the lower surface of the membrane (principle of a Boyden chamber) was quantitated by a fluorescence measurement. Mean values and standard errors were calculated from 16 measurements per cell line. *Statistically significant changes, compared with CRN2-shRNA/GFP cells. #Statistically significant changes, compared with CRN2-shRNA/GFP-CRN2-WT cells. See Table 2 for P values.

Article Snippet: Cell adhesion to a confluent monolayer of primary human aortic endothelial cells (Genlantis, PH30405AK) was measured using the CytoSelect Tumor-Endothelium Adhesion Assay (Cell Biolabs, CBA-215) according to the manufacturer's protocol.

Techniques: Derivative Assay, Fluorescence, Labeling, Membrane, shRNA

Journal: Materials science & engineering. C, Materials for biological applications

Article Title: Polymeric Nanofibrous Scaffolds Laden with Cell-Derived Extracellular Matrix for Bone Regeneration

doi: 10.1016/j.msec.2020.110981

Figure Lengend Snippet: A schematic representation of the electrospinning fabrication process of nanofibrous dual-layer scaffolds with embedded decellularized ECM from osteoblasts and endothelial cell cultures in fibers.

Article Snippet: Primary rat endothelial cells (ECs, Cell Applications, San Diego, CA) were cultured in a supplemented endothelial cell medium without phenol red according to manufacturer’s instructions.

Techniques:

Journal: Materials science & engineering. C, Materials for biological applications

Article Title: Polymeric Nanofibrous Scaffolds Laden with Cell-Derived Extracellular Matrix for Bone Regeneration

doi: 10.1016/j.msec.2020.110981

Figure Lengend Snippet: Scanning electron microscopy images depicting morphology of electrospun (A) PCL, (B) PCL-collagen (COL), (C) PCL-endothelial dECM (ENDO), (D) PCL-osteoblast dECM (OST) fibers. Histogram of fiber diameter distribution for (E) PCL, (F) COL, (G) ENDO, (H) OST fiber groups. Average fiber diameter (I) of PCL fibers and fibers with embedded dECM protein. Percentage of porosity (J) measured for each group in dual-layer scaffolds. ***P<0.0001. n.s. P>0.05. Scale bar 10μm.

Article Snippet: Primary rat endothelial cells (ECs, Cell Applications, San Diego, CA) were cultured in a supplemented endothelial cell medium without phenol red according to manufacturer’s instructions.

Techniques: Electron Microscopy

Journal: Materials science & engineering. C, Materials for biological applications

Article Title: Polymeric Nanofibrous Scaffolds Laden with Cell-Derived Extracellular Matrix for Bone Regeneration

doi: 10.1016/j.msec.2020.110981

Figure Lengend Snippet: Cell attachment and growth response in vitro to PCL-dECM fibers. Cell morphology depicted using scanning electron microscopy of endothelial cells (A-E) and osteoblasts(F-J) attached to dual layer PCL (A,F), COL (B,G), ENDO (C,H), OST (D,I), and EO (E,J)scaffolds. Cell number of osteoblasts (K) cultured for 7, 14, 21, and 28 days on dual layer PCL scaffolds (PCL) with dECM from endothelial cells (ENDO), osteoblasts (OST), and osteoblast and endothelial cell dECM in separate layers (EO). Enzymatic activity measurement of Alkaline Phosphatase (ALP) secreted by osteoblasts cultured on PCL scaffolds with embedded dECM(L) at day 7, 14, 21, and 28. Values are expressed as mean ± SD. *P<0.01. **P<0.001. ***P<0.0001. n.s. P>0.05. Scale bar 10 μm.

Article Snippet: Primary rat endothelial cells (ECs, Cell Applications, San Diego, CA) were cultured in a supplemented endothelial cell medium without phenol red according to manufacturer’s instructions.

Techniques: Cell Attachment Assay, In Vitro, Electron Microscopy, Cell Culture, Activity Assay

Journal: Materials science & engineering. C, Materials for biological applications

Article Title: Polymeric Nanofibrous Scaffolds Laden with Cell-Derived Extracellular Matrix for Bone Regeneration

doi: 10.1016/j.msec.2020.110981

Figure Lengend Snippet: Alizarin Red S (ARS) staining of osteoblast calcium mineral deposits (red) after 28 days of culture (A-K). Cells were cultured tissue culture plate (TCP, F) and on PCL fibrous scaffolds mounted on a coverglass (A,G) as controls. Nanofibrous scaffolds with embedded collagen (COL) (B,H), endothelial dECMs (ENDO)(C,I), osteoblasts dECM (OST) (D,J), or endothelial/osteoblasts dECM in separate fiber layers (EO) (E,K). Images at lower magnification show entire scaffold and the extent of mineralization (A-F).Higher magnification show relative areas and intensity of mineral deposit staining on the scaffold (G-K). For stereomicroscope images (A-F), scale bar 5 mm. For optical microscope images (G-K), scale bar 100 μm. Concentration of ARS abound to calcium mineral deposits extracted from Day 28 cultures with cetylpyridinium chloride (U). Values are expressed as mean ± SD. *P<0.01. **P<0.001.

Article Snippet: Primary rat endothelial cells (ECs, Cell Applications, San Diego, CA) were cultured in a supplemented endothelial cell medium without phenol red according to manufacturer’s instructions.

Techniques: Staining, Cell Culture, Microscopy, Concentration Assay

Journal: Materials science & engineering. C, Materials for biological applications

Article Title: Polymeric Nanofibrous Scaffolds Laden with Cell-Derived Extracellular Matrix for Bone Regeneration

doi: 10.1016/j.msec.2020.110981

Figure Lengend Snippet: Immunostaining of osteocalcin (OC) and osteopontin (OPN) extracellular deposits secreted by osteoblasts cultured on PCL fibers with embedded collagen (COL) (E,F,G,H,V), endothelial dECM (ENDO) (I,J,K,L,W), osteoblast dECM (OST) (M,N,O,P,X), and endothelial/osteoblast dECM in separate layers (EO) (Q,R,S,T,Y). Osteoblasts cultured on PCL scaffolds were stained as controls (A, B, C, D, U). Cells were stained with a nuclear stain DAPI (A,E,I,M,Q) (blue), antibodies against OPN (B,F,J,N,R) (green), and antibodies against OC (C, G, K, O,S) (red).To identify extracellular and intracellular OPN and OC, images were merged (MERGE) (D,H,L,P,T,U,V,W,X,Y). Boxes in D,H,L,P,T indicate regions imaged in U,V,W,X,Y, respectively. Scale bar 200 μm.

Article Snippet: Primary rat endothelial cells (ECs, Cell Applications, San Diego, CA) were cultured in a supplemented endothelial cell medium without phenol red according to manufacturer’s instructions.

Techniques: Immunostaining, Cell Culture, Staining

Journal: Materials science & engineering. C, Materials for biological applications

Article Title: Polymeric Nanofibrous Scaffolds Laden with Cell-Derived Extracellular Matrix for Bone Regeneration

doi: 10.1016/j.msec.2020.110981

Figure Lengend Snippet: Representative histological examination of decalcified femurs 6 weeks postsurgery with different treatments: untreated control (A, Defect), dual-layer PCL scaffold (B, PCL), scaffold with embedded collagen (C, COL), scaffold with embedded endothelial dECM (D, ENDO), scaffold with embedded osteoblast dECM (E, OST), and scaffold with endothelial/osteoblast dECM (F, EO).Sections were stained with Masson’s Trichrome stain. Lines indicate boundaries of defects. # indicates uninjured cortical bone. * indicates regenerate bone. + indicates un-degraded scaffold. Histomorphological analysis of femur sections showing new bone formation per unit area (G), total perimeter of new bone formation per unit area (H), and cortical width (I). For each measurement, the images of 8 sections were used. Values are expressed as mean ± SD. *P<0.01. **P<0.001. ***P<0.0001. Scale bar 1mm.

Article Snippet: Primary rat endothelial cells (ECs, Cell Applications, San Diego, CA) were cultured in a supplemented endothelial cell medium without phenol red according to manufacturer’s instructions.

Techniques: Control, Staining