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

Journal: Nature

Article Title: Epigenetic Silencing by SETDB1 Suppresses Tumor Intrinsic Immunogenicity

doi: 10.1038/s41586-021-03520-4

Figure Lengend Snippet: (a) Proportion of chromatin accessible sites (ATAC-seq) gained in Setdb1 KO LLC or B16 cells that are located within (red) or outside (grey) SETDB1 domains. (b) Proportion of ATAC-seq sites gained in Setdb1 KO LLC or B16 cells that coincide with promoters (light grey), distal TEs (red), or other promoter-distal sites (dark grey). Statistics by permutation testing. (c) Proportion of gained ATAC-seq sites at distal TEs in Setdb1 KO B16 cells that also gain H3K27 acetylation and resemble active enhancers. (d) Coordinate gain of chromatin accessibility and H3K27 acetylation at an example TE-site in Setdb1 KO B16 cells. (e) Activation of genes near (<50kb) gained ATAC-seq sites at distal TEs in Setdb1 KO LLC or B16 cells compared to control genes. Statistics by permutation testing. (f-h) Flow cytometry in control and Setdb1 KO cells showing (f) gating strategy, (g) cell-surface expression (y-axis, median fluorescence intensity (MFI)) for ULBP1 and RAET1 ligands in LLC (left), and MHC-I expression in LLC and B16 (right) +/− induction with IFNγ (10ng/mL, 24hr). Data are mean +/− s.e.m. and reflect 2 independent experiments with 4 biological replicates. Statistics by two-sided Student’s t-test. *P < 0.05; **P < 0.01.

Article Snippet: Cells were stained with hybridoma supernatant against MuLV envelope proteins (ATCC, HB-10392) followed by secondary staining with fluorescent antibodies, or with directly conjugated fluorescent antibodies against ULBP1 (FAB2588P, R&D systems), pan-RAET1 (FAB17582P, R&D systems), or MHC Class I (28–8-6, BioLegend) at 1:100 dilution 50 .

Techniques: Activation Assay, Control, Flow Cytometry, Expressing, Fluorescence

Journal: Journal of Inflammation (London, England)

Article Title: ERK2 and Akt are negative regulators of insulin and Tumor Necrosis Factor-α stimulated VCAM-1 expression in rat aorta endothelial cells

doi: 10.1186/s12950-016-0115-6

Figure Lengend Snippet: Insulin and TNFα increase expression of total VCAM-1 in rat aorta endothelial cells over time. RAEC were cultured in Complete Growth Medium (CGM) until 90 % confluent. Subsequently, cells were stimulated for indicated times with insulin (10 nM) or TNFα (10 ng/mL) alone or in combination. Total VCAM-1 protein was determined by Western blot analysis. Analysis was performed by the Licor Odyssey detection system. Total VCAM-1 is expressed as the percent of controls and represents the mean ± SEM of six independent experiments. *, P < 0.05 vs negative controls. **, P < 0.05 vs TNFα alone

Article Snippet: Rat aorta vascular endothelial cells (RAEC) (CRL-1444) were from ATCC (Manassas, VA) and culture medium was from Life Technologies (Grand Island, NY).

Techniques: Expressing, Cell Culture, Western Blot

Journal: Journal of Inflammation (London, England)

Article Title: ERK2 and Akt are negative regulators of insulin and Tumor Necrosis Factor-α stimulated VCAM-1 expression in rat aorta endothelial cells

doi: 10.1186/s12950-016-0115-6

Figure Lengend Snippet: Different clones of shRNA for Akt and ERK2 differentially downregulate their respective proteins. Akt and ERK2 shRNA clones provided by manufacturer were amplified in E. coli and isolated by plasmid preparation. Individual clones of each shRNA were transfected into different groups of RAEC as described in Methods. (Panel a ) Akt and ERK2 proteins were quantified in unstimulated cells by Western blot analysis in untreated cells. “0” is negative control clone, and numbers 1–4 are different clones provide by the manufacturer. Intensity of bands are proportional to expression of designated protein in the presence of each shRNA clone. (Panel b ) Relative protein expression is compared to controls (negative control clone)

Article Snippet: Rat aorta vascular endothelial cells (RAEC) (CRL-1444) were from ATCC (Manassas, VA) and culture medium was from Life Technologies (Grand Island, NY).

Techniques: Clone Assay, shRNA, Amplification, Isolation, Plasmid Preparation, Transfection, Western Blot, Negative Control, Expressing

Journal: Journal of Inflammation (London, England)

Article Title: ERK2 and Akt are negative regulators of insulin and Tumor Necrosis Factor-α stimulated VCAM-1 expression in rat aorta endothelial cells

doi: 10.1186/s12950-016-0115-6

Figure Lengend Snippet: RAEC transfected with shERK2 (clone #2) and then stimulated with insulin or TNFα alone or in combination demonstrates the negative regulation of ERK2 in VCAM-1 expression. RAEC were stably transfected with shERK2 (ERK2 KD) and then stimulated with insulin (10 nM, 1 h) or TNFα (10 ng/mL, 6 h) alone or in combination. Total VCAM-1 protein was analyzed by SDS-PAGE and determined by Western blot analysis. (Upper Panel) “NST”, cells not stimulated with insulin or TNFα. “Ins” cells stimulated with insulin. “TNFα” cells stimulated with TNFα. “C” control cells not transfected with shRNA, while “sh” cells were transfected with shERK2. (Lower Panel) “C” control RAEC not transfected (open bars). ERK2 KD are RAEC stably transfected with shERK2 (black bars). “NST” cells not stimulated with insulin or TNFα. Total VCAM-1 is expressed as the percent of controls and represents the mean ± SEM of six independent experiments. Alpha-tubulin was used to normalize VCAM-1 profiles. *, P < 0.05 vs non-stimulated cells

Article Snippet: Rat aorta vascular endothelial cells (RAEC) (CRL-1444) were from ATCC (Manassas, VA) and culture medium was from Life Technologies (Grand Island, NY).

Techniques: Transfection, Expressing, Stable Transfection, SDS Page, Western Blot, Control, shRNA

Journal: Journal of Inflammation (London, England)

Article Title: ERK2 and Akt are negative regulators of insulin and Tumor Necrosis Factor-α stimulated VCAM-1 expression in rat aorta endothelial cells

doi: 10.1186/s12950-016-0115-6

Figure Lengend Snippet: RAEC transfected with shAkt (clone #2) and then stimulated with insulin or TNFα alone or in combination demonstrates the negative regulation of Akt in VCAM-1 expression. REAC were stably transfected with shAkt (Akt KD) and then stimulated with insulin (10 nM, 1 h) or TNFα (10 ng/mL, 6 h) alone or in combination. Total VCAM-1 protein was analyzed by SDS-PAGE and determined by Western blot analysis. (Upper Panel) “NST”, cells not stimulated with insulin or TNFα. “Ins” cells stimulated with insulin. “TNFα” cells stimulated with TNFα. “C” control RAEC not transfected with shRNA, while “sh” cells were transfected with shAkt. (Lower Panel) “C” control RAEC not transfected (open bars). Akt KD are RAEC stably transfected with shAkt (black bars). “NST” cells not stimulated with insulin or TNFα. Total VCAM-1 is expressed as the percent of controls and represents the mean ± SEM of six independent experiments. Alpha-tubulin was used to normalize VCAM-1 profiles. *, P < 0.05 vs non-stimulated cells

Article Snippet: Rat aorta vascular endothelial cells (RAEC) (CRL-1444) were from ATCC (Manassas, VA) and culture medium was from Life Technologies (Grand Island, NY).

Techniques: Transfection, Expressing, Stable Transfection, SDS Page, Western Blot, Control, shRNA

Journal: Journal of Inflammation (London, England)

Article Title: ERK2 and Akt are negative regulators of insulin and Tumor Necrosis Factor-α stimulated VCAM-1 expression in rat aorta endothelial cells

doi: 10.1186/s12950-016-0115-6

Figure Lengend Snippet: RAEC transfected with shAkt and shERK2 and then stimulated with insulin or TNFα alone or in combination exhibit increased VCAM-1 protein expression greater than stimulated, mock transfected cells, but not greater than cells transfected with shAkt alone. RAEC were stably transfected with shAKT and then transiently transfected with shERK2. Subsequently, cells were stimulated with insulin (10 nM, 1 h) or TNFα (10 ng/mL, 6 h) or in combination. (Upper Panel) C, control RAEC, no shRNA. A, cells transfected with shAkt alone. AE, cells stably transfected with shAkt and then transiently transfected with shERK2. (Lower Panel) Open bars are control cells not transfected. Stippled bars (Akt KD) RAEC stably transfected with shAkt. Black bars (Akt KD + shERK2) RAEC stably transfected with shAkt and transiently transfected with shERK2. “NST”, cells not stimulated with insulin or TNFα. Total VCAM-1 is expressed as fold increase above negative controls and represents the mean ± SEM of five independent experiments. Alpha-tubulin was used to normalize VCAM-1 profiles. #, P < 0.05 verses non-stimulated controls. *, P < 0.05 versus stimulated controls of similar treatment group without shRNA

Article Snippet: Rat aorta vascular endothelial cells (RAEC) (CRL-1444) were from ATCC (Manassas, VA) and culture medium was from Life Technologies (Grand Island, NY).

Techniques: Transfection, Expressing, Stable Transfection, Control, shRNA

Journal: Journal of Inflammation (London, England)

Article Title: ERK2 and Akt are negative regulators of insulin and Tumor Necrosis Factor-α stimulated VCAM-1 expression in rat aorta endothelial cells

doi: 10.1186/s12950-016-0115-6

Figure Lengend Snippet: RAEC transfected with shERK2 and shAkt and then stimulated with insulin or TNFα alone or in combination exhibit increased VCAM-1 protein expression greater than stimulated mock transfected cells, but not greater than cells transfected with one shERK2 alone. RAEC were stably transfected with shERK2 and then transiently transfected with shAkt. Subsequently, cells were stimulated with insulin (10 nM, 1 h) or TNFα (10 ng/mL, 6 h) or in combination. (Upper Panel) C, control RAEC, no shRNA. E, cells transfected with shERK2 alone. EA, cells stably transfected with shERK2 and then transiently transfected with shAkt. (Lower Panel) Open bars are control cells not transfected. Stippled bars (ERK2 KD) RAEC stably transfected with shERK2. Black bars (ERK2 KD + shAkt) RAEC stably transfected with shERK2 and transiently transfected with shAkt. “NST”, cells not stimulated with insulin or TNFα. Total VCAM-1 is expressed as fold increase above negative controls and represents the mean ± SEM of five independent experiments. Alpha-tubulin was used to normalize VCAM-1 profiles. #, P < 0.05 verses non-stimulated controls. *, P < 0.05 versus stimulated controls of similar treatment group without shRNA

Article Snippet: Rat aorta vascular endothelial cells (RAEC) (CRL-1444) were from ATCC (Manassas, VA) and culture medium was from Life Technologies (Grand Island, NY).

Techniques: Transfection, Expressing, Stable Transfection, Control, shRNA

Journal: Journal of Inflammation (London, England)

Article Title: ERK2 and Akt are negative regulators of insulin and Tumor Necrosis Factor-α stimulated VCAM-1 expression in rat aorta endothelial cells

doi: 10.1186/s12950-016-0115-6

Figure Lengend Snippet: RAEC transfected with shAkt and shERK2 exhibit increased insulin and TNFα-stimulated VCAM-1 at the cell surface as determined by flow cytometery. (Panel a ) RAEC were stably transfected with shAkt (Akt KD) or (Panel b ) shERK2 (ERK2 KD) and then transiently transfected with shERK2 or shAkt, respectively. Subsequently, cells were stimulated without or with insulin (10 nM) or TNFα (10 ng/mL) or in combination and surface VCAM-1 was determined by flow cytometry. C, non-transfected RAEC (no shRNA). NST, no stimulation. Akt KD, stable cell lines with shAkt. ERK2 KD, stable cell lines with shERK2. Akt KD + shERK2, stable shAkt cells lines transiently transfected with shERK2. ERK2 KD + shAkt, stable cell lines with shERK2 transiently transfected with shAkt. Surface VCAM-1 is expressed as percent of respective controls (not stimulated, No Stim) and represents the mean ± SEM of five independent experiments. *, P < 0.05 compared to their respective unstimulated controls. #, P < 0.05 compared to stimulated non-transfected controls

Article Snippet: Rat aorta vascular endothelial cells (RAEC) (CRL-1444) were from ATCC (Manassas, VA) and culture medium was from Life Technologies (Grand Island, NY).

Techniques: Transfection, Stable Transfection, Flow Cytometry, shRNA

Journal: Journal of Inflammation (London, England)

Article Title: ERK2 and Akt are negative regulators of insulin and Tumor Necrosis Factor-α stimulated VCAM-1 expression in rat aorta endothelial cells

doi: 10.1186/s12950-016-0115-6

Figure Lengend Snippet: RAEC transfected with shAkt and shERK2 exhibit increased insulin and TNFα-stimulated cell surface VCAM-1 as determined by confocal microscopy. RAEC were plated and stimulated as described in Methods. Surface VCAM-1 was detected by immunocytochemistry as described in Methods and visualized by confocal microscopy. CON, RAEC controls, no shRNA; No Stim, no stimulation by insulin or TNFα; Akt KD, stable cell lines transfected with shAkt. ERK2 KD, stable cell lines transfected with shERK2. shAkt, cells transiently transfected with shAkt. shERK2, cells transiently transfected with shERK2. Scale bar is 50 μm

Article Snippet: Rat aorta vascular endothelial cells (RAEC) (CRL-1444) were from ATCC (Manassas, VA) and culture medium was from Life Technologies (Grand Island, NY).

Techniques: Transfection, Confocal Microscopy, Immunocytochemistry, shRNA, Stable Transfection

Journal: Annals of Translational Medicine

Article Title: Endothelial cells promote the proliferation and migration of Schwann cells

doi: 10.21037/atm-22-81

Figure Lengend Snippet: Endothelial cells promote the migration of Schwann cells. (A) Images of the cells in each group were captured under a light microscope (×4). As the proportion of vascular endothelial cells increased, the area of scratches decreased. (B) Compared to the single Schwann cell group, the migration ability of Schwann cells co-cultured with 15% RAOECs was significantly enhanced. (C) For the Transwell assay, the migrated cells were stained with crystal violet at 8 and 24 h after incubation. (D) The relative number of cells peaked in 15% RAOECs at 8 and 24 h (1.13±0.01 and 1.55±0.02, respectively). ***, P<0.001; ****, P<0.0001. RAOECs, rat aortic endothelial cells.

Article Snippet: Complete medium and the RAOECs filled the upper portion of the 0.4-μm-pore co-culture Transwell chamber (Corning; NY, USA) in proportions of 5%, 10%, and 15% to create a non-direct contact co-culture model. Only the complete medium for RAOEC was added to the upper chamber for the control group.

Techniques: Migration, Light Microscopy, Cell Culture, Transwell Assay, Staining, Incubation