Journal: Pharmaceuticals

Article Title: Testing Mitochondrial-Targeted Drugs in iPSC-RPE from Patients with Age-Related Macular Degeneration

doi: 10.3390/ph15010062

Figure Lengend Snippet: iPSC-RPE derived after reprogramming conjunctival cells obtained from patients with AMD. ( A ) Phase microscopy image showing that confluent iPSC-RPE lines form a monolayer with cobblestone appearance and have pigmentation. Scale bar = 100 µm. ( B ) Confocal microscopy image of iPSC-RPE cultured on a transwell insert. En face views of the RPE monolayer shown as maximum intensity projection through the z-axis. Bestrophin (red) is expressed on the basal surface. ZO-1 (green) marks cell borders. Nuclei are stained with DAPI (blue). Scale bar = 40 µm. ( C ) Results from ELISA analysis of pigment epithelium-derived factor (PEDF) and vascular endothelial growth factor A (VEGF-A) content measured in apical (top) and basal (bottom) media from iPSC-RPE ( n = 5) grown on transwells. Mean ± SEM. ( D ) iPSC-RPE cultures express prototypic RPE proteins as demonstrated on Western immunoblots. Molecular mass for each protein is shown on the left. HR is a homogenate of RPE tissue from a human donor. Stain-free image is loading control. ( E ) Representative data from FACS analysis measuring the phagocytosis of FITC-labeled OS by RPE. Dot plots and histograms for cells without and with the addition of OS are shown.

Article Snippet: ELISA for vascular endothelial growth factor A (VEGF-A) (Thermo Fisher Scientific; Waltham, MA, USA; BMS277/2) and pigment epithelium-derived factor (PEDF) (R & D Systems; Minneapolis, MN, USA; DY1177-05) were performed as described [ ].

Techniques: Derivative Assay, Microscopy, Confocal Microscopy, Cell Culture, Staining, Enzyme-linked Immunosorbent Assay, Western Blot, Labeling

Journal: Chemical & Pharmaceutical Research

Article Title: Inhibition of Corneal Neovascularization with the Combination of Aflibercept and Plasmid Pigment Epithelium-Derived Factor-Synthetic Amphiphile INTeraction-18 Vector

doi: 10.33425/2689-1050.1038

Figure Lengend Snippet: Figure 2: Slit-lamp photographs of Sprague-Dawley rat corneas showing corneal NV for the four experimental groups on day 20. (A) 0 μg + 0 μg of aflibercept + p-PEDF-SAINT-18, respectively (control group; the substitute was purified water); (B) 0.1 μg + 0.1 μg of aflibercept + p-PEDF- SAINT-18, respectively; (C) 1 μg + 1 μg of aflibercept + p-PEDF–SAINT-18, respectively; (D) 10 μg + 10 μg of aflibercept + p-PEDF–SAINT-18, respectively; (E) Subconjunctival administration of 1 μg of p-PEDF–SAINT-18 alone; (F) Subconjunctival administration of 1 μg of aflibercept alone. * 2 μg of the p-bFGF–SAINT-18 complex.

Article Snippet: The membrane was then incubated with anti-bFGF (purified anti-human FGF-basic antibody; BioLegend, San Diego, CA, USA), anti-PEDF antibody (anti-human serpin F1/PEDF antibody; R&D Systems, inc., USA) and anti-VEGF (Santa Cruz Biotechnology inc. CA.USA) for 90 min at room temperature.

Techniques: Control, Purification

Journal: Chemical & Pharmaceutical Research

Article Title: Inhibition of Corneal Neovascularization with the Combination of Aflibercept and Plasmid Pigment Epithelium-Derived Factor-Synthetic Amphiphile INTeraction-18 Vector

doi: 10.33425/2689-1050.1038

Figure Lengend Snippet: Figure 4: Levels of bFGF, VEGF and PEDF protein were estimated by western blot analysis. Visualization of the α-tubulin band was used as the control for normalization. (A) BFGF bands (molecular weight, approximately 18 kDa) were produced with 2 μg p-bFGF-encoded plasmids after day 20. (B) Visualization of the PEDF bands (molecular weight, about 50 kDa) was produced with 0 μg, 0.1 μg, 1 μg, and 10 μg PEDF-encoding plasmids, and this band was also noted in the 0 μg PEDF group due to endogenous PEDF. Inhibition of VEGF expression was more significant in the 10μg + 10 μg of aflibercept + p-PEDF–SAINT-18 group. (C) On day 90 after administration, the PEDF and VEGF levels within the corneal and subconjunctival substantia propria were determined.

Article Snippet: The membrane was then incubated with anti-bFGF (purified anti-human FGF-basic antibody; BioLegend, San Diego, CA, USA), anti-PEDF antibody (anti-human serpin F1/PEDF antibody; R&D Systems, inc., USA) and anti-VEGF (Santa Cruz Biotechnology inc. CA.USA) for 90 min at room temperature.

Techniques: Western Blot, Control, Molecular Weight, Produced, Inhibition, Expressing

Journal: Oncology reports

Article Title: The pigment epithelial-derived factor gene loaded in PLGA nanoparticles for therapy of colon carcinoma.

doi: 10.3892/or_00000905

Figure Lengend Snippet: Figure 1. The characterizations of PLGANPs. (A) Scanning electron micrograph (SEM) of pDNA-PLGANPs. (B) Size distribution of PLGANPs by volume (%). (C) Agarose gel electrophoresis was used to evaluate the integrity and purity of pAAV2-PEDF extracted from PLGANPs. They were pAAV2-PEDF extracted from PLGANPs (P-P) and untreated pAAV2-PEDF (U-P), respectively. (D) MTT assay was used to examine the cell cytotoxicity of bPLGANPs and PEI at different concentrations against CT26s, 48 h post-treatment (n=3). The data are expressed as mean ± SD.

Article Snippet: Expressed PEDF protein was probed with a primary monoclonal antibody mouse against human PEDF (3:1000, mAb; R&D Systems, Boston, MA, USA) and a peroxidaseconjugated secondary antibody goat anti-mouse IgG (1:10,000, ZSGB-BIO, Beijing, China).

Techniques: Agarose Gel Electrophoresis, MTT Assay

Journal: Oncology reports

Article Title: The pigment epithelial-derived factor gene loaded in PLGA nanoparticles for therapy of colon carcinoma.

doi: 10.3892/or_00000905

Figure Lengend Snippet: Figure 2. The effects of PEDF-PLGANPs in vitro. (A) The supernatants from CT26s of NS, AAV2-PLGANPs and PEDF-PLGANPs group were, respectively, detected by SDS-PAGE. PEDF protein (50 kDa) was only probed in CT26s treated with PEDF-PLGANPs. (B) Apoptotic CT26s of PEDF-PLGANPs, AAV2-PLGANPs and NS group were evaluated by flow cytometry with the values of 61.9, 15.2 and 13.2%, respectively. (C) The inhibitory effect of PEDF-PLGANPs on the proliferation of HUVECs was performed with a series of 1/2 dilutions from 1:2 to 1:64. The inhibitory rates of PEDF-PLGANP treated group declined with the increase of dilutions while AAV2-PLGANPs or NS group had no significantly change.

Article Snippet: Expressed PEDF protein was probed with a primary monoclonal antibody mouse against human PEDF (3:1000, mAb; R&D Systems, Boston, MA, USA) and a peroxidaseconjugated secondary antibody goat anti-mouse IgG (1:10,000, ZSGB-BIO, Beijing, China).

Techniques: In Vitro, SDS Page, Flow Cytometry

Journal: Oncology reports

Article Title: The pigment epithelial-derived factor gene loaded in PLGA nanoparticles for therapy of colon carcinoma.

doi: 10.3892/or_00000905

Figure Lengend Snippet: Figure 3. The study of PEDF-PLGANPs on the mice bearing CT26 tumors. (A) Tumor volumes of the mice bearing CT26 tumors in each group. (B) The tumors of experimental mice were weighed on the day 33 after CT26 inoculation when the mice were sacrificed. The values are expressed as mean ± SD. *P<0.05 compared with the tumor weight of AAV2-PLGANPs or NS group.

Article Snippet: Expressed PEDF protein was probed with a primary monoclonal antibody mouse against human PEDF (3:1000, mAb; R&D Systems, Boston, MA, USA) and a peroxidaseconjugated secondary antibody goat anti-mouse IgG (1:10,000, ZSGB-BIO, Beijing, China).

Techniques:

Journal: Oncology reports

Article Title: The pigment epithelial-derived factor gene loaded in PLGA nanoparticles for therapy of colon carcinoma.

doi: 10.3892/or_00000905

Figure Lengend Snippet: Figure 4. The expression of PEDF gene loaded in PLGANPs in vivo. PEDF protein (50 kDa) could be only detected in CT26 tumors treated with PEDF-PLGANPs (P-P) while none was found in CT26 tumors from AAV2- PLGANPs (A-P) or NS treated mice.

Article Snippet: Expressed PEDF protein was probed with a primary monoclonal antibody mouse against human PEDF (3:1000, mAb; R&D Systems, Boston, MA, USA) and a peroxidaseconjugated secondary antibody goat anti-mouse IgG (1:10,000, ZSGB-BIO, Beijing, China).

Techniques: Expressing, In Vivo

Journal: Oncology reports

Article Title: The pigment epithelial-derived factor gene loaded in PLGA nanoparticles for therapy of colon carcinoma.

doi: 10.3892/or_00000905

Figure Lengend Snippet: Figure 5. Related factors to the growth inhibition of CT26 tumors. (A) Apoptotic cells within CT26 tumors were detected by TUNEL assay. Apoptotic CT26s (green) of NS, AAV2-PLGANPs and PEDF-PLGANPs group were, respectively, observed under a fluorescence microscope (original magnification, x200). (B) The apoptotic indexes (%) of CT26 tumors treated with NS, AAV2-PLGANPs and PEDF-PLGANPs. (C) CT26 tumors from NS, AAV2-PLGANPs and PEDF-PLGANP-treated mice were stained with H&E (original magnification, x400). (D) CD31 immunoreactive microvessels were seen from the sections of NS, AAV2-PLGANPs and PEDF-PLGANP-treated tumors. (E) The quantitative data manifested that MVD per 400 field of PEDF-PLGANP- treated tumors had a significant decrease compared with that of NS or AAV2-PLGANP-treated tumors. The data are expressed as mean ± SD. *P<0.05 compared with AAV2-PLGANPs or NS group.

Article Snippet: Expressed PEDF protein was probed with a primary monoclonal antibody mouse against human PEDF (3:1000, mAb; R&D Systems, Boston, MA, USA) and a peroxidaseconjugated secondary antibody goat anti-mouse IgG (1:10,000, ZSGB-BIO, Beijing, China).

Techniques: Inhibition, TUNEL Assay, Fluorescence, Microscopy, Staining

Journal: Cell Death & Disease

Article Title: Pigment epithelium-derived factor mediates retinal ganglion cell neuroprotection by suppression of caspase-2

doi: 10.1038/s41419-019-1379-6

Figure Lengend Snippet: a Fold change in CASP2 mRNA relative to β-actin reference gene in retinal cells freshly isolated and after 3d exposure to NBA, siCNL, 50 nM siCASP2 and PEDF/PEDF-34/PEDF-44. b % of surviving βIII-tubulin + RGC after 3d in culture in NBA, and after treatment with siCNL, 50 nM siCASP2 and PEDF/PEDF-34/PEDF-44. c Fold change in CASP2 mRNA relative to β-actin reference gene in retinal cells freshly isolated and after 3d exposure to suboptimal doses of siCASP2 (5–20 nM) with and without PEDF/PEDF-34/PEDF-44. d % of surviving βIII-tubulin + RGC after 3d in culture in with suboptimal concentrations of siCASP2 (5–20 nM) with and without PEDF/PEDF-34/PEDF-44 ( n = 9 wells/treatment)

Article Snippet: In a preliminary dose response experiment, the optimal concentration of goat polyclonal anti-rat PEDF antibody (Cat No. AF1177; R&D Systems Europe) to antagonise the effects of pre-optimised PEDF and PEDF-34 on RGC survival in vitro was determined , .

Techniques: Isolation

Journal: Cell Death & Disease

Article Title: Pigment epithelium-derived factor mediates retinal ganglion cell neuroprotection by suppression of caspase-2

doi: 10.1038/s41419-019-1379-6

Figure Lengend Snippet: a Fold change in CASP2 mRNA at 7d after ONC + PBS, ONC + siCNL and ONC + siCASP2 and ONC + PEDF treatment. b, c Western blot and subsequent densitometry to show that ONC-induced C-CASP2 (p12 fragment) was significantly suppressed by siCASP2 and PEDF treatment ( n = 18 retinae/treatment)

Article Snippet: In a preliminary dose response experiment, the optimal concentration of goat polyclonal anti-rat PEDF antibody (Cat No. AF1177; R&D Systems Europe) to antagonise the effects of pre-optimised PEDF and PEDF-34 on RGC survival in vitro was determined , .

Techniques: Western Blot

Journal: Cell Death & Disease

Article Title: Pigment epithelium-derived factor mediates retinal ganglion cell neuroprotection by suppression of caspase-2

doi: 10.1038/s41419-019-1379-6

Figure Lengend Snippet: a Fold change in CASP2 mRNA after ONC + saline and ONC + PEDF-34 daily eye-drops over 21d. b , c Western blot and subsequent densitometry to show that ONC-induced rise in C-CASP2 (p12 fragment) is significantly suppressed at 1d and 3d after injury and treatment, without changes in C-CASP3 or C-CASP6 levels. d Immunohistochemistry to show that ONC-induced localisation of C-CASP2 (red) in βIII-tubulin + (green) RGC (arrowheads) after ONC + saline treatment is suppressed by 1d and 3d after ONC + PEDF-34 eye-drops. Scale bar = 50 µm. GCL = ganglion cell layer. (n = 18 retinae/treatment)

Article Snippet: In a preliminary dose response experiment, the optimal concentration of goat polyclonal anti-rat PEDF antibody (Cat No. AF1177; R&D Systems Europe) to antagonise the effects of pre-optimised PEDF and PEDF-34 on RGC survival in vitro was determined , .

Techniques: Saline, Eye Drops, Western Blot, Immunohistochemistry

Journal: Cell Death & Disease

Article Title: Pigment epithelium-derived factor mediates retinal ganglion cell neuroprotection by suppression of caspase-2

doi: 10.1038/s41419-019-1379-6

Figure Lengend Snippet: a Fold change in CASP2 mRNA in 5-FDU-treated cultures showing no change in CASP2 mRNA after siCASP2 or PEDF treatment compared to cultures with glia. b % of surviving βIII-tubulin + RGC in 5-FDU treated cultures in siCASP2 treated cells was unaffected but survival was reduced in the absence of glia. c Fold change in CASP2 mRNA in purified RGC cultures showing no change in CASP2 mRNA after siCASP2 or PEDF treatment. d % of surviving βIII-tubulin + RGC in purified RGC cultures in siCASP2 treated cells was unaffected but survival was reduced in the absence of glia ( n = 9 wells/treatment)

Article Snippet: In a preliminary dose response experiment, the optimal concentration of goat polyclonal anti-rat PEDF antibody (Cat No. AF1177; R&D Systems Europe) to antagonise the effects of pre-optimised PEDF and PEDF-34 on RGC survival in vitro was determined , .

Techniques: Purification

Journal: Cell Death & Disease

Article Title: Pigment epithelium-derived factor mediates retinal ganglion cell neuroprotection by suppression of caspase-2

doi: 10.1038/s41419-019-1379-6

Figure Lengend Snippet: a ELISA to determine levels of BDNF, CNTF, GDNF, NGF and NT-3 released into the culture medium. Treatment of retinal cultures with ELISA isolated levels of BDNF, GDNF and NGF or CNTF either alone or in combination partially suppresses b CASP2 mRNA and C-CASP2 protein c , d by western blot and subsequent densitometry. e Representative images to demonstrate morphology of βIII-tubulin + RGC (note: neurite outgrowth occurs in surviving βIII-tubulin + RGC in wells containing PEDF, BDNF/GDNF/NGF and CNTF). (n = 9 wells/treatment). Scale bar in f = 20 µm

Article Snippet: In a preliminary dose response experiment, the optimal concentration of goat polyclonal anti-rat PEDF antibody (Cat No. AF1177; R&D Systems Europe) to antagonise the effects of pre-optimised PEDF and PEDF-34 on RGC survival in vitro was determined , .

Techniques: Enzyme-linked Immunosorbent Assay, Isolation, Western Blot

Journal: Cell Death & Disease

Article Title: Pigment epithelium-derived factor mediates retinal ganglion cell neuroprotection by suppression of caspase-2

doi: 10.1038/s41419-019-1379-6

Figure Lengend Snippet: a – c Pre-treatment of PEDF and PEDF-34 with a polyclonal antibody to PEDF fails to reduce CASP2 mRNA ( a ) and protein levels ( b ) whilst PEDF is suppressed in the absence of PEDF polyclonal antibody treatment, as confirmed by densitometry ( c ). d Pre-treatment with PEDF polyclonal antibody also suppresses the RGC neuroprotective effects of PEDF and PEDF-34. (n = 9 wells/treatment). e Representative images of the treatments in d to show morphology of βIII-tubulin + RGC. Scale bar in e = 20 µm

Article Snippet: In a preliminary dose response experiment, the optimal concentration of goat polyclonal anti-rat PEDF antibody (Cat No. AF1177; R&D Systems Europe) to antagonise the effects of pre-optimised PEDF and PEDF-34 on RGC survival in vitro was determined , .

Techniques:

Journal: Cell Death & Disease

Article Title: Pigment epithelium-derived factor mediates retinal ganglion cell neuroprotection by suppression of caspase-2

doi: 10.1038/s41419-019-1379-6

Figure Lengend Snippet: a CASP2-dependent apoptosis requires the formation of the PIDDosome (Adapted from ). b PEDF treatment suppresses CASP2, RAIDD but not PIDD and downstream Bim mRNA. c , d PEDF treatment suppresses C-CASP2, RAIDD, phopho-c-Jun and downstream Bim as detected by western blot and densitometry. e , f Immunohistochemistry to show changes in Bim protein colocalised to caspase-2 in the ganglion cell layer (GCL), inner plexiform layer (IPL), occasional cells of the inner nuclear layer (INL) whilst high power views of the GCL showed that Bim and caspase-2 were colocalised to RGC. Scale bars in e and ( f ) = 50 µm ( n = 18 retinae/treatment)

Article Snippet: In a preliminary dose response experiment, the optimal concentration of goat polyclonal anti-rat PEDF antibody (Cat No. AF1177; R&D Systems Europe) to antagonise the effects of pre-optimised PEDF and PEDF-34 on RGC survival in vitro was determined , .

Techniques: Western Blot, Immunohistochemistry

Journal: Journal of Cellular and Molecular Medicine

Article Title: The anti-angiogenic factor PEDF is present in the human heart and is regulated by anoxia in cardiac myocytes and fibroblasts

doi: 10.1111/j.1582-4934.2009.00731.x

Figure Lengend Snippet: PEDF expression in human cardiac tissue. PEDF and HIF-1α determination by Western blot of homogenized cardiac tissue isolated from two healthy hearts (lanes 1 and 2) and four explanted hearts from patients suffering from ischemic (lanes 3 and 4) and dilatative (lanes 5 and 6) cardiomyopathy (A). PEDF mRNA expression in human cardiac tissue isolated from the same hearts used for Western blot in (A), e.g. two healthy hearts (lanes 1 and 2) and four explanted hearts from patients suffering from ischemic (lanes 3 and 4) and dilatative (lanes 5 and 6) cardiomyopathy; GAPDH served as a loading control (B). Immunohistochemical staining of PEDF, troponin I and actin in paraffin embedded heart tissue from a healthy heart and explanted hearts from patients suffering from ischemic and dilatative cardiomyopathy, respectively (C). mRNA was isolated from the left ventricle of healthy human hearts ( n = 4) and from hearts of patients suffering from ischemic ( n = 8; * P = 0.014) or dilatative cardiomyopathy ( n = 17; n.s., P = 0.287); real-time PCR was performed employing specific primers for PEDF. Values represent mean values ± S.D. Values are given as x -fold of control and were normalized using GAPDH levels (D).

Article Snippet: We used an anti-human PEDF goat antibody (15 μg/ml; R&D Systems) for PEDF staining, an anti-human cardiac troponin I rabbit monoclonal antibody (1/250 dilution; Abcam, Cambridge, UK) for cardiac muscle staining and an anti-human smooth muscle α-actin mouse monoclonal antibody (ready to use, BioGenex).

Techniques: Expressing, Western Blot, Isolation, Control, Immunohistochemical staining, Staining, Real-time Polymerase Chain Reaction