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: 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