Journal: EMBO Molecular Medicine

Article Title: Photoreceptor glucose metabolism determines normal retinal vascular growth

doi: 10.15252/emmm.201707966

Figure Lengend Snippet: The mouse neonates received intraperitoneal injection of STZ and schematic graph shows mouse retinal vessel development after birth (left). GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. Representative images of the deep vascular network in isolectin‐stained (red, top) retinal whole mounts of HAR and controls. Scale bar, 50 μm (top) and 1 mm (bottom). The total retinal area is outlined in yellow and deep vascular coverage is highlighted in red (bottom, grayscale). Quantification of deep vascular network of HAR and controls. n = 17–27 retinas/group. Data presented as mean ± SEM, unpaired t ‐test. Blood glucose ( n = 19–27 retinas/group), serum insulin (ELISA) ( n = 4–7 pooled samples/group) and triglyceride (TG) concentrations ( n = 7/group) of HAR and controls. Data presented as mean ± SEM, unpaired t ‐test. Data information: See also Fig .

Article Snippet: All serum samples were diluted 1:306 and APN levels assayed using a human adiponectin ELISA kit (E091M, Mediagnost, Reutlingen, Germany).

Techniques: Injection, Staining, Enzyme-linked Immunosorbent Assay

Journal: EMBO Molecular Medicine

Article Title: Photoreceptor glucose metabolism determines normal retinal vascular growth

doi: 10.15252/emmm.201707966

Figure Lengend Snippet: A Left: serum APN levels (ELISA) ( n = 8–9 retinas/group); right: retinal apn (qRT–PCR) ( n = 6 retinas/group) of HAR and controls. B Left: retinal cross‐sectional layers for laser capture microdissection (LCM: DAPI for nuclei, blue; isolectin for vessels, red); center: mRNA levels of AdipoR1 and AdipoR2 in retinal neuronal layers and vessels. n = 3 pooled retinas/group. Right: AdipoR1 mRNA in HAR and control retinas. n = 5 retinas/group. C Control and HAR eyes in WT and Apn −/− mice. Left: representative images of DAPI‐stained hyaloid vessels (blue). Right: quantification of preserved hyaloid vessels branching from the hyaloid artery (white arrow). Scale bar, 1 mm. n = 8–30 retinas/group. D Left: Fundus photograph focused to show persistent hyaloid vessels (white arrows) (green, fluorescein AK‐FLUOR) in WT and Apn −/− mice at P30 ( n = 37–48 retinas/group); right: pie graph of percentage of eyes examined with persisting hyaloid. E Left: In WT and Apn −/− representative images of deep retinal vasculature (lectin, red) in whole‐mounted retinas; right: quantification of deep vessels ( n = 14–22 retinas/group). F, G Left: Representative images of deep retinal vasculature (lectin, red) in whole‐mounted retinas of WT hyperglycemic (HAR) mice with recombinant mouse APN treatment (F) ( n = 6–10 retinas/group) or with AdipoRon treatment (G). Right: quantification of deep retinal vasculature ( n = 7–14 retinas/group). Data information: Scale bars, 50 μm (B, E–G) or 1 mm (C). Data presented as mean ± SEM; unpaired t ‐test (A, B, E–G) or ANOVA (C). See also Fig .

Article Snippet: All serum samples were diluted 1:306 and APN levels assayed using a human adiponectin ELISA kit (E091M, Mediagnost, Reutlingen, Germany).

Techniques: Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Laser Capture Microdissection, Control, Staining, Recombinant

Journal: Gene Therapy

Article Title: A macrophage-specific synthetic promoter for therapeutic application of adiponectin

doi: 10.1038/gt.2014.3

Figure Lengend Snippet: Constructs of plasmid vectors for APN expression. ( a ) SP146-C1 was selected to develop a therapeutic vector. The original luciferase gene in the pGL3-basic plasmid was replaced by the APN gene. ( b ) Cells were transfected and mRNA expression of APN with CMV-APN and SP146-C1-APN (SP-APN) was detected 24 h later by RT-PCR in 293 T and RAW264.7 cells. CMV-APN was used as a positive control. Densities of bands were measured using Alpha easy FC software and normalized to that obtained with actin or glyceraldehyde 3-phosphate dehydrogenase (GAPDH). ( c ) Concentrations of APN in the media were measured 48 h later using a human APN ELISA Kit in 293 T and RAW264.7 cells. Data shown are means±s.d.'s. ( d ) The ratio of expressions of SP-APN and CMV-APN was calculated and expressed in graphs. Both mRNA level of APN and released APN showed high ratio in RAW264.7 cells. ** P <0.01 and *** P <0.001. APN, adiponectin; ELISA, enzyme-linked immunosorbent assay; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Article Snippet: Cells were transfected with APN-encoding plasmid vector (CMV-APN) or APN-encoding SP plasmid vector (SP-APN) and the concentration of APN in media was determined using a human APN ELISA Kit (AbFrontier, Seoul, Korea) 48 h later according to the manufacturer's manual.

Techniques: Construct, Plasmid Preparation, Expressing, Luciferase, Transfection, Reverse Transcription Polymerase Chain Reaction, Positive Control, Software, Enzyme-linked Immunosorbent Assay