Expression of integrins in retinal AC under different glucose conditions.

<p>Integrins α5β1 (A) and αvβ3 (B) levels were determined by FACS analysis. Shaded areas show staining in the absence of primary antibody. Please note the dramatic decrease (2-fold) in mean florescence intensity of α5β1 integrin in AC under high glucose conditions compared to normal glucose and osmolarity control conditions (P<0.05, n = 3). This was mainly attributed with a nearly 2-fold decrease in the fluorescence intensity of α5 integrin, while the fluorescence intensity of β1 integrin did not change under various glucose conditions (not shown). The mean fluorescence intensity of αvβ3 was increased (1.5-fold) under high glucose or osmolarity control conditions compared to normal glucose conditions (P<0.05 n = 3), consistent with enhanced adhesion of AC to fibronectin and vitronectin under high glucose conditions.</p

The impact of high glucose conditions on the level of ROS and nuclear localization of Nrf2 in retinal AC.

<p>(A) Retinal AC cultured under high glucose or osmolarity control exhibited a significantly elevated level of ROS compared to normal glucose conditions. Data are presented as mean ± SEM. n≥60; *<i>p</i><0.05 (NG vs. HG and NG vs. NG+L-Glu). (B) Nuclear localization of Nrf2 in retinal AC under different glucose conditions was examined by immunofluorescence staining. Scale bar indicates 20 µm. (C) Nuclear localization of Nrf2 was assessed by measuring intensity of fluorescence in cell nucleus as described in Methods. Data are presented as mean ± SEM. n≥150 cells; *<i>p</i><0.05 (NG vs. HG).</p

Additional file 1 of MicroRNA-125a-5p regulates the effect of Tregs on Th1 and Th17 through targeting ETS-1/STAT3 in psoriasis

Additional file 1: Table S1. Patient characteristics. Table S2. Primer sequences for reverse-transcription quantitative polymerase chain reaction in humans and mice. Figure S1. The upregulated Th17 cells and downregulated Tregs in peripheral blood of psoriatic patients. a The proportion of CD4+IL17 + T cells in peripheral blood of psoriatic patients and healthy controls by flow cytometry (n=10 per group, p<0.001). b The proportion of CD25+Foxp3+ T cells in peripheral blood of psoriatic patients by flow cytometry (n=10 per group, p<0.01). Data are representative of three independent experiments (mean±SEM). *P<0.05, **P<0.01, ***P<0.001. Two-tailed unpaired Student’s t-test (a, b). Th17, T helper cell 17; Treg, regulatory T cell; IL, interleukin; Foxp3, Forkhead transcription factor3

High glucose conditions had no effect on apoptosis of retinal AC but enhanced their DNA synthesis and GFAP expression.

<p>(A) The rate of apoptosis was determined by TdT-dUTP Terminal Nick-End Labeling (TUNEL) staining. Positive cells were counted using a fluorescence microscope and calculated as percentage of total cell number per field. (B) Level of cleaved caspase-3 was determined by FACS analysis. Shaded areas show staining in the absence of primary antibody. (C) The percentage of retinal AC undergoing active DNA synthesis was determined using FACScan flow cytometery. Data are presented as Mean ± SEM. *<i>p</i><0.05; <i>n</i> = 3 (NG vs. HG and NG vs. NG+L-Glu). EdU is 5-ethynyl-2′-deoxyuridine. (D) Level of GFAP in retinal AC was determined by FACS analysis. High glucose conditions increased the mean fluorescence intensity of GFAP by 1.5-fold in retinal AC compared with normal glucose and osmolarity control conditions (P<0.05, n = 3). Shaded areas show staining in the absence of primary antibody.</p

Attenuation of retinal AC migration and network formation in Matrigel under high glucose conditions.

<p>(A) High glucose conditions inhibited the organization and network formation of retinal AC in Matrigel. (B) The quantitative assessment of the data. Data are the mean number of branch points from 5 high-power fields (×100) ± SEM. *<i>p</i><0.05; n = 5 (NG vs. HG and NG vs. NG+L-Glu). (C) Transwell migration of retinal AC under different glucose conditions. High glucose conditions significantly inhibited migration of retinal AC compared with normal glucose and osmolarity control. Data are presented as mean ± SEM. n = 3,*<i>p</i><0.05 (NG vs. HG). (D) Conditioned medium was collected from retinal AC incubated under normal glucose, high glucose, or osmolarity control for five days. The effects of these conditioned medium on the migration of retinal EC was determined in a transwell migration assay. Please note a significant decrease in migration of retinal EC incubated with conditioned medium from retinal AC cultured under high glucose conditions. Data are presented as mean ± SEM. n = 3; *<i>p</i><0.05 (NG vs. HG)</p

The effect of different glucose conditions on the expression of antioxidant enzymes.

<p>The expression of HO-1 (A) and Prdx2 (B) was determined by Western blot analysis. The quantification of data is also shown. The β-actin was used as loading control. Data are presented as mean ± SEM. n = 3; *<i>p</i><0.05 (NG vs. HG and NG vs. NG+L-Glu).</p

The impact of high glucose conditions on various down-stream signaling pathways.

<p>The status of various signaling pathways in retinal AC under various glucose conditions was determined as described in Methods. Levels of active Src (A), Akt (B), ERK (C), p38 (D), JNK1 (E), Fyn (F) and p65 (G) in retinal AC under different glucose conditions were determined by Western blot analysis from equal amounts of cell lysates. The total level for each protein is shown in the lower part of each panel. The quantitative assessment of the data for each blot is shown below the blot. Data are presented as mean ± SEM. n = 3; *<i>p</i><0.05 (NG vs. HG and NG vs. NG+L-Glu). Levels of active Fyn (F) in retinal AC under different glucose conditions was determined by Western blot analysis of immunoprecipitates from equal amounts of retinal AC lysates.</p

High glucose conditions enhanced the adhesion of retinal AC to extracellular matrix (ECM) proteins.

<p>Adhesion of retinal AC to fibronectin (A) and vitronectin (B) was determined by measuring the number of adherent cells in each well coated with different concentration of ECM proteins. Number of adherent cells was quantified by measuring the cellular phosphatase activity as described in Methods. Data are presented as mean ± SEM. n = 3; *<i>p</i><0.05 (NG vs. HG). No significant adhesion to collagen I or laminin was observed (not shown). The impact of different glucose conditions on formation of actin stress fibers and focal adhesions in retinal AC. (C) Examination of actin stress fibers and focal adhesions in retinal AC. Retinal AC were stained with anti-vinculin (red), phalloidin (green), and DAPI (×630). Scale bar  = 20 µm. (D) Coherency of stress fibers was assessed using image J software. Data are presented as Mean ± SEM. (E) The number of focal adhesions was quantified per cell. Cells (5 to 10) in each condition were analyzed. Data are presented as Mean ± SEM. n ≥5; <i>p</i>>0.05 (NG vs. HG).</p

The effects of various glucose conditions on PDGF-mediated migration of retinal AC.

<p>Migration of retinal AC incubated under different glucose conditions for five days was determined using transwell assay. Transwell assay was performed with basal medium or medium containing PDGF-AA (A) or PDGF-BB (B) in lower compartment. Data are presented as mean ± SEM. n = 3; *<i>p</i><0.05 (Basal vs. PDGF-AA or PDGF-BB). Please note PDFG-AA only enhanced the migration of AC under high glucose conditions. However, PDGF-BB enhanced AC migration under various glucose conditions.</p

Effects of NAC on the migration of retinal AC under different glucose conditions and capillary morphogenesis of retinal EC.

<p>(A) Incubation of retinal AC cultured under high glucose with NAC restored basal migration. Data are presented as mean ± SEM. n = 3; *<i>p</i><0.05 (NG vs. HG). (B) Retinal EC were resuspended in conditioned medium collected from retinal AC incubated under different glucose conditions with or without NAC and plated on Matrigel to analyze capillary morphogenesis. (C) The quantitative assessment of capillary morphogenesis of retinal EC. Data are the mean number of branch points from 8 high-power fields (×100) ± SEM. *<i>p</i><0.05; n = 8 (NG vs. HG and HG vs. HG+NAC). NAC had no effect on capillary morphogenesis of retinal EC in the absence of AC conditioned medium (not shown).</p