Journal: Acta Neuropathologica Communications

Article Title: EphrinB/EphB forward signaling in Müller cells causes apoptosis of retinal ganglion cells by increasing tumor necrosis factor alpha production in rat experimental glaucomatous model

doi: 10.1186/s40478-018-0618-x

Figure Lengend Snippet: EphrinB/EphB forward signaling is activated in retinal Müller cells by IOP elevation and ephrinB1-Fc treatment. a , Representative immunoblots showing the changes in EphB1 and phosphorylated EphB (p-EphB) expression in sham-operated (control, Ctr) and COH retinal extracts from G1w to G4w. b-d , Bar charts summarizing the average densitometric quantification of immunoreactive bands of p-EphB ( b ), EphB1 ( c ) and the average p-EphB/EphB1 ratios ( d ) obtained in COH retinal extracts from G1w to G4w. n = 5 for all groups. e , Representative immunoblots showing the changes in EphB1 and p-EphB levels in Müller cell extracts treated with IgG-Fc (500 ng/ml) (Ctr) and those treated with ephrinB1-Fc (500 ng/ml) for different periods of time (1–24 h). f-h , Bar charts summarizing the average densitometric quantification of immunoreactive bands of p-EphB ( f ), EphB1 ( g ) and the average p-EphB/EphB1 ratios ( h ) in Müller cells treated with ephrinB1-Fc for different periods of time, as compared to those in control condition. n = 5 for all groups. i , Representative immunoblots showing the changes in EphB1 and p-EphB expression in normal saline-injected retina (Ctr), and ephrinB1-Fc-injected retinas (0.5 μg/μl, 2 μl) at different post-injection times. j-l , Bar charts summarizing the average densitometric quantification of immunoreactive bands of p-EphB ( j ), EphB1 ( k ) and the average ratios of p-EphB/EphB1 ( l ) in retinal extracts under control condition and those of ephrinB1-Fc-injected retinas at different post-injection times (3 d and 7 d). n = 4 for all groups. All the data are normalized to their corresponding β-actin and then to Ctr. * P < 0.05, ** P < 0.01 and *** P < 0.001 vs. Ctr

Article Snippet: The following primary antibodies were used: monoclonal mouse anti-β-actin (A5316, 1:10000, Sigma-Aldrich, St. Louis, MO, USA), polyclonal rabbit anti-phosphoY594 EphB1/EphB2 (ab61791, 1:500, Abcam, Cambridge, MA, USA), polyclonal goat anti-EphB1 (AF542, 1:500, R&D systems), monoclonal mouse anti-PI3K p85α (sc-1637, 1:1000; Santa Cruz Biotechnology), polyclonal rabbit anti-PI3K p110 (sc-7189, 1:1000, Santa Cruz Biotechnology), polyclonal rabbit anti- Akt1/2/3 phospho Ser473 (sc-7985-R, 1:1000, Santa Cruz Biotechnology), monoclonal mouse anti-Akt1 (sc-5298, 1:1000, Santa Cruz Biotechnology), polyclonal rabbit anti-NF-κB p65 (sc-372, 1:1000, Santa Cruz Biotechnology), monoclonal mouse anti-NMDAR1 (#556308, 1:500, BD Pharmingen, Franklin Lakes, NJ, USA), polyclonal rabbit anti-NMDA NR2B phospho Tyr1472 (#454583, 1:500, Calbiochem), polyclonal rabbit anti-NMDAR2B (ab65783, 1:500, Abcam), monoclonal mouse anti-GFAP (G6171, 1:500, Sigma-Aldrich).

Techniques: Western Blot, Expressing, Saline, Injection

Journal: Acta Neuropathologica Communications

Article Title: EphrinB/EphB forward signaling in Müller cells causes apoptosis of retinal ganglion cells by increasing tumor necrosis factor alpha production in rat experimental glaucomatous model

doi: 10.1186/s40478-018-0618-x

Figure Lengend Snippet: Changes in GFAP protein levels in ephrinB1-Fc treated Müller cells and ephrinB1-Fc injected retinas. a , Representative immunoblots showing the changes in GFAP protein levels in Müller cell extracts treated with IgG-Fc (500 ng/ml) (Ctr) and those treated with ephrinB1-Fc (500 ng/ml) for different periods of time (1–24 h). b , Bar charts summarizing the average densitometric quantification of immunoreactive bands of GFAP under the conditions as shown in A . n = 6 for all groups. c , Immunofluorescence labeling showing GFAP expression in rat retinal vertical slices taken from normal saline-injected retina (c1) (Ctr) and ephrinB1-Fc-injected retinas on 1 and 2 weeks (c2 and c3) after the injection. Scale bar, 20 μm, for all the images. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer

Article Snippet: The following primary antibodies were used: monoclonal mouse anti-β-actin (A5316, 1:10000, Sigma-Aldrich, St. Louis, MO, USA), polyclonal rabbit anti-phosphoY594 EphB1/EphB2 (ab61791, 1:500, Abcam, Cambridge, MA, USA), polyclonal goat anti-EphB1 (AF542, 1:500, R&D systems), monoclonal mouse anti-PI3K p85α (sc-1637, 1:1000; Santa Cruz Biotechnology), polyclonal rabbit anti-PI3K p110 (sc-7189, 1:1000, Santa Cruz Biotechnology), polyclonal rabbit anti- Akt1/2/3 phospho Ser473 (sc-7985-R, 1:1000, Santa Cruz Biotechnology), monoclonal mouse anti-Akt1 (sc-5298, 1:1000, Santa Cruz Biotechnology), polyclonal rabbit anti-NF-κB p65 (sc-372, 1:1000, Santa Cruz Biotechnology), monoclonal mouse anti-NMDAR1 (#556308, 1:500, BD Pharmingen, Franklin Lakes, NJ, USA), polyclonal rabbit anti-NMDA NR2B phospho Tyr1472 (#454583, 1:500, Calbiochem), polyclonal rabbit anti-NMDAR2B (ab65783, 1:500, Abcam), monoclonal mouse anti-GFAP (G6171, 1:500, Sigma-Aldrich).

Techniques: Injection, Western Blot, Immunofluorescence, Labeling, Expressing, Saline

Journal: Acta Neuropathologica Communications

Article Title: EphrinB/EphB forward signaling in Müller cells causes apoptosis of retinal ganglion cells by increasing tumor necrosis factor alpha production in rat experimental glaucomatous model

doi: 10.1186/s40478-018-0618-x

Figure Lengend Snippet: Activation of ephrinB/EphB forward signaling increases TNF-α production in cultured Müller cells and retinal tissues. The cells were treated by IgG-Fc (500 ng/ml) (Ctr) or ephrinB1-Fc (500 ng/ml) for different periods of time (1–24 h). a-c , Cumulative data summarizing the changes in mRNA levels of BDNF ( a ), GDNF ( b ) and NGF ( c ) in Müller cell extracts obtained in Ctr and those with ephrinB1-Fc-treatment for different periods of time. n = 6 for all groups. d-f , Cumulative data summarizing the changes in mRNA levels of IL-1β ( d ), IL-6 ( e ) and TNF-α ( f ) in Müller cell extracts obtained in Ctr and those with ephrinB1-Fc-treatment for different periods of time. The relative abundance of mRNA was quantified using 2 -ΔΔct calculation method and expressed as fold change. n = 6 for all groups.* P < 0.05 and *** P < 0.001 vs. Ctr. g , Bar chart showing the average extracellular TNF-α concentrations in IgG-Fc-treated group (Ctr) and groups of ephrinB1-Fc-treatment for different periods of time. n = 6 for all groups. * P < 0.05 and *** P < 0.001 vs. Ctr. h , Summary data showing that the ephrinB1-Fc-treatment induced increase in TNF-α mRNA levels in cultured Müller cells was blocked by pre-incubation with PP2. PP2 (10 μM/50 μM) was added to the medium 30 min before a 6 h ephrinB1-Fc treatment. n = 6 for all groups. *** P < 0.001 vs. IgG-Fc treated group (Ctr). i , Bar chart showing that pre-incubation with PP2 inhibited the ephrinB1-Fc-treatment induced increase in TNF-α protein levels in cultured Müller cells. PP2 (10 μM) was added to the medium 30 min before a 24 h ephrinB1-Fc treatment. n = 6 for all groups. *** P < 0.001 vs. IgG-Fc treated group (Ctr). j , Cumulative data summarizing the changes in TNF-α mRNA levels in normal saline-injected retinas (Ctr) and retinas with ephrinB1-Fc-injection at different post-injection times. EphrinB1-Fc (0.5 μg/μl, 2 μl) was intravitreally injected and retinas were collected at different post-injection times (3 d and 7 d). n = 6 for all groups. k , Bar chart showing the changes in TNF-α protein levels in Ctr retinas and ephrinB1-Fc injected retinas at different post-injection times (3 d and 7 d). n = 4 for all groups. * P < 0.05 and *** P < 0.001 vs. Ctr

Article Snippet: The following primary antibodies were used: monoclonal mouse anti-β-actin (A5316, 1:10000, Sigma-Aldrich, St. Louis, MO, USA), polyclonal rabbit anti-phosphoY594 EphB1/EphB2 (ab61791, 1:500, Abcam, Cambridge, MA, USA), polyclonal goat anti-EphB1 (AF542, 1:500, R&D systems), monoclonal mouse anti-PI3K p85α (sc-1637, 1:1000; Santa Cruz Biotechnology), polyclonal rabbit anti-PI3K p110 (sc-7189, 1:1000, Santa Cruz Biotechnology), polyclonal rabbit anti- Akt1/2/3 phospho Ser473 (sc-7985-R, 1:1000, Santa Cruz Biotechnology), monoclonal mouse anti-Akt1 (sc-5298, 1:1000, Santa Cruz Biotechnology), polyclonal rabbit anti-NF-κB p65 (sc-372, 1:1000, Santa Cruz Biotechnology), monoclonal mouse anti-NMDAR1 (#556308, 1:500, BD Pharmingen, Franklin Lakes, NJ, USA), polyclonal rabbit anti-NMDA NR2B phospho Tyr1472 (#454583, 1:500, Calbiochem), polyclonal rabbit anti-NMDAR2B (ab65783, 1:500, Abcam), monoclonal mouse anti-GFAP (G6171, 1:500, Sigma-Aldrich).

Techniques: Activation Assay, Cell Culture, Incubation, Saline, Injection

Journal: Acta Neuropathologica Communications

Article Title: EphrinB/EphB forward signaling in Müller cells causes apoptosis of retinal ganglion cells by increasing tumor necrosis factor alpha production in rat experimental glaucomatous model

doi: 10.1186/s40478-018-0618-x

Figure Lengend Snippet: Inhibition of TNF-α reduces RGC apoptosis in ephrinB1-Fc-injected retinas. a , Representative TUNEL staining images of RGC apoptosis in normal saline-injected (control) (a), ephrinB1-Fc-injected (b), and ephrinB1-Fc plus Xpro1595-injected (c) whole flat-mounted retinas on 7 d after the injections in the regions at angle 0°. Scale bar, 50 μm, for all the images. b , Bar chart showing the average number of TUNEL-positive cells in whole flat-mounted retinas under different conditions. XPro1595 (50 μg/μl, 2 μl) was pre-injected 1 d before the ephrinB1-Fc injection. n = 5 for all groups. * P < 0.05 and *** P < 0.001 vs. control; # P < 0.05 vs. ephrinB1-Fc alone group

Article Snippet: The following primary antibodies were used: monoclonal mouse anti-β-actin (A5316, 1:10000, Sigma-Aldrich, St. Louis, MO, USA), polyclonal rabbit anti-phosphoY594 EphB1/EphB2 (ab61791, 1:500, Abcam, Cambridge, MA, USA), polyclonal goat anti-EphB1 (AF542, 1:500, R&D systems), monoclonal mouse anti-PI3K p85α (sc-1637, 1:1000; Santa Cruz Biotechnology), polyclonal rabbit anti-PI3K p110 (sc-7189, 1:1000, Santa Cruz Biotechnology), polyclonal rabbit anti- Akt1/2/3 phospho Ser473 (sc-7985-R, 1:1000, Santa Cruz Biotechnology), monoclonal mouse anti-Akt1 (sc-5298, 1:1000, Santa Cruz Biotechnology), polyclonal rabbit anti-NF-κB p65 (sc-372, 1:1000, Santa Cruz Biotechnology), monoclonal mouse anti-NMDAR1 (#556308, 1:500, BD Pharmingen, Franklin Lakes, NJ, USA), polyclonal rabbit anti-NMDA NR2B phospho Tyr1472 (#454583, 1:500, Calbiochem), polyclonal rabbit anti-NMDAR2B (ab65783, 1:500, Abcam), monoclonal mouse anti-GFAP (G6171, 1:500, Sigma-Aldrich).

Techniques: Inhibition, Injection, TUNEL Assay, Staining, Saline

Journal: Acta Neuropathologica Communications

Article Title: EphrinB/EphB forward signaling in Müller cells causes apoptosis of retinal ganglion cells by increasing tumor necrosis factor alpha production in rat experimental glaucomatous model

doi: 10.1186/s40478-018-0618-x

Figure Lengend Snippet: EphrinB1-Fc treatment induces an increase in p-NR2B protein levels and increases TNF-α mRNA and protein levels in cultured Müller cells. a , Representative immunoblots showing the protein levels of NR1 in IgG-Fc-treated cells (Ctr), and those treated with ephrinB1-Fc for different periods of time. b , Bar chart summarizing the average densitometric quantification of immunoreactive bands of NR1. n = 4 for all groups. c , Representative immunoblots showing the protein levels of phosphorylated NR2B (p-NR2B) and NR2B in Müller cells treated with ephrinB1-Fc for different periods of time. d , Bar chart summarizing the average p-NR2B/NR2B ratios in Müller cells treated with ephrinB1-Fc for different periods of time. n = 6~ 7. * P < 0.05 and ** P < 0.01 vs. Ctr. e , Representative immunoblots showing that pre-incubation with PP2 eliminated the ephrinB1-Fc-treatment induced changes in p-NR2B protein levels in Müller cells. PP2 (10 μM) was added to the medium 30 min before a 3 h ephrinB1-Fc-treatment. f , Bar charts summarizing the average p-NR2B/NR2B ratios obtained in the presence of ephrinB1-Fc, with or without 30 min pre-incubation of 10 μM PP2. All the data are normalized to the ratio obtained in IgG-Fc treated group (Ctr). n = 4 for all groups. *** P < 0.001 vs. IgG-Fc treated group (Ctr). g , Summary data showing that relative TNF-α mRNA levels in Müller cells, represented as fold changes, were significantly elevated by ephrinB1-Fc treatment, but the elevation was abolished by addition of RO25–6981 (5 μM) 30 min before the ephrinB1-Fc treatment. n = 5 for all groups. ** P < 0.01 vs. Ctr (IgG-Fc treated group). h , Summary data showing that the ephrinB1-Fc treatment induced increase in TNF-α protein levels of cultured Müller cells was blocked by adding RO25–6981 (5 μM) to the medium 30 min before the ephrinB1-Fc treatment. n = 5 for all groups. *** P < 0.001 vs. Ctr (IgG-Fc treated group)

Article Snippet: The following primary antibodies were used: monoclonal mouse anti-β-actin (A5316, 1:10000, Sigma-Aldrich, St. Louis, MO, USA), polyclonal rabbit anti-phosphoY594 EphB1/EphB2 (ab61791, 1:500, Abcam, Cambridge, MA, USA), polyclonal goat anti-EphB1 (AF542, 1:500, R&D systems), monoclonal mouse anti-PI3K p85α (sc-1637, 1:1000; Santa Cruz Biotechnology), polyclonal rabbit anti-PI3K p110 (sc-7189, 1:1000, Santa Cruz Biotechnology), polyclonal rabbit anti- Akt1/2/3 phospho Ser473 (sc-7985-R, 1:1000, Santa Cruz Biotechnology), monoclonal mouse anti-Akt1 (sc-5298, 1:1000, Santa Cruz Biotechnology), polyclonal rabbit anti-NF-κB p65 (sc-372, 1:1000, Santa Cruz Biotechnology), monoclonal mouse anti-NMDAR1 (#556308, 1:500, BD Pharmingen, Franklin Lakes, NJ, USA), polyclonal rabbit anti-NMDA NR2B phospho Tyr1472 (#454583, 1:500, Calbiochem), polyclonal rabbit anti-NMDAR2B (ab65783, 1:500, Abcam), monoclonal mouse anti-GFAP (G6171, 1:500, Sigma-Aldrich).

Techniques: Cell Culture, Western Blot, Incubation

Journal: Acta Neuropathologica Communications

Article Title: EphrinB/EphB forward signaling in Müller cells causes apoptosis of retinal ganglion cells by increasing tumor necrosis factor alpha production in rat experimental glaucomatous model

doi: 10.1186/s40478-018-0618-x

Figure Lengend Snippet: EphrinB1-Fc treatment activates PI3K/Akt signaling in cultured Müller cells. a , Representative immunoblots showing the changes in PI3K p85 and p110 protein levels in IgG-Fc-treated cells (Ctr), and those treated with ephrinB1-Fc for different periods of time. b-c , Bar chart summarizing the average densitometric quantification of immunoreactive bands of PI3K p85 ( b ) and PI3K p110 ( c ) under different conditions as shown in A . n = 4 for all groups. All the data are normalized to Ctr. * P < 0.05, ** P < 0.01 and *** P < 0.001 vs. Ctr. d , Representative immunoblots showing that pre-incubation with RO25–6981 eliminated the ephrinB1-Fc-treatment induced changes in PI3K p85 and p110 protein levels in Müller cells. RO25–6981 (0.5 μM/5 μM) was added to the medium 30 min before a 6 h ephrinB1-Fc treatment. n = 5 for all groups. e-f , Bar chart summarizing the average densitometric quantification of immunoreactive bands of PI3K p85 ( e ) and p110 ( f ) under different conditions as shown in D . n = 5 for all groups. * P < 0.05, ** P < 0.01 and *** P < 0.001 vs. Ctr (IgG-Fc treated group). g , Representative immunoblots showing the changes in p-Akt and Akt protein levels in IgG-Fc-treated cells (Ctr), and those treated with ephrinB1-Fc for different periods of time. h , Bar chart summarizing the average p-Akt/Akt ratios in Müller cells treated with ephrinB1-Fc for different periods of time. n = 5 for all groups. * P < 0.05, vs. Ctr. i , Representative immunoblots showing that ephrinB1-Fc treatment induced changes in p-Akt and Akt protein levels were blocked by adding PP2 (10 μM) to the medium 30 min before a 3 h ephrinB1-Fc treatment. j , Bar chart summarizing the average p-Akt/Akt ratios of Müller cells under different conditions as shown in I . n = 5 for all groups. ** P < 0.01 vs. Ctr (IgG-Fc treated group)

Article Snippet: The following primary antibodies were used: monoclonal mouse anti-β-actin (A5316, 1:10000, Sigma-Aldrich, St. Louis, MO, USA), polyclonal rabbit anti-phosphoY594 EphB1/EphB2 (ab61791, 1:500, Abcam, Cambridge, MA, USA), polyclonal goat anti-EphB1 (AF542, 1:500, R&D systems), monoclonal mouse anti-PI3K p85α (sc-1637, 1:1000; Santa Cruz Biotechnology), polyclonal rabbit anti-PI3K p110 (sc-7189, 1:1000, Santa Cruz Biotechnology), polyclonal rabbit anti- Akt1/2/3 phospho Ser473 (sc-7985-R, 1:1000, Santa Cruz Biotechnology), monoclonal mouse anti-Akt1 (sc-5298, 1:1000, Santa Cruz Biotechnology), polyclonal rabbit anti-NF-κB p65 (sc-372, 1:1000, Santa Cruz Biotechnology), monoclonal mouse anti-NMDAR1 (#556308, 1:500, BD Pharmingen, Franklin Lakes, NJ, USA), polyclonal rabbit anti-NMDA NR2B phospho Tyr1472 (#454583, 1:500, Calbiochem), polyclonal rabbit anti-NMDAR2B (ab65783, 1:500, Abcam), monoclonal mouse anti-GFAP (G6171, 1:500, Sigma-Aldrich).

Techniques: Cell Culture, Western Blot, Incubation

Journal: Acta Neuropathologica Communications

Article Title: EphrinB/EphB forward signaling in Müller cells causes apoptosis of retinal ganglion cells by increasing tumor necrosis factor alpha production in rat experimental glaucomatous model

doi: 10.1186/s40478-018-0618-x

Figure Lengend Snippet: Activation of ephrinB/EphB forward signaling induces translocation of NF-κB p65 from the cytoplasm into the nucleus. a , Confocal laser microphotographs of cultured Müller cells, stained with the antibody against NF-κB p65 (green), showing the changes in NF-κB p65 protein expression in IgG-Fc treatment (Ctr) and ephrinB-Fc-treatment for different periods of time (a1-a7). b1-b7 are DAPI images. c1-c7 are merged images. Scale bar: 10 μm, for all the images. b , Representative immunoblots showing the changes in NF-κB p65 protein levels in whole cell extracts obtained from IgG-Fc-treated cells (Ctr) and those treated with ephrinB1-Fc for different periods of time. c , Bar chart summarizing the average densitometric quantification of immunoreactive bands of NF-κB p65 in Müller cells treated with ephrinB1-Fc for different periods of time. n = 5 for all groups. ** P < 0.01 vs. Ctr. d , Representative immunoblots showing the changes of NF-κB p65 in nucleus component of Müller cells treated with IgG-Fc (Ctr), and with ephrinB1-Fc for different periods of time. e , Bar chart summarizing the average densitometric quantification of immunoreactive bands of nucleus NF-κB p65 under the conditions as shown in D . n = 6 for all groups. * P < 0.05, and *** P < 0.001 vs. Ctr. f , Representative immunoblots showing the changes in NF-κB p65 protein levels in the nucleus component of Müller cells obtained in the presence of ephrinB1-Fc, with or without in the presence of LY294002. LY294002 (2 μM/10 μM) was added to the medium 30 min before a 3 h ephrinB1-Fc treatment. g , Bar chart summarizing the average densitometric quantification of immunoreactive bands of nucleus NF-κB p65 under the conditions as shown in F . n = 4 for all groups. ** P < 0.01 vs. Ctr (IgG-Fc treated group). h , Summary data showing that pre-incubation with PDTC inhibited the ephrinB1-Fc treatment induced increase in TNF-α mRNA levels in Müller cells. PDTC (10 μM/50 μM) was added to the medium 30 min before the ephrinB1-Fc treatment. n = 4 for all groups. i , Summary data showing that PDTC blocked the ephrinB1-Fc treatment induced increase in TNF-α protein levels in Müller cells. PDTC (50 μM) was added to the medium 30 min before the ephrinB1-Fc treatment. n = 4 for all groups. ** P < 0.01 and *** P < 0.001 vs. Ctr (IgG-Fc treated group)

Article Snippet: The following primary antibodies were used: monoclonal mouse anti-β-actin (A5316, 1:10000, Sigma-Aldrich, St. Louis, MO, USA), polyclonal rabbit anti-phosphoY594 EphB1/EphB2 (ab61791, 1:500, Abcam, Cambridge, MA, USA), polyclonal goat anti-EphB1 (AF542, 1:500, R&D systems), monoclonal mouse anti-PI3K p85α (sc-1637, 1:1000; Santa Cruz Biotechnology), polyclonal rabbit anti-PI3K p110 (sc-7189, 1:1000, Santa Cruz Biotechnology), polyclonal rabbit anti- Akt1/2/3 phospho Ser473 (sc-7985-R, 1:1000, Santa Cruz Biotechnology), monoclonal mouse anti-Akt1 (sc-5298, 1:1000, Santa Cruz Biotechnology), polyclonal rabbit anti-NF-κB p65 (sc-372, 1:1000, Santa Cruz Biotechnology), monoclonal mouse anti-NMDAR1 (#556308, 1:500, BD Pharmingen, Franklin Lakes, NJ, USA), polyclonal rabbit anti-NMDA NR2B phospho Tyr1472 (#454583, 1:500, Calbiochem), polyclonal rabbit anti-NMDAR2B (ab65783, 1:500, Abcam), monoclonal mouse anti-GFAP (G6171, 1:500, Sigma-Aldrich).

Techniques: Activation Assay, Translocation Assay, Cell Culture, Staining, Expressing, Western Blot, Incubation

Journal: Molecular Vision

Article Title: EphB1 causes retinal damage through inflammatory pathways in the retina and retinal Müller cells

doi:

Figure Lengend Snippet: EphB1 protein levels are increased in the retinas of diabetic patients and diabetic mice. Western blotting was done for EphB1 in whole retinal lysates from (A) control and diabetic patients and (B) control and diabetic mice. All diabetic patients had had the disease for over 10 years. *p<0.05 versus control. n=7 for both panels. Data are presented as mean ± standard error of mean (SEM).

Article Snippet: Some cells in high glucose were treated with siRNA to EphB1 (Origene) or scrambled siRNA transfected into the cells using RNAiMax.

Techniques: Western Blot, Control

Journal: Molecular Vision

Article Title: EphB1 causes retinal damage through inflammatory pathways in the retina and retinal Müller cells

doi:

Figure Lengend Snippet: EphB1 is increased in rMC-1 Müller cells. Panel A shows the localization of EphB1 in rMC-1 cells in culture in normal glucose (top) and high glucose (bottom). Cells are stained with green for EphB1 and DAPI for nuclear localization. Panel B is western blot data from rMC-1 cells grown in normal (5 mM) or high (25 mM) glucose. *p<0.05 versus normal glucose (NG), n=5 for the western blot. Data are presented as mean ± SEM. Scale bar is 25 µm.

Article Snippet: Some cells in high glucose were treated with siRNA to EphB1 (Origene) or scrambled siRNA transfected into the cells using RNAiMax.

Techniques: Staining, Western Blot

Journal: Molecular Vision

Article Title: EphB1 causes retinal damage through inflammatory pathways in the retina and retinal Müller cells

doi:

Figure Lengend Snippet: EphB1 siRNA reduced inflammatory mediators in rMC-1 cells. rMC-1 cells were grown in normal (5 mM) or high (25 mM) glucose. Some cells in high glucose were treated with EphB1 siRNA or scrambled siRNA. Western blotting was performed for (A) EphB1, (B) TNFα, and (C) NLRP3. An ELISA was done for IL-1β in panel D. *p<0.05 versus NG, #p<0.05 versus high glucose (HG). n=5. Data are presented as mean ± SEM.

Article Snippet: Some cells in high glucose were treated with siRNA to EphB1 (Origene) or scrambled siRNA transfected into the cells using RNAiMax.

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Molecular Vision

Article Title: EphB1 causes retinal damage through inflammatory pathways in the retina and retinal Müller cells

doi:

Figure Lengend Snippet: Ephrin B1 increased inflammatory mediators in rMC-1 cells. rMC-1 cells were grown in normal (5 mM) or high (25 mM) glucose. Some cells in each glucose condition were treated with ephrin B1-Fc. Western blotting was performed for (A) HMGB1 and (B) NLRP3. An ELISA was done for IL-1β. *p<0.05 versus NG, #p<0.05 versus HG. n=5. Data are presented as mean ± SEM.

Article Snippet: Some cells in high glucose were treated with siRNA to EphB1 (Origene) or scrambled siRNA transfected into the cells using RNAiMax.

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Molecular Vision

Article Title: EphB1 causes retinal damage through inflammatory pathways in the retina and retinal Müller cells

doi:

Figure Lengend Snippet: EphB1 can be expressed by AAV in Müller cells. To express EphB1 in retinal Müller cells in mice, mice were injected equal amounts of either the EphB1 overexpression AAV or control AAV. The left panel (green) shows HA staining (as a control), the middle panel is EphB1 (red), and the merged image is on the right with yellow staining demarcated by arrows showing co-localization. Five mice in each group were used for the staining experiments. Scale bar is 50 nm.

Article Snippet: Some cells in high glucose were treated with siRNA to EphB1 (Origene) or scrambled siRNA transfected into the cells using RNAiMax.

Techniques: Injection, Over Expression, Control, Staining

Journal: Molecular Vision

Article Title: EphB1 causes retinal damage through inflammatory pathways in the retina and retinal Müller cells

doi:

Figure Lengend Snippet: Ischemia/reperfusion damage was worsened by overexpression of EphB1 by AAV. The top panels show retinal thickness and cell loss in the ganglion cell layer. Quantification of cell loss and retinal thickness is shown in graphs below the images. *p<0.05 versus AAV control, #p<0.05 versus AAV control I/R. n=5 for all groups. Data are mean ± SEM.

Article Snippet: Some cells in high glucose were treated with siRNA to EphB1 (Origene) or scrambled siRNA transfected into the cells using RNAiMax.

Techniques: Over Expression, Control