5 research outputs found

    TAK1 regulates RPE cells EMT phenotype upon TGF-β stimulation.

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    <p><b>A:</b> RPE cells were serum-starved for 16 hours, pretreated with 5Z-7-oxozeaenol (1μM) or left untreated for 1 hour. The cells were then treated with TGF-β1 (2.5ng/ml) as described in Materials and Methods for 24h and immunostained with α-SMA antibodies (red) and DAPI (blue). Representative photographs of three independent experiments. The histogram represent quantification of pixel intensity <b>B:</b> RPE cells were serum-starved for 16 hours, then treated with mitomycin C (10ng/ml) for 3h. Thereafter, pretreated with 5Z-7-oxozeaenol (1μM) or Dimethyl sulfoxide (DMSO) for 1 hour. Following this process a scratch was performed in the cell monolayer and the serum free medium was supplemented with TGF-β1 (2.5ng/ml) or left unsupplemented. Scratches were photo-documented at the indicated times (top panel) and their width was measured using Image-J software. The histogram (bottom panel) demonstrates the percentage of remaining gap at each time point, relative to initial gap width, based on three independent experiments. Bars are Mean±SD. <b>C:</b> RPE cells were serum-starved for 16 hours then pretreated with 5Z-7-oxozeaenol (1μM) or SB431542 (10μM), or DMSO for 1 hour. Finally, the medium was replaced with serum free medium with or without TGF-β1 (2.5ng/ml) and supernatants from the different treatments were collected after 24 hours. Total MMP-9 activities were processed by gelatin zymography (top panel) and the band intensity values were calculated by Quantity One 1-D analysis software. Histogram demonstrating secretion levels of MMP-9 in the different treatments (bottom panel). Statistics were computed using student t-test (Two tailed distribution equal variance). Data is expressed as the Mean±SD.</p

    TGF-β regulates morphological and transcription changes in RPE cells through TAK1.

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    <p><b>A:</b> RPE cells were plated on fibronectin-coated glass coverslips, serum-starved for 16 hours, pretreated with 5Z-7-oxozeaenol (1μM) or SB431542 (10μM), or DMSO for 1 hour, then treated with or without TGF-β1 for 2 days. Following treatment the cells were stained with rhodamine-phalloidin (actin fibers-red) and DAPI (blue) and visualized by confocal microscopy. Scale bars: 20μM. The histogram represents quantification of cells size <b>B:</b> Serum-starved RPE cells pretreated with 5Z-7-oxozeaenol (1μM) DMSO for 1 hour, were exposed to TGF-β as in A. Total RNA was extracted at each time point and qPCR was performed. Transcription levels of CTGF were determined after 6h, 16h and 24h. Bars represent the specific mRNA amount relative to GAPDH mRNA in the same samples. All experiments were performed in triplicates. Representative histogram from two independent experiments. Statistics were computed using student t-test (Two tailed distribution equal variance). Data is expressed as the Mean±SD.</p

    Inhibition of TAK1 abolishes the activation of TGF-β cascades.

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    <p><b>A</b>: Serum-starved RPE cells were pretreated with or without with 5Z-7-oxozeaenol (1μM) for 1 hour and then with TGF-β (2.5ng/ml) for the indicated times. Total protein extracts were analyzed by western blot using the indicated antibodies. The blot shows a representative result of four independent experiments. <b>B:</b> Levels of phospho-Smad2/3 were quantified and normalized to total Smad2/3 <b>C:</b> Statistical analysis of p-p38 and p-Smad3 activation normalized to p38 and Smad3 respectively, with or without TGF-β stimulation. The histograms present results of 5 independent experiments. Statistics were computed using student t-test (Two tailed distribution equal variance). Data is expressed as the Mean±SD.</p

    TAK1 is activated upon TGF-β1 stimulation in RPE cells.

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    <p><b>A:</b> RPE cells were treated with TGF-β1 (2.5ng/ml) for the indicated times or left untreated. The cells were then immunostained with phospho-Thr 187 TAK1 antibodies (green) and DAPI (blue) as described in Materials and Methods. Representative photographs of three independent experiments. Scale bar is 10μm for all images. <b>B</b>: The histogram demonstrating pixel intensity, measured using Image-J software, is based on three independent experiments. (Number of cells: Control 0 = 24, Control 4 hours = 28, control 24 hours = 21, control 48 hours = 25; TGF-β1 4 hours = 26, TGF-β1 24 hours = 21, TGF-β1 48 hours = 22). Statistics were computed using student t-test (Two tailed distribution equal variance). Data is expressed as the Mean±SD.</p

    TAK1 is a general regulator of the EMT process in RPE cells.

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    <p><b>A:</b> RPE cells were pre-treated with or without 5Z-7-oxozeaenol (1μM) and seeded in collagen lattices in full medium. The experiments were performed in triplicates. Lattices were photo-documented after 24 hours and measured using Image-J software. <b>B:</b> The histogram demonstrates the percentage of lattice area (marked by white line) relative to initial gel area, based on three independent experiments. Statistics were computed using student t-test (Two tailed distribution equal variance). Data is expressed as the Mean±SD.</p
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