1. From: Light Induces Programmed Cell Death by Activating Multiple Independent Proteases in a Cone Photoreceptor Cell Line
Invest. Ophthalmol. Vis. Sci ;48(1): doi: /iovs
Figure Legend:
(A) Western blot analysis of calpain 2 activation in 661W cells after light stress. Percent activation was determined by dividing the densitometric readings of the calpain band by the corresponding actin band. Lane 4D: 4 hours of dark incubation; lanes 1L, 2L, 3L, and 4L: are 1, 2, 3, and 4 hours of exposure to light, respectively. (B) Western blot analysis of cathepsin D in 661W cells after light stress. A procathepsin D band was observed in samples incubated with 9-cis retinal in the dark for 4 hours (black arrow). Light caused the processing of the procathepsin D band to a mature form (gray arrow) within 1 hour of exposure to light (lane 1L). This processed band was also observed at 2, 3, and 4 hours after exposure to light, indicating cathepsin D activation in the system. Numbers below the actin blot only, to indicate lane numbers. (C) Protection from light stress with protease inhibitors, individually and in combination. The cathepsin D inhibitor pepstatin A (2 μM), the pancaspase inhibitor zVAD-fmk (10 μM), and/or the calpain inhibitor MDL (25 μM) were used to determine the roles of different proteases in the apoptotic cell death of light-stressed 661W cone photoreceptor cells. Viability was assessed by MTT assay by measuring absorbance of the blue formazan product at 570 nm. The graph was plotted by calculating the percentage of viable cells incubated with different concentrations of inhibitors and 10 μM 9-cis retinal after exposure to light versus the dark control, with dark control cells being 100% viable. A DMSO control (without 9-cis retinal) was included because 9-cis retinal was dissolved in DMSO. Statistical analysis using the Bonferroni multiple comparison was performed and significant probabilities are: A versus B-I, <0.001; B versus D, <0.01; B versus E–I, <0.001; C versus D, <0.05; C versus E–I, <0.001; D versus G, <0.01; D versus H and I, <0.001; E versus G, <0.05; E versus H, <0.01; E versus I, <0.001; F versus I, <0.001; G versus I, <0.001; and H versus I, < (D) Inhibitory effects of the pancaspase inhibitor zVAD-fmk and/or the calpain 2 inhibitor MDL on caspase-3 cleavage after 4 hours of exposure to light (4L). There was a reduction of cleaved caspase-3 in the presence of the pancaspase inhibitor compared with no inhibitor or MDL alone. Samples incubated with 9-cis retinal in the dark (4D) showed no caspase activation. The ratio was obtained by dividing the densitometric reading of the band for cleaved caspase-3 by that for actin. Actin controls are shown below each blot.
Date of download: 11/3/2017
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