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Abstract
Purpose
To determine whether the expression of mutant myocilin can lead to death of human trabecular meshwork (HTM) cells and to determine whether the mechanism by which this occurs is apoptosis.
Methods
HTM cells were transduced with a recombinant adenovirus expressing human mutant (Q368X) myocilin. The apoptotic death of HTM cells caused by expression of mutant myocilin was examined using a cell proliferation assay, flow cytometry, Western blot analysis, and immunocytochemistry.
References
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Figure 1.
Effect of mutant myocilin expression on cell viability. HTM cells were transduced at an MOI indicated with an adenovirus expressing mutant (Q368X) myocilin (Ad-Q368X), and 2, 3, and 4 days after transduction, subjected to cell proliferation assay. Cells treated for 48 hours with Tg were included as positive controls. Each column rep-resents the means from three different wells of three independent experiments with the bars denoting the standard deviation. ∗ p < 0.01 vs. control (Dunnet's test).
Figure 2.
Quantitative analysis of apoptotic cell death caused by mutant myocilin expression. HTM cells were transduced at an MOI of 50 pfu per cell with AdQ368X, and 48 hours later, stained with Annexin V-APC or propi-dium iodide (PI) and then analyzed by flow cytometry. Cells treated for 48 hours with 400 uM Tg served as positive controls. Shown are representative of three independent experiments with similar results.
Figure 3.
Western blots showing the effect of mutant myocilin expression on the level of Grp78, p-JNK, and cleaved cas-pase-3. HTM cells transduced at an MOI indicated with Ad-Q368X. After 48 hours, cell lysates were assayed for Grp78, p-JNK and cleavaged caspase 3 by Western blot analysis. Data were normalized with α-tubulin expression.
Figure 4.
Effect of mutant myocilin expression on the activation of caspsae-3. HTM cells were transduced at an MOI of 50 (C, D) or 100 pfu (E, F) per cell with Ad-Q368X, and after 48 hours, stained with aspartylglutamy-lalanylaspartic acid (DEVD) coupled to cresyl violet, MR-(DEVD)2 (A, B, C, and E). Cells untreated (A) or treated (B) with 400 uM of Tg served as controls. Red fluorescence and green fluorescence represent caspase-3 activity and GFP-tagged myocilin expression, respectively. Blue fluorescence corresponds to nuclei stained with Hoechst 33342. Bar: 100 um.
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