Research Paper Volume 13, Issue 6 pp 8628—8642

Activation of ATF4 triggers trabecular meshwork cell dysfunction and apoptosis in POAG

Suppression of ATF4 prevented tunicamycin-induced apoptosis and ROS generation, and rescued the phagocytotic activity of HTMC. HTMCs were exposed to media in the absence (Mock) or presence of 100 ng/ml of tunicamycin (Tuni) for 24 h. Alternatively, cells were transfected with si-ATF4 or si-sc for 48 h, followed by incubation with 100 ng/ml of tunicamycin (Tuni) for additional 24 h. (A) Expression of cleaved caspase-3 was determined by Western blot analysis and quantified by densitometry (mean ± SEM, n = 3). (B) Representative images of ROS production in cells incubated with DCFH-DA (green. scale bar, 60 μm). (C) Quantification of intracellular ROS production. Values are expressed as the fold increase from Mock in ROS content evaluated by fluorescence intensity (mean ± SEM, n = 3). (D) Representative images of subcellular expression of ELAM-1 by indirect immunofluorescence (green, ELAM-1. blue, DAPI. scale bar, 40 μm). (E) Levels of cellular immunofluorescence of ELAM-1 were measured and expressed as corrected total cell fluorescence (mean ± SEM, n = 3). (F) Phagocytosis of colloidal gold by HTMCs was examined by dark field microscope (gold, colloidal gold). Representative images were mounted in upper panels (scale bar, 20 μm). Lower panels show magnified images of individual cells. *P**P #P

Figure 4. Suppression of ATF4 prevented tunicamycin-induced apoptosis and ROS generation, and rescued the phagocytotic activity of HTMC. HTMCs were exposed to media in the absence (Mock) or presence of 100 ng/ml of tunicamycin (Tuni) for 24 h. Alternatively, cells were transfected with si-ATF4 or si-sc for 48 h, followed by incubation with 100 ng/ml of tunicamycin (Tuni) for additional 24 h. (A) Expression of cleaved caspase-3 was determined by Western blot analysis and quantified by densitometry (mean ± SEM, n = 3). (B) Representative images of ROS production in cells incubated with DCFH-DA (green. scale bar, 60 μm). (C) Quantification of intracellular ROS production. Values are expressed as the fold increase from Mock in ROS content evaluated by fluorescence intensity (mean ± SEM, n = 3). (D) Representative images of subcellular expression of ELAM-1 by indirect immunofluorescence (green, ELAM-1. blue, DAPI. scale bar, 40 μm). (E) Levels of cellular immunofluorescence of ELAM-1 were measured and expressed as corrected total cell fluorescence (mean ± SEM, n = 3). (F) Phagocytosis of colloidal gold by HTMCs was examined by dark field microscope (gold, colloidal gold). Representative images were mounted in upper panels (scale bar, 20 μm). Lower panels show magnified images of individual cells. *P<0.05 vs. Mock, **P <0.01 vs. Mock, #P<0.05 vs. Tuni+si-sc.