Research Paper Volume 13, Issue 6 pp 8628—8642

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

TBHP activated the expression of transcription factor ATF4 and induced apoptosis in HTMCs. HTMCs were exposed to media in the absence (Mock) or presence of 50 μM of TBHP for 12 h. (A–C) Western blot analysis for phospho-eIF2α, total eIF2α, ATF4, CHOP and GAPDH. Intensities were quantified and normalized against the level of total proteins (eIF2α) or GAPDH (mean ± SEM, n = 3). (D) Representative images of immunostaining for apoptotic (TUNEL-positive, red) HTMCs. Nuclei were labeled with DAPI (blue). Scale bar represents 100 μm. (E) Quantification of apoptotic nuclei by Image-Pro Plus software. Values are expressed as the percentage of TUNEL-positive cells to total (DAPI-positive) cells (mean ± SEM, n = 3). (F) Western blot analysis for cleaved caspase-3. Intensities were quantified and normalized against the level of total caspase-3 (mean ± SEM, n = 3). *P**P

Figure 1. TBHP activated the expression of transcription factor ATF4 and induced apoptosis in HTMCs. HTMCs were exposed to media in the absence (Mock) or presence of 50 μM of TBHP for 12 h. (AC) Western blot analysis for phospho-eIF2α, total eIF2α, ATF4, CHOP and GAPDH. Intensities were quantified and normalized against the level of total proteins (eIF2α) or GAPDH (mean ± SEM, n = 3). (D) Representative images of immunostaining for apoptotic (TUNEL-positive, red) HTMCs. Nuclei were labeled with DAPI (blue). Scale bar represents 100 μm. (E) Quantification of apoptotic nuclei by Image-Pro Plus software. Values are expressed as the percentage of TUNEL-positive cells to total (DAPI-positive) cells (mean ± SEM, n = 3). (F) Western blot analysis for cleaved caspase-3. Intensities were quantified and normalized against the level of total caspase-3 (mean ± SEM, n = 3). *P<0.05 and **P <0.01 vs. Mock.