Fig. 5: ATF4 increases protein synthesis and ER client protein load in TM cells and tissues.
From: ATF4 leads to glaucoma by promoting protein synthesis and ER client protein load
a GTM3 cells were transduced with Ad5.Empty, ATF4, and CHOP for 36 h. and cellular lysates were examined for markers of chronic ER stress by Western blot analysis. Expression of ATF4 increased XBP-1 (s) and GADD34 while ATF4 reduced p-eIF2α (n = 3 replicates). b GTM3 cells transfected with ATF4 or CHOP were incubated with cycloheximide (CHX) (10 μg/ml) for 16 h. Puromycin (10 µg/ml) was added to cells for 30 min before harvesting cell lysates. Total cellular lysates were subjected to Western blot analysis using anti-puromycin and GAPDH antibodies. Increased puromycin incorporation observed in the total ER fractions of ATF4 transfected cells (n = 3 replicates), which was blocked by CHX signifying a higher rate of de novo protein synthesis. c and d GTM3 cells stably expressing DsRed-tagged WT c or mutant d MYOC were transfected with plasmids expressing Empty and ATF4 for 48 h. Cells were fixed and stained with calreticulin (ER marker). ATF4 increased intracellular WT and mutant myocilin accumulation in the ER as evident from a strong colocalization of myocilin with calreticulin in ATF4-transfected cells (n = 3 replicates). Scale bar is 25 µm. e 3-month-old C57BL/6J mice were intravitreally injected with Ad5.Empty, Ad5.ATF4, and Ad5.CHOP (2 × 107 pfu/eye). After 3 weeks of injections, anterior segment lysates were subjected to Western blot (Supplementary Fig. 15) and densitometric analyses e of ECM (fibronectin, collagen-1) and ER stress (Grp78, ATF4, and CHOP) markers (n = 4 mice; data are presented as mean ± SEM, one-way ANOVA, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). f Increased ATF4 and KDEL staining in TM of Ad5.ATF4-injected mice compared to Ad5.Empty-injected mice (n = 3 mice). Scale bar is 100 µm.
