Increased ECM accumulation and ER stress induction in the TM of Dex-treated cultured corneoscleral segments.
A) Immunostaining for myocilin and fibronectin, B) GRP78 (ER stress marker) and collagen IV in quadrants cultured for 7 days treated with the vehicle (0.1% ethanol) and Dex (100nM). Dex treatment prominently increased myocilin, fibronectin, collagen IV and GRP78 staining in the TM region. (n = 4 biological replicates, scale bar is 50μm). Western blot and densitometric analysis for FN (ECM marker) and ATF4, CHOP and GRP78 (ER stress markers) in TM tissue lysates (C-D) of vehicle- and Dex- (100nM) treated cultured corneoscleral segments. Dex treatment led to a significant increase in the ECM marker, FN (n = 3 biological replicates) and ER stress markers CHOP and ATF4 but not GRP78 (n = 4 biological replicates). Note that densitometric analysis included only Dex responders. Similarly, conditioned medium (E-F) from Dex-treated corneoscleral segments showed a significant increase in ECM proteins FN and Col IV (n = 8); unpaired t-test, *P<0.05, **P<0.01, ***P<0.001). Arrows indicate the TM region.