Figure 4.

Synthesis and maturation of cathepsin-D is compromised by NaIO_3. (A) Quantitative RT-qPCR for cat-D showed 0.75-fold downregulation in lysates from NaIO₃-treated ARPE-19 cells relative to controls. Human GAPDH was amplified in parallel. (B) Probing of NaIO₃-treated cell lysates for cat-D showed decreased expression of pro-cat-D and its mature form relative to controls (lanes 2, 4 vs. 1, 3). (C) Densitometry after normalization with β-actin showed 0.7- and 0.5-fold decreases in the levels of pro-cat-D and mature cat-D in NaIO₃-treated samples relative to the controls. (D) The densitometry ratio of mature cat-D relative to pro-cat-D was significantly lower in NaIO₃-treated cell lysates relative to control. (E) Probing of ARPE-19 cell lysates for cat-D showed the expected reactivity of the pro-cat-D form at 52 kDa in control cells transfected with scrambled siRNA, and minimal reaction in cells exposed to cat-D siRNA (lane 2 vs. 1). This further led to lowered levels of mature cat-D levels in cat-D siRNA-treated cell lysates. Probing for ferritin showed significant upregulation in the absence of cat-D relative to control (lanes 2 vs. 1). (F) Quantification by densitometry after normalization with β-actin showed 2.7-fold upregulation of ferritin due to downregulation of cat-D. Full blots and their details are provided in Supplementary Figure S1. Values are means ± SEM of the indicated n. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001.