Figure 4.

Inhibition of apoE proteolytic fragmentation by protease inhibitors. SK-N-SH cells were cultured for 9 days in the presence of 10 μm ATRA. The culture medium was then replaced with fresh medium alone (Con) or with fresh medium containing either a broad-spectrum PIM or the serine protease inhibitor AEBSF and cultured for a further 24-h period. The addition of PIM inhibited the formation of apoE fragments in the supernatant (S/N) (A). The addition of PIM had no impact on cellular apoE levels (B). A similar result was observed when AEBSF was used to treat the cells (D and E). Quantitative assessment of the full-length apoE (35 kDa) and apoE fragments at 28 and 25 kDa indicates a significant inhibition of apoE fragment formation in the presence of either PIM (C; light gray bars) or AEBSF (F; light gray bars)). Data in C and F are relative optical density measurements, in which the vehicle control (DMSO 1:1000) sample (dark gray bars) is defined as 1.0. Data shown are derived from one experiment performed in triplicate and representative of three independently performed experiments. The histogram bars represent mean values, and the error bars represent S.D. *, p < 0.05; †, p < 0.01 compared with the respective control for each fragment; two-tailed t test.