Figure 8.

Recombinant expression of PAI-1 or PAI-2 increases autophagy in fibroblasts. To assess the effects of PAI-1 and PAI-2 overexpression on autophagy in fibroblasts, the levels of a variety of autophagic markers were assessed in control fibroblasts and in PAI-1/2(+)-overexpressing fibroblasts. (A) Determination of AVOs (acidic vesicular organelles) using acridine orange. In orange-acridine stained cells, cytoplasm fluorescences green, whereas acid compartments fluorescence red. Note that fibroblasts overexpressing PAI-1 or PAI-2 display a larger amount of AVOs compared with control fibroblasts. Images captured at original magnification of 63x. Nuclei were stained with Hoechst (blue). (B) Localization of Beclin-1. Note that overexpression of PAI-1 or PAI-2 upregulates the expression of Beclin-1, an autophagic marker. Images were captured at an original magnification of 63x. Nuclei were stained with Hoechst (blue). (C) Immunoblotting for LAMP-1 and LAMP-2. Note that the expression of autophagic markers, LAMP-1 and LAMP-2, is increased in fibroblasts overexpressing PAI-1 or PAI-2. Immunoblotting with β-tubulin is shown as a control for equal loading. (D) Proliferation of fibroblasts overexpressing PAI-1 or PAI-2. Note that BrdU incorporation is significantly reduced in fibroblasts overexpressing PAI-1 or PAI-2 as compared with control fibroblasts. Results are represented as the mean ± SEM. An asterisk indicates that p ≤ 0.05, compared with controls. Ctrl, fibroblasts containing empty vector alone; PAI-1, fibroblasts overexpressing PAI-1; PAI-2, fibroblasts overexpressing PAI-2.