Accumulation of endosomal/lysosomal proteins and proteasomes upon cysteine cathepsin inhibition. (A) Representative Lamp-1 immunofluorescence images of untreated −/+E64d and four days TGFβ-1 −/+E64d treated iPL32 cells are shown (Lamp-1: green, Hoechst nuclear stain: blue). (B) Lamp-1, Rab5 Ctsl, Ctsb, and Ctsd protein levels were analyzed by Western blot with α-tubulin and actin as loading controls in whole cell lysates of untreated −/+E64d and four days TGFβ-1 −/+E64d treated iPL32 cells. Ctsl heavy (h) and light (l) chains indicate the fully processed mature protease. (C) Flow cytometry analysis of Acridine-Orange (AO) stained iPL32 cells pretreated with or without TGFβ-1 −/+E64d for four days: Representative histograms for FL-3 height (orange) and FL-1 height (green) are shown in the upper panel, statistical analysis of independent experiments in the lower panel. Geometric mean orange or green fluorescence was normalized to untreated control cells (n = 3, *p ≤ 0.05, **p ≤ 0.01). (D) Proteasome activity of untreated −/+E64d and four days TGFβ-1 −/+E64d treated iPL32 cells was measured by Suc-LLVY-AMC cleavage (n = 3). (E) Ctsb, Ctsl, Ctsd, Lamp-1, and proteasome subunit 4 “Psmb4” mRNA levels in untreated and four days TGFβ-1 −/+E64d treated iPL32 cells were analyzed by qRT-PCR. Starting quantity values were normalized to the untreated group. Data are shown as the mean ± SEM (n = 3, *p ≤ 0.05 **p ≤ 0.01 by two sample two sided t-test).