Figure 3

Uptake and degradation of denatured collagen by rat HSCs as a function of cell culture time and the effect of inhibitors of lysosomal function on these processes. (A) At each day, cells cultured in 6-well plates were washed, pre-incubation for 45 min at 37°C, and then incubated for 2 h at 37°C with 10 μg/ml ¹²⁵I-TC-collagen. After washing, cells were lysed to determine total cell-associated radioactivity (= the sum of acid-soluble and acid-precipitable radioactivity), which was taken as a measure of total uptake of ¹²⁵I-TC-collagen. Acid-soluble radioactivity was taken as a measure of degradation of ¹²⁵I-TC-collagen. (B) The calculated percentage of internalized ¹²⁵I-TC-collagen degraded by cells. Results are shown as means ± SD for triplicate wells and represent one of three similar experiments. Data are normalized for cell number. Cells were counted on the indicated days as described in Materials and methods. (C) Sub-cultured (first passage) HSCs were washed, pre-incubation for 45 min at 37°C, and then incubated at 37°C with ¹²⁵I-TC-collagen for either 30 min in the absence or presence of excess unlabeled denatured collagen (100-fold) or for 2 h in the absence or presence of E64d (100 μM) or concanamycin A (Con A, 1.5 μM). Uptake and degradation were determined as described above. Data represent either average of duplicate determinations (unlabeled collagen) or means ± SD for triplicate determinations (E64d and Con A) and are representative for at least two such experiments performed.