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To determine whether the increased capacity to ingest apoptotic and necrotic cells by KIM1-PK1 cells was a function of the neomycin resistance used to maintain the expression of KIM-1, we prepared a KIM1-PK1 cell line that expressed a KIM-1 construct with only the neomycin resistance cassette (KIM1-NR) and untransfected LLC-PK1 cells as a control (Figure 6F). KIM1-NR cells had a 40% increase in necrotic cell ingestion compared with the control cells, as assessed by microscopy (Figure 6G). In addition, intracellular LC3B-II expression was reduced in KIM1-NR cells compared with the control cells, consistent with the reduction in LC3B-II expression in KIM1-PK1 cells (Figure 6H). These findings indicate that the ability of KIM1-PK1 cells to internalize necrotic cells is not dependent upon the neomycin resistance cassette.
In this study, we demonstrate that KIM1-PK1 cells have an increased capacity to internalize apoptotic and necrotic cells. KIM1-PK1 cells exhibit greater levels of KIM1 expression, decreased LC3B-II expression, and increased capacity to ingest cells. The expression of LC3B-II is a hallmark of autophagy, the process of cytoplasmic degradation. This process involves autophagosome formation followed by fusion of the lysosome with the autophagosome and degradation of the contents by lysosomal enzymes (reviewed in [8]). In normal conditions, the cellular level of LC3B-II is relatively constant. However, autophagy is upregulated during starvation and a variety of stresses to provide a temporary supply of nutrients to the cell. LC3B-II is phosphatidylethanolamine conjugated to LC3B-I, and the level of LC3B-II can be used as a marker of autophagy (reviewed in [9]). The decrease in LC3B-II expression in KIM1-PK1 cells is consistent with a model where the increased internalization of apoptotic and necrotic cells is mediated by autophagy. Thus, KIM1-PK1 cells may exert a protective effect against cell death by undergoing autophagy.
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