FIGURE 10.

KLA-induced autophagy is dependent on de novo sphingolipid biosynthesis. RAW264.7 cells stably expressing GFP-LC3 were incubated for 24 h with vehicle control (PBS), KLA (100 ng/ml), ISP1 (1 μm), or KLA + ISP1. For cells treated with KLA + ISP1, ISP1 was added 1 h prior to the addition of KLA. A, representative images. B, number of cells displaying GFP-LC3 puncta (upper panel) and the number of GFP-LC3 puncta/cell were quantified using ImageJ. For each experimental condition, a minimum of 195 cells/experiment were counted. Data represent mean ± S.E. (n = 4). *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001.