FIGURE 11.

KLA promotes the co-localization of ceramide with the autophagosome. 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, following treatment, the extra- and intracellular Cer was stained for all conditions. Representative images. B, number of cells displaying GFP-LC3 puncta co-localized with ceramide (upper panel) was quantified using ImageJ. For each experimental condition, a minimum of 150 cells/experiment were analyzed for ceramide co-localization. Data represent mean ± S.E. (n = 4). Lower panel, number of autophagic cells displaying GFP-LC3 puncta co-localized with Cer was quantified using ImageJ. For each experimental condition, a minimum total of 40 autophagic cells were analyzed for Cer co-localization. Data represent mean ± S.E. (n = 4). *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001. C, RAW264.7 cells stably expressing GFP-LC3 were incubated for 24 h with vehicle control (PBS) or KLA (100 ng/ml). Following treatment, the Golgi complex and endoplasmic reticulum were stained using antibodies against GM130 and BiP, respectively.