Figure 9.

Degradation of PyrSM in human fibroblasts and in Hrs + Tsg101- or NPC1-depleted HeLa cells. (A) Partial colocalization of Pyr₄SM or Pyr₁₄SM with DiI-LDL chased into late endosomes in human fibroblasts. Cells were first labeled with DiI-LDL for 30 min and then with PyrSM for 5 min and chased for 1 to 1.5 h at 37°C. PyrSM was imaged after TNP-LPE quenching of PM fluorescence at RT. Arrows indicate colocalization of DiI and pyrene fluorescence. (B) Pyr₄SM and Pyr₁₄SM hydrolysis in control and NPA fibroblasts after 1–2 h of chase (n = 10 for control and 5 h for NPA cells). (C) Hydrolysis of Pyr₄SM and Pyr₁₄SM after a 30-min labeling and 2-h chase in HeLa cells transfected with control (GL2), Hrs + Tsg101, or NPC1 RNAi (n = 3–7). Error bars are SEM. ***p < 0.001.