Figure 7.

NGF depletion activates KIF1Bβ and YME1L1 in PC12 cells leading to apoptosis. A, NGF depletion results in apoptotic cell death in PC12 cells. PC12 cells were treated with NGF for 6 days and then were incubated with or without NGF for additional 2 days. Cells were subjected to FACS or immunoblotting analyses. B, NGF depletion promotes mitochondrial fragmentation in PC12 cells. Mitochondrial morphology in the cells in (A) was observed by indirect immunofluorescence utilizing BacMam‐GFP (green). The mitochondrial length was measured, and average values are shown (n = 100). Membrane potential was quantified by the JC‐1 red fluorescence measurement assay and was shown as the normalized values by the value obtained from NGF‐treated cells. *P < 0.05 (n = 3). C, NGF depletion activates KIF1Bβ and YME1L1 in PC12 cells. At indicated time points, cells were collected and subjected for immunoblotting. Endogenous rat YME1L1 is indicated by the arrowhead. D, Knockdown of either KIF1Bβ or YME1L1 prevents apoptosis mediated by NGF depletion. At 6 days after incubation with NGF, NGF was depleted from culture medium. Simultaneously, the knockdown of KIF1Bβ or YME1L1 utilizing independent siRNAs for each gene was performed for 2 days. Cells were used for FACS or immunoblotting analyses. C, cytoplasm; FACS, fluorescence‐activated cell sorting; M, mitochondria; siRNA, small interfering RNA [Color figure can be viewed at wileyonlinelibrary.com]