Figure 3.

Endogenous XIAP is essential for FAIM-L to protect Type II cells. A, PC12 cells were transduced with Scrambled shRNA (Scr), shRNA XIAP R1, or shRNA XIAP R2 lentiviral particles for 3 d and then transfected with pcDNA3, 3xHA-Bcl-xL, 6xMyc-XIAP, or 3xFLAG-FAIM-L expression plasmids and treated with sFasL (100 ng/ml) and AD (1 nm) for 24 h. Percentage of apoptosis was assessed and Student test was performed, comparing Bcl-xL, XIAP, and FAIM-L-overexpressing cells treated with sFasL+AD with their control (pcDNA3)-treated counterpart. *p ≤ 0.01. B, PC12 cells were transduced as in A with Scrambled shRNA (Scr) or shRNA FAIM-L lentiviral particles and then transfected with pcDNA3, 3xHA-Bcl-xL, or 6xMyc-XIAP and treated as in A. Conditions of Scrambled-transduced cells are identical in A and B. Percentage of apoptosis was assessed and Student test was performed, comparing Bcl-xL, XIAP, and FAIM-L-overexpressing cells treated with sFasL+AD with their control (pcDNA3)-treated counterpart. **p ≤ 0.001. *p ≤ 0.01. C, RT-PCR using rat XIAP or L27 primers was performed to control XIAP knock-down efficiency. D, Immunoblot analysis followed by Naphtol Blue staining of the PVDF membrane confirmed FAIM-L knockdown efficiency. E, Cortical neurons were transduced with Scrambled shRNA (Scr) or shRNA XIAP R1 lentiviral particles for 3 d and then transduced with empty, Bcl-xL, or FAIM-L overexpression lentiviral particles for 3 additional days. Primary neurons were treated with sFasL (100 ng/ml) and cycloheximide (CHX; 1 μg/ml) for 24 h. Percentage of apoptosis was assessed and Student test was performed, comparing Bcl-xL and FAIM-L-overexpressing neurons treated with sFasL+CHX with their control (Empty)-treated counterpart. *p ≤ 0.01. All conditions represent the mean of three independent experiments.