Figure 3. PI3K-III activity is necessary for NR4A1-mediated vesicular cell death.

HEK293 cells were transfected with either the empty vector pcDNA3, NK₁R and exposed or not to SP, or IGF1R as indicated. A, Pharmacological inhibition of PI3K with LY294002 reduces cell death. DMSO was added as vehicle control. B, Specific down regulation by RNAi of PI3K-III inhibits cell death. HEK293 cells were transfected with the indicated siRNAs. Cells death was determined by trypan blue exclusion 24 hr after induction. Bars represent standard error. ***, p<0.001, n = 4. C, Western blot to show the efficiency of RNAi. Luc refer to siRNAs targeting the luciferase gene, as irrelevant siRNA. GAPDH was detected as loading reference. D, induction of NR4A1 expression upon cell death activation is upstream of autophagy activation. HEK293 cells were transfected with NK₁R expression vector and exposed or not SP, and the expression of NR4A1 was monitored by Western blot in the presence or absence of the autophagy inhibitor LY294002, as indicated.