Figure 3.

Bex3 protein levels modulate the survival of NGF-dependent neurons and the differentiation of PC12 cells. A, bex3 mRNA downregulation in DRG neurons by shRNA. Cultured DRG neurons were infected at DIV5 with lentivirus-expressing GFP and control shRNA (c) or GFP and Bex3 shRNA (b). RNA was obtained 5 d later, and semiquantitative RT-PCRs were performed as described in Materials and Methods. Actin was used as a control. Two representative RT-PCR experiments are shown (left). Quantification of bex3 and actin mRNA was performed using ImageJ software (right). The intensity of the DNA bands was quantified and normalized to that of actin in the control (100% vs 33.1 ± 5.9%, control vs Bex3 shRNA, respectively). Data are presented as the mean ± SEM (n = 7). The p value was calculated using a two-tailed Student's t test. B, Bex3 protein downregulation in DRG neurons. Cultured DRG neurons were infected as described in A, and extracts were obtained to analyze Bex3 protein by Western blot. A representative Western blot showing a decrease in Bex3 protein is depicted. Tubulin was used as a loading control. Arrows indicate Bex3 monomer and dimer. C, Bex3 protein levels modulate the survival of NGF-dependent neurons in vitro. Cultured DRG neurons were transfected with plasmids expressing either GFP and control shRNA (c) or GFP and Bex3 shRNA (b). Neurons were deprived of NGF for 48–72 h and stained with Hoechst 33342, indicating which ones were apoptotic (red arrowhead) or non-apoptotic (white arrowhead) within GFP-positive population. Left, Representative images of NGF-dependent DRG neurons transfected with control or Bex3 shRNA plasmids. Scale bar, 10 μm. Right, Quantification of apoptosis in GFP-positive cells. Data are shown as the mean ± SEM. The total number of quantified neurons was 249 and 464 in control and Bex3 shRNA-transfected groups respectively from four independent experiments (16.8 ± 3.1% vs 28.9 ± 2.8%, control vs Bex3 shRNA, respectively; right). The p value was calculated using a two-tailed Student's t test. D, Bex3 downregulation impairs neurite outgrowth of PC12. Cells were infected with lentivirus expressing GFP and control shRNA (c) or GFP and Bex3 shRNA (b) for 5 d, and then stimulated with NGF (100 ng/ml) for 72 h. Scale bar, 10 μm. Neurite outgrowth was quantified by assessing the percentage of GFP-positive cells bearing neurites at least twice the length of the cell body. Data are shown as the mean ± SEM. The total number of cells in the control and Bex3 shRNA-infected groups were respectively 204 and 210 from four independent experiments (32.5 ± 6.5% vs 19.3 ± 6.1%, control vs Bex3 shRNA, respectively). The p value was calculated using a two-tailed Student's t test. E, The downregulation of Bex3 impairs NGF-mediated signaling pathways in DRG neurons. DRG neurons were infected as indicated in A, and 5 d later they were treated with NGF (100 ng/ml) for 1 h. Cell extracts were obtained, and active TrkA, Akt, and MAPK were assessed using antibodies that recognize specific phosphorylated residues. Representative blots are shown (n = 6).