Fig. 4.
hNGF exerts neuroprotective effects in PC12 cells in vitro. (a) PC12 cells express the NGF receptors TrkA and p75NTR (green). DAPI identifies nuclei (blue). Scale bar, 50 μm. (b) Schematic showing the experimental model. Rat fibroblasts were transduced with Ad.hNGF or Ad.βGal; non-transduced cells served as the control. Conditioned medium from fibroblasts was collected after 48 h and used to mimic the paracrine action of circulating NGF on target PC12 cells in a basal- or high-glucose environment, or with mannitol as osmotic control. Endpoints of the experiments were NGF intracellular signalling, apoptosis assay and neural differentiation. (c) V5-tagged hNGF was detected by western blotting in fibroblast total cellular extracts and conditioned medium. Only the mature NGF was detected, indicating that preproNGF was successfully cleaved to the mature form. (d) ELISA detected 540 pg/ml mature NGF in the fibroblast-conditioned medium used for the experiments. No proNGF was detected. (e) Blots for phospho-proteins and corresponding non-phosphorylated forms associated with pro-survival TrkA and pro-apoptotic p75NTR signalling. (f–k) Graphs showing blot densitometry (n = 1). In (g–k), values are expressed as fold change of the basal glucose non-virus-conditioned medium. (l) Bar graph showing caspase 3/7 activity in PC12 exposed to either basal or high glucose or mannitol, for 48 h, in the presence of fibroblast-conditioned medium. Caspase activity, measured as relative luminescence units, is expressed as fold change of the basal glucose non-virus-conditioned-medium group (n = 4 per group). Data are expressed as means ± SEM. *p < 0.05 and **p < 0.01 between groups, as indicated. BG, basal glucose; CM, conditioned medium; Ctrl, control; E, total cellular extract; HG, high glucose; n.d., not detected; NV, non-virus; RLU, relative luminescence units