Figure 5.

CXCL12 induces the expression of a transcriptionally active Rb that regulates its prosurvival effect in neurons. Neurons were transfected with the Rb-TA-Luc plasmid and treated with CXCL12 (20 nM; 24 h post transfection) for the indicated time (a). Data are shown as mean±S.E.M. (*P<0.05 versus control; n = 3). Images in (b) are from neurons transfected with the Rb shRNA vector, psi(Rb), and a GFP expression vector, fixed after 24 h and immunostained for Rb (red); Hoechst 33342 (blue) was used to stain neuronal nuclei. Arrows point to an example of transfected neuron identified by GFP. Immunoblots from neurons transfected with psi(Rb) or a control vector carrying a scrambled sequence are also shown in (b). The western blot shows three separate psi(Rb) transfections in the same gel. The densitometric analysis from five independent experiments is reported in the graph (mean±S.E.M.; *P<0.05 versus scrambled). The graph in (c) shows survival data from cortical neurons transfected with either psi(Rb) or a control vector carrying scrambled sequence (along with a GFP expression vector as transfection marker). After 24 h post transfection, the neurons were treated with NMDA (100 μM) with or without CXCL12 (20 nM); data are expressed as mean±S.E.M. from three independent experiments containing at least three coverslips per experiment (*P<0.01 versus control; only this is shown in graph for clarity purposes). Multiple comparison analyses showed that both scrambled and psi(Rb) NMDA are different than controls (P<0.01); the control scrambled and control psi(Rb) are not different; within the scrambled group, CXCL12 and CLCL12 + NMDA are not different than control, but different than NMDA alone (P<0.01); within the psi(Rb) group the CXCL12 and CXCL12 +NMDA are different than control (P<0.05) and not different than NMDA alone; the scrambled NMDA +CXCL12 is different than the psi(Rb) NMDA + CXCL12 and the scrambled CXCL12 is different than the psi(Rb) CXCL12 (P<0.05). Scale bars =50 μm