Figure 1.
NSE is down-regulated and tPA is up-regulated during and following TTX application.
(a–c) Real-time PCR analysis showing the change in the mean mRNA expression level of the neuronal marker NSE, TNF and tPA after short (4D) and long (14D) term TTX treatment and following 4 days of TTX treatment after which it was washed out and the cells were left to grow in regular medium for 10 more days (wash condition), relative to control (means±SEM; * = P<0.05; *** = P<0.001). (d) Immunoflorescence images of control, long term TTX-treated and TTX-washed cultures, stained for the neuronal marker NeuN (green - left panel, bar = 50 µm) or double-labeled for NeuN and for tPA (red - right panel, bar = 20 µm), showing the elevation in tPA protein levels among the treated neurons and its cellular localization.
Figure 2.
The effect of TTX and conditioned medium on the cell survival and tPA expression level in neuronal cultures.
(a) Mean cell survival rates of primary neuronal cultures following the application of 3 days TTX-conditioned medium or fresh TTX for 7 days. (b) Cell survival rates of primary neuronal cultures following the application of conditioned medium of cells that were treated for 4 days with TTX (‘wash conditioning’) and left for 3 more days in fresh medium, with or without the addition of fresh TTX, for 7 days (c) The change in tPA mRNA expression level in primary neuronal cultures following a week of incubation in TTX- or wash-conditioned medium (means±SEM; * P<0.05).
Figure 3.
Intrinsic blockade of tPA protects neurons from TTX-mediated neuronal cell death.
(a) Representative images of control and TTX-treated neuronal cultures stained for the neuronal marker NeuN (red), and for the tPA inhibitor PAI-1 (green), transfected with either GFP, PAI-1 or non-transfected controls (bar = 100 µm). (b) Control and TTX-treated neurons were stained for NeuN and for PAI-1. Cell survival rates of the NeuN-positive cells and of the PAI-1-positive cells out of the NeuN-positive population were estimated. (c) Cell survival rates of double-labeled cells of the three groups - Non-transfected, GFP and PAI-1, demonstrating the protection exerted by the over-expression of PAI-1.