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Fig 3. Netrin-1 dynamically regulates NFPC in retinal growth cones.
(A-D) Cultured stage 24 retinal neurites were stimulated with Netrin-1 or a vector-only control for 0 (A), 10 (B), 30 (C) or 60 (D) min, and then assayed for NFPC expression via immunofluorescence labelling. Panels E-H reveal the quantification of immunofluorescence as an indicator of total NFPC levels within the growth cone (open bars–vector only control; black bars–Netrin-1 treatment). In each case, data were normalized to the 0 min control treatment. (E) Netrin-1 induced a significant decrease in the levels of NFPC within the growth cone after 10 min. By 30 min, however, NFPC localized to the growth cone had returned to levels comparable to that in the control. (F) The decrease in NFPC immunoreactivity localized to the growth cone after 10 min was abolished when explants were pre-treated with the proteasomal inhibitors lactacystin (Lacta) or LLnL. (G) The decrease in NFPC localized to the growth cone after 10 min was also abolished when the explants were treated with the endocytosis inhibitors phenylarsine oxide (PAO) or monodansylcadaverine (MDC). (H) Blocking protein translation with anisomycin (Aniso) in isolated retinal neurites suppressed the recovery in growth cone NFPC levels seen after 30 min of Netrin-1 exposure. However, inhibition of transcription with α-amanatin (α-aman) did not prevent the recovery of growth cone NFPC levels. *** p < 0.001, Kruskal Wallis test. Numbers within the bars indicate the number of growth cones assayed. Scale bar in A: 5 μm.
https://doi.org/10.1371/journal.pone.0141290.g003 |
