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. 2014 Mar 25;9(3):e92917. doi: 10.1371/journal.pone.0092917

Figure 4. Light-induced Raf/MEK/ERK activation leads to significant neurite outgrowth in PC12 cells.

Figure 4

(A) Three days of light stimulation induced significant neurite outgrowth in cells co-transfected with CIBN-GFP-CaaX and CRY2PHR-mCherry-Raf1 (red arrow head). Non-transfected cells in the same culture showed no marked neurite outgrowth (black arrow head). (B) When kept in the dark, cells co-transfected with both plasmids failed to grow long neurites after the same period of time, which is similar to that of non-transfected cells in the same culture (blue arrow head). (C) Quantitative analysis of neurite outgrowth in PC12 cells under various conditions. Under continuous light stimulation at 0.08 mW/cm2 (gray bars), co-transfected cells grew neurites significantly longer than those of singly transfected cells, non-transfected cells, or co-transfected cells in the dark. Removal of the Raf1 domain from CRY2PHR-mCherry-Raf1 abrogated the ability of co-transfected cells to grow out long neurites. ***p<0.001. The number of cells in each condition is n = 51 (light+co-transfection), n = 45 (dark+co-transfection), n = 23 (light+CRY2PHR-mCh-Raf1 single transfection), n = 18 (dark+CRY2PHR-mCh-Raf1 single transfection), n = 42 (light+CIBN-GFP-CaaX single transfection), n = 37 (dark+CIBN-GFP-CaaX single transfection), n = 30 (light+no transfection), n = 30 (dark+no transfection), n = 23 (light+co-transfection(ΔRaf1)), n = 23 (dark+co-transfection ΔRaf1). Data are presented with mean ± standard error (s.e.m.). Each condition was repeated for two sets of independent experiments. (D) Light-induced neurite outgrowth was blocked by U0126, a MEK inhibitor, but not by K252A, a TrkA inhibitor. (E) Quantifications of NGF and light induced neurite outgrowth with or without inhibitors. ***p<0.001. The number of cells for each condition is n = 55 (light), n = 117 (light + K252A), n = 18 (light + U0126), n = 69 (light + dnRaf1), n = 64 (NGF), n = 107 (NGF + K252A), n = 79 (NGF + U0126), n = 144 (No NGF). The bar graph is presented with mean ± standard error (s.e.m.). Each condition was repeated for at least three independent sets of experiments.