Fig. 2

Combinatorial screen of 9 transcription factors for neurite growth promotion and synergy in F11 cells. F11 cells were transfected with dual promoter plasmids expressing GFP and a TF or the control vector expressing GFP only (No-TF), singly or in combinations. After 24, 48 and 72 h cells were fixed and automated microscopy and neurite tracing (Cellomics) was carried out on GFP-positive neurites and the total neurite length in µm calculated. Measurements at 72 h were compared using a linear mixed model with plate as a random effect and Dunnett’s post-hoc test. Shown are screens with 1, 2, 3 and 4 factors. Each time the number of factors was increased by one, combinations were compared with the best condition with one less factor. Control groups for each comparison are shown as the first condition, indicated by the letter C and grey background shading. The grid indicates which factors were present in each condition. (A) Screen of individual TFs and all pairs of TFs expressed together. Of the single TFs, KLF7 overexpression significantly increased total neurite length in comparison with GFP while CEBPD and STAT3 significantly reduced total neurite length. Among the co-expressed pairs, KLF7/MEF2 overexpression significantly increased total neurite length in comparison with KLF7. (B) Screen of selected combinations of 3 TFs and 4 TFs expressed together. ATF3/KLF7/MEF2 overexpression significantly increased total neurite length in comparison with KLF7/MEF2. No combinations of 4 factors are better than ATF3/KLF7/MEF2 while ATF3/JUN/KLF7/MEF2 and ATF3/KLF7/MEF2/SRF overexpression significantly decreased total neurite length in comparison with ATF3/KLF7/MEF2. (C) Maximum outgrowth achieved for each number of TFs in combination. Outgrowth begins to plateau with 3 TFs and starts to decline when more than 6 TFs are used. (D) Synergistic power of each TF. Each data point shows the synergistic additional neurite outgrowth (i.e. the growth increase beyond simple additive effects) at 72 h obtained by adding each individual TF to all other combinations that do not include it. All combinations from A-D and Supp. Fig. S2 were included. Horizontal bars indicated the mean. MEF2 and KLF7 show the greatest mean synergistic effects at 72 h. Key: *,# p < 0.05; **, ## p < 0.01; *** p < 0.001 (linear mixed model, Dunnett’s post-hoc test; n ≥ 6). Stars indicate increased outgrowth compared with the control condition, hashes indicate decreased outgrowth compared with controls