Fig. 5: DAP5 regulates axonal outgrowth of hippocampal neurons by enhancing DSCR1.4 expressions.

a DSCR1.4 overexpression results in the increment of phospho-cofilin/cofilin ratio. EGFP Mock or EGFP DSCR1.4 plasmids were transfected into N2A cells. Phospho-cofilin and cofilin levels were detected by Western blot. DSCR1.4 overexpression was confirmed by anti-EGFP antibody. b Inhibition of DSCR1.4 expression reduces phospho-cofilin/cofilin ratio. siCon or siDSCR1.4 were transfected into N2A cells. Phospho-cofilin and cofilin levels were detected by Western blot. Anti-DSCR1 antibody was used to confirm DSCR1.4 knockdown.c Increased DAP5 expression raises phospho-cofilin/cofilin ratio by accelerating DSCR1.4 mRNA translation. N2A cells were transfected with Flag Mock or Flag DAP5 vectors and incubated for 24 h. DAP5 overexpression was confirmed by Western blot using anti-Flag antibody. d Phospho-cofilin/cofilin ratio was reduced by suppressed DAP5 expression and the reduction was recovered by DSCR1.4 overexpression. N2A cells were co-transfected with designated siRNA and plasmids and incubated for 24 h. The numbers at the bottom indicate the fold relative to siCon and EGFP Mock co-transfected cells. e, f Increased DSCR1.4 expression facilitates axon outgrowth. EGFP Mock or EGFP DSCR1.4 plasmids were transfected into DIV1 mouse hippocampal neurons. Tau1 was detected to identify axon. e Representative image obtained by confocal microscopy. f The graph shows relative axon length measured by Image J. The bars represent the mean ± SEM (EGFP Mock; n = 25, EGFP DSCR1.4; n = 18). g, h Induction of DAP5 increases axon length. Flag Mock or Flag DAP5 plasmids were transfected into DIV1 mouse hippocampal neurons. g Representative image obtained by confocal microscopy. h The graph shows relative axon length measured by Image J. The bars represent the mean ± SEM (Flag Mock; n = 29, Flag DSCR1.4; n = 25). i Reduced DAP5 expressions inhibit axon outgrowth. shRNAs were transfected into DIV1 hippocampal neurons. EGFP signal was only detected on cells with shRNAs. i Representative image. j The graph shows relative axon length measured by Image J. Axon length of shMock plasmid transfected cells was set as 1. The bars represent the mean ± SEM (shMock; n = 28, shDAP5; n = 26). Scale bar, 20 μm. Data information: In f, h, j, ^****P < 0.0001 (Student’s t test)