Knockdown of endogenous Nischarin expression promotes neurite outgrowth in Neuro-2a cells.

<p>Neuro-2a cells were transfected with Nis-shRNA1-4 or control-shRNA. Nischarin expression was determined by real time quantitative RT-PCR <b>(A)</b> and western blot assay <b>(B)</b>. Expression of the Nischarin gene and protein after transfection with Nis-shRNA1-4 decreased to some degree. The knockdown efficiency of Nis-shRNA-3 could reach up to ~60% inhibition. <b>(C)</b> The Nischarin signal came back to ~90% level when loading double amount of the lysates. Asterisks indicate a significant difference compared with control-shRNA (*<i>p</i><0.05, n = 3). <b>(D)</b> Representative images of Neuro-2a cells 48 h after transfection with Nis-shRNA-2, Nis-shRNA-3 or control-shRNA. Cells were differentiated with 20 μM retinoic acid in DMEM for 24 h. For the analysis of the neurite length, pictures were taken with an inverted microscope from five fields of view per well. The neurite length for each individual cell was determined by manual tracing and measured using NIH ImageJ software. Neurites were defined as a process with lengths equivalent to one diameters of a cell body. The percentage of neurite-bearing cells was calculated from the total number of counted cells (n = 3, ~1200 cells measured). Scale bar: 20 μm. Suppressing expression of Nischarin increased the number of neurite-bearing cells <b>(E)</b>, the mean length of the longest neurite <b>(F)</b>, and the total neurite length <b>(G)</b>. Asterisks indicate significant differences from the control shRNA cells (*<i>p</i><0.05). Data presented are the mean ± SEM.