MIDN is necessary for NGF-induced neurite outgrowth in PC12 cells. (a) MIDN-positive (+) and -negative (−) PC12 cells were generated using CRISPR/Cas9 technology, then MIDN protein expression was examined by Western blotting. (b) The viability of the MIDN-positive (+) and -negative (−) PC12 cells was examined using an MTT assay, measuring absorbance at 570 nm (n = 5). (c) The MIDN-positive and -negative PC12 cells were incubated in the presence or absence of 100 ng/ml NGF in serum-free medium for one day. Cells were fixed in 4% paraformaldehyde, and the actin filaments and nuclei were stained with rhodamine-phalloidin (5 U/ml) or Hoechst-33258 (1 μg/ml), respectively. Forty randomly selected areas per well were photographed, then the total neurite length was divided by the number of nuclei to give the neurite length per cell (μm/cell). While NGF significantly promoted neurite outgrowth in MIDN-positive cells compared with drug-free control (*p < 0.05, n = 6), the neurite outgrowth in MIDN-negative cells was attenuated compared with MIDN-positive cells (#
p < 0.05, n = 6).