Inhibition of neuronal differentiation of P19 cells by Rbfox3 knockdown. (A–L) Decreased axonal outgrowth in Rbfox3 knockdown cells. P19 cells expressing shRNAs indicated at the top were treated with retinoic acid. Phase-contrast images and immunostaining for proteins indicated on the left are shown. Overlay image includes nuclear staining (DAPI). Arrowheads and an arrow indicate axonal processes and Rbfox3⁺ cells, respectively. Bars: (C) 50 µm; (L) 100 µm. (M) Immunoblot analysis of Rbfox3 and pNF. GAPDH serves as a loading control. (N) Rescuing the effects of Rbfox3 shRNA by exogenous expression of Rbfox3 containing silent mutations. The expression constructs for myc-smRbfox3-L and -S contain silent mutations in the region targeted by shRbfox3-T2. Immunoblots detecting proteins indicated at the right are shown.