Supplementary Figure 6: Reducing YTHDF3 expression in hippocampal neurons causes excessive dendritic filopodia, in place of mature spines, and APC protein expression in YTHDF1-knockdown neurons is reduced.

a. Confocal images of DIV17 dissociated hippocampal neurons expressing shScram-GFP (top) or shYTHDF3-GFP (bottom). DAPI (blue), GFP (green), YTHDF3 (red). Arrowheads point to GFP(+) shScram or shYTHDF3 transfected neurons. Scale bar, 10 μm (n=3 independent experiments). b. Confocal images on dendritic shaft of GFP (green) and YTHDF3 (red). Scale bar, 10 μm (n=3 independent experiments); c. Quantification of YTHDF3 protein expression by immuno-staining in shScram and shYTHDF3 cells (Scram, sh1, and sh2). YTHDF3 protein in sh1 and sh2 samples decreased over a 4-day time course after transfection (n=3 independent experiments). d. Confocal images of dendritic shaft and spines of shScram and shYTHDF3-2 cells (n=3 independent experiments). Left, GFP labels morphology of dendritic shaft and spines; Right, PSD-95 staining in the same sample (magenta). Contour of affected dendrites was traced as the white lines through GFP expression. Fluorescence signals outside of the contour were masked to restrict quantification to the affected neurons. Scale bar, 5 μm (n=3 independent experiments). e. Confocal images of APC immunostaining using APC N-terminus antibody (top) or APC C-terminus antibody (bottom). GFP labels neurons expressing the shRNAs (shScram or shYTHDF1). Both antibodies consistently detected decreased APC protein expression in YTHDF1-KD neurons; Arrowheads point to GFP-positive neurons. DAPI (blue), GFP (green), APC protein (magenta). Scale bar, 10 μm (n=3 independent experiments).