FIG. 6.

The N-terminal domain of Dnmt3b restores NGF-induced PC12 cell differentiation. (A) Schematic representation of the different mutants of Dnmt3b used in the present study and Western blot analysis for analyzing their expression in PC12 cells. Anti-Myc antibody (α-myc) was used to detect Myc-tagged wild-type (WT) and catalytic-domain mutant Dnmt3b (C6547S), and anti-Flag antibody (α-flag) was used to show the overexpression of ΔATRX, ΔC, and ΔNDnmt3b. Corresponding vectors were used as negative controls. The expected peptides are indicated (arrows). aa, amino acids. (B) Cotransfection of wild-type Dnmt3b or different mutant constructs, along with 3bsi. Cells were transfected with different DNA constructs (as indicated) with Lipofectamine reagent. After 24 h, the cells were treated with NGF and allowed to grow for an additional 6 days with two intermediate cotransfections every 72 h. Bar, 100 μm. (C) Ability of the wild type or different mutants of Dnmt3b to recover NGF-induced neurite outgrowth. The cells bearing neurites at least two cell bodies in length were counted. About 200 cells were counted in each sample. The error bars represent standard deviations of triplicate experiments (t test; P < 0.01). Con, control; CS, C657S. (D) Overexpression of N-terminal domain of Dnmt3b facilitates differentiation. (D-1) Quantitative analysis of cells generating neurites at least two cell bodies in length (t test; P < 0.01). d, day(s). (D-2) Microscopy images of vector-transfected and ΔCDnmt3b-transfected cells. Bar, 250 μm.