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. 2005 Jan;25(2):751–766. doi: 10.1128/MCB.25.2.751-766.2005

FIG. 2.

FIG. 2.

Targeteddown-regulation of Dnmt3b inhibits PC12 cell differentiation. (A) Selective down-regulation of Dnmt3b with 3bsi. Total RNA was isolated from PC12 cells transfected with scrambled siRNA (scramble) (40 nM) or 3bsi (20 to 60 nM) for 48 h, converted to cDNA (equivalent to 300 ng of RNA from each sample), and subjected to RT-PCR analysis with primers specific for Dnmt3b, Dnmt1, Dnmt3a, and Cox-1 cDNAs. (B and C) Inhibition of NGF-induced neurite outgrowth in PC12 cells treated with Dnmt3b siRNA. PC12 cells transfected with 3bsi RNA (40 nM) or scrambled siRNA (scramble) (40 nM) were treated with (+) 50 ng of NGF/ml for up to 6 days. The cells were viewed under a phase-contrast microscope, and images were captured with a digital camera. (B) Quantification of differentiating cells to assess the percentage of cells generating neurites at least two cell bodies in length. Approximately 200 cells were counted for each sample. The error bars represent the standard deviation of triplicate results. +, present; −, absent. (C) Microscopy image of cells transfected with either scrambled RNA or 3bsi. Bar, 250 μm. (D) Specific reduction of Dnmt3b level in stable cell lines expressing antisense Dnmt3b, as shown by immunoblot analysis. Total proteins isolated from two cell lines expressing antisense Dnmt3b (#21 and #28), untransfected PC12 cells (P), and pcDNA3.1 vector-transfected cells (V) were subjected to Western blot analysis using antibodies against Dnmt1, Dnmt3a, Dnmt3b, and Ku-70 autoantigen (N3H10). (E and F) Suppression of NGF-induced differentiation in stable cell lines expressing antisense Dnmt3b. Parental (P) and vector-transfected (V) cells and clones (#28 and #21) were allowed to differentiate in the presence of 50 ng of NGF/ml for 6 days. (E) Quantitative analysis of the percentage of cells bearing neurites. This figure represents the number of cells with neurites at least two cell bodies in length. The error bars represent the standard deviations of triplicate results. Approximately 200 cells were counted in each sample. (F) Representative microscopy image of control and Dnmt3b antisense cell lines. Bar, 50 μm.