Figure 6.

Fyn distribution and Fyn-dependent process outgrowth are dependent on one-carbon metabolism in N2a cells.A, immunoblot analysis of Fyn and pY416-SFK (pSFK) in total lysates (total) and detergent-insoluble (insoluble) fractions from N2a cells that were incubated for ∼16 h with 100-μM SAM, 100-μM Hcy or vehicle (control). B, representative immunoblots of Fyn and pY416–SFK (pSFK) in total lysates and detergent-insoluble fractions from N2a cells that were incubated for ∼16 h with 50-μM 3-deazaadenosine (3-DZA), 100-μM SAH, or vehicle (control). A subset of cells was incubated for 4 h in a folate-deficient (FD) medium. Quantitative analyses of the immunoblots from n = 3 to 4 separate experiments revealed that incubation with either SAM, Hcy, SAH, 3-DZA, or FD did not induce any statistically significant changes (p > 0.05) in total Fyn protein expression levels, or total or detergent-insoluble Fyn phosphorylation levels, relative to vehicle-treated N2a cells. C, detergent-insoluble Fyn levels were quantified in these cells. Data shown are mean ± SEM from n = 3 to 4 independent experiments and were analyzed using one-way ANOVA (F (5, 17) = 69.37, p < 0.0001) with post hoc Dunnett’s test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001, versus control. D, time-dependent changes in detergent-insoluble levels of Fyn, methylated PP2Ac, and LCMT1 enzymes in N2a cells switched for the indicated time from normal folate-containing medium to FD medium. E, immunoblot analysis of detergent-insoluble Fyn levels in control or WT-expressing N2a cells incubated for 16 h with 100-μM SAM, 100-μM SAH, or a combination of 100-μM SAM and 5-nM okadaic acid (OA). F, immunoblot analysis of detergent-insoluble Fyn and actin levels in L309Δ- and PME1-expressing N2a cells incubated for ∼16 h with 100-μM SAM or vehicle. For panels D–F, similar results were observed in three separate experiments. G, representative confocal images of GFP–Fyn in transfected N2a cells that were incubated for ∼18 h in a low-serum medium in the presence of 100-μM SAM, 50-μM 3-DZA, or vehicle (control) before fixation. Scale bars, 10 μm. H, N2a cells transfected with either EV or GFP–Fyn were incubated for ∼18 h in the differentiation medium in the absence or presence of 5-μM PP2 and labeled with anti-βIII-tubulin antibodies. Scale bars, 10 μm. I, quantification of the neurite length in these cells. Data shown are mean ± SD from cells from 3 separate experiments and were analyzed with two-way ANOVA (effect of SAM: F = 1152, p < 0.0001; effect of Fyn–PP2: F =1600, p < 0.0001; interaction: F = 342.1, p < 0.0001) with post hoc Tukey’s test. ∗∗∗∗p < 0.0001, versus control + EV or control + Fyn; ^####p < 0.0001. EV, empty vector; FD, folate-deficient; Hcy, homocysteine; LCMT1, leucine carboxyl methyltransferase 1; N2a, Neuro-2a; PP2Ac, catalytic “C” subunit of PP2A.