Figure 5

WWOX regulates neuronal differentiation through its interaction with GSK3β. (a) SH-SY5Y cells were transfected with GFP, GFP–GSK3β WT, S9A, K8586MI and R96A for 24 h. The cells were then treated with 10 μM RA for 24 h. Cells with neurites longer than twice the length of the cell body were counted. Data are presented as the mean±S.E. from three independent experiments. ^**P<0.01 compared with the GFP control. (b) Western blot analysis confirmed the expression of the GFP-fusion protein by the anti-GFP antibody. (c) SH-SY5Y cells were transfected with pre-designed siRNA targeting GSK3β for 24 h and treated with 10 μM RA for 1 to 4 days. Cells with neurites longer than twice the length of the cell body were counted. Bright-field results are shown to indicate post-differentiation status at 24 h. Data are presented as the mean±S.E. from three independent experiments. ^*P<0.05 compared with mock control. (d) Cells were treated with siRNA and lysed in 2 × sample buffer. Western blot analysis was carried out using antibodies against GSK3β. The blot was stained for actin to verify equal loading. (e) Tau protein was incubated with GST, GST–WWOX or GST–WWOX L404A in the presence of GSK3β for the microtubule assembly assay. The reaction mixture also contained tubulin protein, GTP, and reaction buffer. The reaction mixture was incubated at 37 °C in a thermostatic spectrophotometer, and the change in reflectance over time was measured at 350 nm. (f) SH-SY5Y cells were transfected with empty vector, GFP–WWOX or GFP–WWOXL404A, as indicated. After transfection, cells were washed, and the medium was replaced with 1% serum and treated with 10 μM RA. The photos were taken on day 1. (g) The percentage of differentiated cells with neurites longer than twice the length of the cell body was calculated. The data are presented as the mean±S.E. from three independent experiments. ^*P<0.05 compared with the mock-treated control