Figure 1.

Cbl and Cbl-b regulate SH-SY5Y neuroblastoma cell differentiation by decreasing ERK phosphorylation levels and inhibiting neurite outgrowth

(A–E) SH-SY5Y cells were treated with siRNA (as indicated) for 48-72 hr.

(A) Lysates were subjected to immunoblotting (left panel) with antibodies against phospho-ERK/ERK or Cbl/Cbl-b. Right panel shows quantification relative to siGFP control (n = 4).

(B) Representative images of siGFP and siCbl/Cbl-b-treated cells for neurite outgrowth (indicated by white arrows) visualization (scale bar, 50 μm).

(C) Representative raw and segmented images of Cbl/Cbl-b knockdown SH-SY5Y cells. Segmented images (bottom panel) show neurites (white) and nuclei (cyan). Neurites and nuclei were quantified by “Neurite Outgrowth Quantification” software.

(D) Immunoblotting (left panel) detecting indicated proteins in lysates of SH-SY5Y treated with Cbl/Cbl-b siRNA in conjunction with U0126 and quantification (right panel) relative to siGFP control.

(E) Corresponding quantification of neurite outgrowth.

(F) Graphical model of neuroblastoma differentiation and study design. Data in bar graphs are shown as means ± SEM and in boxplots as median with 95% confidence interval (CI) and representative of n = 3 independent experiments. ∗, ∗∗, ∗∗∗ and ∗∗∗∗ indicate p < 0.05, 0.01, 0.001, and <0.0001, respectively, compared to the corresponding control determined by one-sample t test (A), t test (C), or one-way ANOVA (D and E).