Fig. 6

Depletion of USP3 promotes RA-induced neuronal differentiation in neuroblastoma cells. (A-B) Box plot showing the difference between (A) USP3 and (B) REST mRNA expression levels in undifferentiated and differentiated neuroblastoma tissues in the TARGET neuroblastoma dataset. Data are presented as the mean and standard deviation. Student’s t-test was used, and P values are indicated. (C) The effect of RA-induced differentiation on USP3 and REST protein expression. The neuroblastoma cell lines were treated with 10 µM all trans-RA to undergo differentiation for the indicated time. The cells were harvested at a given time point and the effect of RA during neuroblastoma differentiation on USP3, REST, and differentiation marker proteins were assessed by western blot analysis. GAPDH was used as an internal loading control. (D) The effect of RA-induced differentiation on REST ubiquitination. The neuroblastoma cells were induced to differentiate by treating with 10 µM all trans- RA for 5 days, and then treated with MG132 for 6 h before harvest. Cell lysates were immunoprecipitated with anti-REST antibody and immunoblotting with an anti-ubiquitin antibody. (E-F) The morphology of the cells was examined under (E) a bright field microscope and (F) phalloidin staining showing depletion of USP3 promotes neurite outgrowth during RA-induced differentiation in neuroblastoma. The SH-SY5Y cells were either transfected with scrambled shRNA (Mock) or USP3 shRNA and treated with 10 µM all trans- RA for indicated days. The red arrow indicates neurite extension indicating differentiation. (G) The effect of RA-induced differentiation on neuroblastoma cells were evaluated by immunofluorescence analysis using indicated antibodies. (H) The immunofluorescence staining data from (G) was quantified. The number of TUJ1 and NESTIN positive cells were quantified in USP3-depleted cells through immunofluorescence analysis. Data are presented as the mean and standard deviation of three independent experiments. Student’s t-test was used, and P values are indicated. (I) The effect of USP3 depletion on mRNA expression levels of REST, USP3, TUJ1, and NESTIN during RA-induced differentiation of neuroblastoma cells were measured by quantitative real-time PCR. Data are presented as the mean and standard deviation of three independent experiments (n = 3). One-way ANOVA followed by Tukey’s post hoc test was used, and P values are indicated