Figure 4
From: Novel ubiquitin-independent nucleolar c-Myc degradation pathway mediated by antizyme 2
Knockdown of AZ2 increases nucleolar localization of c-Myc and stimulates pre rRNA expression. (a) Panc-1 cells were treated with either control, AZ2 or NPM1 siRNA and collected after 48 h. Endogenous c-Myc levels in the cell extracts were examined by immunoblotting using anti-c-Myc antibody. (b) Quantitative analysis of data in (a). c-Myc/β-actin ratio calculated from three independent experiments. (c) Panc-1 cells were treated with indicated siRNA for 48 h and then with MG132 for 60 min. Cells were fixed and endogenous c-Myc was visualized using anti-c-Myc antibody and secondary antibody conjugated with AlexaFluor 488. The brief treatment with MG132 was necessary to visualize nucleolar c-Myc. Distribution of c-Myc was displayed as monochrome images. Nuclei was stained with Hoechst 33342 and colored in cyan. Phase-contrast images were shown to confirm the location of nucleoli. Three independent experiments were performed. Scale bars, 20 μm. (d) Percentages of the cells in which nucleolar localization of c-Myc were observed. Three hundred cells were scored from random fields for each siRNA. (e) Panc-1 cells were treated with either control or AZ2 or NPM1 siRNA for 48 h and total RNA was prepared from the cells. qRT-PCR was performed using two probes of 47S pre-rRNA (ETS-1 and ETS-2) as described in Methods. Relative pre-rRNA expressions were represented with bar graph Data in b, d and e represent the mean ± SD calculated from three independent experiments. *P < 0.05, **P < 0.01 versus the control (t test).
