Fig. 4: The effect of NAF1 on ribosome biosynthesis in glioma cells.

a The electron tomography of the ribosomes. The nucleus is shown in the left panels (×8000), and the ribosomes are shown in the middle panels (×100,000). The magnifications of the area indicated by the black squares (right images). The red dotted circle indicates polyribosomes, while the black dotted circle indicates a single ribosome. b, c The expression of U17 H/ACA small nucleolar RNA (snoRNA) and 5′ETS-18S rRNA in SF295 and U87 cells were analyzed by the qRT-PCR assay. U6 H/ACA snoRNA and β-actin were used as endogenous controls. **P < 0.01; ***P < 0.001 (n = 3). d, e Sucrose density gradient analysis of 40S subunits in SF295 cells. The matched curves of the absorbance of different components of the ribosomes (left panels). The quantitative of abundance of 40S subunits (right panels). ***P < 0.001 (n = 3). f Total proteins extracted from the same amount of cells were visualized in SF295 cells by the PAGE-silver nitrate staining assay (left panel). The right panels are the quantification of the total density of proteins. *P < 0.05; **P < 0.01 (n = 3). g, h The synthesis ability of GFP proteins was evaluated in SF295 cells by western blot analysis. GAPDH was used as an endogenous control. Shown in the right panels are the quantitative illustration of expression levels of GFP proteins. **P < 0.01; ***P < 0.001 (n = 3). i Measurement of protein synthesis in SF295 cells. Images were obtained using a confocal microscope (left panels). Green color represents staining of newly synthesized proteins, and the nuclei were stained by Hoechst 33342 into blue. Scale bars, 200 μm. The right panels are the fluorescence quantification of Alexa Fluor 488 relative to DAPI in cells using ImageJ. Data are shown as mean ± SD. **P < 0.01; ***P < 0.001 (n = 5)