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Figure 1


Fig. 1. RANBP1 downregulation in human U20S cells. (A) Western blotting of the indicated proteins in asynchronously cycling U2OS cultures interfered with RANBP1-specific 202 (+) or GL2 (–) siRNA. OF, oligofectamine only; NT, non-transfected cells; RanBP1(i), RANBP1-interfered. (B) Representative RANBP1(i) cells (lower panels) with virtually no RANBP1 signal (green) and elevated RAN-GTP content (red). Bars, 10 µm. The graph (right) shows the distribution of RAN-GTP signal intensity in RANBP1(i) and in GL2-interfered cells. A total of 30 cells per group were randomly selected; the signal was quantified by densitometry and normalized to the cell area; cells were grouped in classes of fluorescence intensity expressed in arbitrary units (a.u.); mean values in the RANBP1(i) (red arrow) and GL2 control (blue arrow) groups are shown. (C) Representative fields from U2OS cultures (40x objective) show morphological changes in cell shape in RANBP1(i) compared with GL2-interfered cultures. Bar, 20 µm. (D) The MI is consistently lower in RANBP1(i) cultures compared with samples incubated with non-specific siRNAs. P values indicate statistically significant (*) or highly significant (**) differences between control versus RANBP1(i) cultures using the {chi}2 test. (E) FACS analysis of apoptosis in RANBP1(i) and control cultures after 64 hours of RNAi: RANBP1(i) cells accumulate in the <2C region after propidium iodide (PI) staining (upper panels) and show an increased fluorescence intensity after incubation with annexin V (lower panels; viable and apoptotic cells are distributed, respectively, before and after the threshold value of 10 in the logarithmic scale). (F) A representative apoptotic cell from RANBP1(i) cultures (bottom panel); compare to the control cell in the top panel. The graph shows that the percentage of apoptotic cells increases over time in RANBP1(i) cultures and is consistently higher compared with samples incubated with non-specific siRNAs at all time-points studied. P values indicate statistically significant (*) or highly significant (**) differences between control versus RANBP1(i) cultures using the {chi}2 test. Quantitative results in D and F were obtained from three experiments using two RANBP1-specific (202 and 459) and two control (GL2 and 116) siRNAs. A total of 1000-1500 cells were scored for each time-point.