Fig. 9. Loss of RSL1D1 expression decreases the compartmental size and protein amount of nucleostemin and B23 in the nucleolus. (A1) The knockdown efficiency of the RSL1D1-specific siRNA duplex (siRSL1D1) was examined at the RNA and protein levels. Compared with samples treated with the control siRNA duplex (siNEG), siRSL1D1 can reduce the endogenous RSL1D1 mRNA by 73% (top panel), and the exogenously expressed Myc-tagged RSL1D1 protein by 43% in HEK293 cells (bottom panel). The siRSL1D1 treatment does not affect the total amount of nucleostemin protein (A2). Tub, ß-tubulin for northern blots (NB), and -tubulin for western blots (WB). The effect of a partial loss of RSL1D1 expression on the nucleolar distribution of nucleostemin was measured in U2OS cells double-labeled with anti-nucleostemin (Ab2438) and anti-B23 antibody. Quantitative analyses show that a partial knockdown of RSL1D1 expression decreased the total nucleolar area (No) occupied by nucleostemin (B1) (P<0.001, n=130). A similar effect was seen in the B23-containing regions (B2). On the y-axis the percentage of cells at or below the size indicated on the x-axis is shown. On the x-axis the nucleoluar area in units of 100 pixels (equals 0.89 µm²) is shown. (C) Immunofluorescence images representative of the average of each group are shown. Dashed lines delineate the nucleo-cytoplasmic boundaries. Bars, 5 µm.