Nucleic Acids Research, 2011, Vol. 39, No. 1 287 Figure 5. Localization of pre-rRNAs by FISH in bystin- or hTsr1-depleted cells. (A) HeLa cells transfected for 48 h with bystin, tsr1 or rps15 siRNAs were ﬁxed, hybridized with the 50ITS1 probe labeled with Cy3 and counterstained with Hoechst 33342. Precursors to the 18S rRNA accumulate in the nucleolus upon bystin depletion, whereas they are found in the whole nucleus in hTsr1 and RPS15 depleted cells. Bar = 10 mm. (B) Nucleoplasmic accumulation of pre-40S particles after hTsr1 and RPS15 knockdown was measured on a minimum of 30 cells in each condition. Intensity of the FISH labeling (average gray value) was quantiﬁed in eight regions of interest, equally dispatched upon the nucleoplasm and the cytoplasm. The graph on the right shows an increase of the labeling ratio between nucleoplasm and cytoplasm in hTsr1 and RPS15 depleted cells. In bystin-depleted cells, the presence of bright nucleoplasmic dots made it difﬁcult to reliably quantify ﬂuorescence in the nucleoplasm. ***P < 0.001. (C) HeLa cells transfected for 48 h with bystin, tsr1 or rps15 siRNAs were hybridized with the 5.8S-ITS2 probe labeled with Cy3, the ETS1b or the ETS1-1399 probe labeled with Cy5. The schematic shows position of the sequences complementary to the probes in the pre-rRNA. Bar = 10 mm.