This image is one of a group of 4 images that supports the conclusion that hRio2 kinase activity is required for cytoplasmic 18S-E pre-rRNA processing. This image shows the distribution of all 18S rRNA precursors (white), via fluorescence in situ hybridization (FISH) with a 5' ITS1 probe, upon depletion of a protein important in rRNA processing, hRio2, and subsequent attempted rescue by transfection with an EGFP-tagged kinase-dead version of hRio2, in Hela cells. Depletion of hRio2 leads to the accumulation of 18S-E pre-rRNA in the cytoplasm (white), and this is rescued only by WT hRio2 (a companion image in this group), but not by hRio2 protein that lacks kinase activity, seen here in green. Note that the 5' ITS1 probe shows the localization of all 18S rRNA precursors, but only 18S-E pre rRNA is detected in the cytoplasm. HeLaK cells were transfected with Rio2-d siRNA and then the rescue construct was transfected after 48 h of RNAi. Cells were fixed after 72 h of RNAi, followed by FISH analysis. In the set of four images in this group, FISH pictures were processed in parallel by enhancing levels, followed by setting gamma correction to 0.75.