Fig 2: High-content siRNA screening to identify proteins involved in nucleolar formation.(A) Experimental scheme of the siRNA screen. HeLa cells in 96-well plates were transfected with siRNAs for 745 genes and cultured for 48 h. The cells were immunostained with anti-NPM1 antibodies, and their images were captured by high-throughput microscopy. NPM1-labeled nucleoli were automatically recognized, and their signal intensities and areas were measured with the image analysis software. (B) Quantification of the nucleolar morphology (each n > 100 cells). The total intensity and the average area per cell are shown in the upper and lower panels, respectively. The x axis represents each knockdown of the 745 target genes. The 16 knocked down genes that were further studied are shown as red bars in the graphs. The Y axis indicates values relative to the siControl (set to 1.0). (C) Validation of siRNA-mediated knockdowns of the factors in Fig 1A and B. Relative mRNA expression levels in HeLa cells were measured by qRT-PCR at 48 h after the transfection of each siRNA. The values from the control treatment (siControl) were set to one. β-Actin was used as an internal control. Values are the means ± SD. (D) HeLa cells treated with single siRNAs against RPL5 or RPL21 show similar nucleolar morphologies to those of cells treated with the pooled siRNAs, accompanied with efficient depletions of their protein and mRNA. Two separate siRNA sequences (#1, #2) were tested against RPL5 or RPL21. The nucleolar morphologies in HeLa cells treated with single siRNAs (#1 or #2) against the indicated proteins are shown. Scale bar, 10 μm. (upper). RPL5 or RPL21 protein (middle) and mRNA expression (lower) levels are shown following the knockdown of the indicated siRNAs. Histone H3 protein levels (H3) are shown as a loading control.