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1.
Figure 4.

Figure 4. From: Mammalian HCA66 protein is required for both ribosome synthesis and centriole duplication.

The homologues of HCA66 in S. cerevisiae and S. pombe cells are not detectable at the SPB. (A) Immunofluorescence microscopy using an S. cerevisiae strain expressing Utp6p-3HA (detected with anti-HA antibodies, red signal) and GFP-tagged Spc97p (green signal). (B) Fluorescence microscopy using an S. pombe strain expressing SpUtp6-mCherry (red signal) and Cdc11-GFP (green signal).

Chrystelle Bonnart, et al. Nucleic Acids Res. 2012 July;40(13):6270-6289.
2.
Figure 2.

Figure 2. From: Mammalian HCA66 protein is required for both ribosome synthesis and centriole duplication.

Depletion of HCA66 in HeLa cells affects early cleavages of the pre-rRNA and the production of the mature 18S rRNA. (A) Western-blot analysis showing the accumulation levels of endogenous HCA66 and actin (loading control) in HeLa cells 48 h after transfection with H2O (mock), scrambled siRNAs (Scramb.) or HCA66 siRNAs (HCA66). (B) Northern-blot analysis showing the accumulation levels of the (pre-)rRNAs in HeLa cells 48 h after transfection with H2O (mock), scrambled siRNAs (Scramb.) or HCA66 siRNAs (HCA66). The probes used to detect the different species (described in Figure 2C and Supplementary Table S1) are indicated below each panel. (C) Outline of the pre-rRNA processing pathway in HeLa cells. Positions of the oligonucleotide probes used in this study (a–c) are indicated and their sequences are described in Supplementary Table S1.

Chrystelle Bonnart, et al. Nucleic Acids Res. 2012 July;40(13):6270-6289.
3.
Figure 3.

Figure 3. From: Mammalian HCA66 protein is required for both ribosome synthesis and centriole duplication.

Depletion of HCA66 in HeLa cells delays the production of the mature 18S rRNA. (A) Western-blot analysis showing the accumulation levels of endogenous HCA66 and actin (loading control) in HeLa cells 48 h after transfection with H2O (−siRNA) or HCA66 siRNAs (+siRNA). (B) Neo-synthesized RNAs from HeLa cells transfected with HCA66 siRNAs (lanes 7–12) or mock-transfected (lanes 1–6) were pulse labeled with l-methyl 3H methionine 48-h post-transfection and cells were harvested after the indicated chase times. Total RNAs extracted from these cells were separated by electrophoresis, transferred to a nylon membrane and labeled RNAs were detected by autoradiography.

Chrystelle Bonnart, et al. Nucleic Acids Res. 2012 July;40(13):6270-6289.
4.
Figure 5.

Figure 5. From: Mammalian HCA66 protein is required for both ribosome synthesis and centriole duplication.

Depletion of Utp6p does not arrest cells in mitosis but induces pre-rRNA processing defects that delay G1 phase progression. (A) Western-blot analysis showing the depletion of 3HA-Utp6p and 3HA-Spc97p in strains GAL::3HA::UTP6 and GAL::3HA::SPC97, respectively, shifted from a galactose- (GAL) to a glucose-based medium (GLU). (B) FACS analysis of cells undergoing 3HA-Utp6p or 3HA-Spc97p depletion, or wild-type cells (BY4741) as a control, showing the distribution of cells in the different cell cycle stages (G1, S and G2/M). (C) Northern-blot analysis of the pre-rRNA processing pathway in wild-type cells (BY4741) or cells undergoing 3HA-Utp6p or 3HA-Spc97p depletion using the indicated oligonucleotide probes (Supplementary Table S1).

Chrystelle Bonnart, et al. Nucleic Acids Res. 2012 July;40(13):6270-6289.
5.
Figure 1.

Figure 1. From: Mammalian HCA66 protein is required for both ribosome synthesis and centriole duplication.

HCA66 is detected both at the centrosome and in the nucleoli in HeLa cells and is required for normal accumulation of 40S ribosomal subunits. (A) Immunofluorescence microscopy using HeLa cells expressing EGFP-HCA66 and antibodies detecting γ-tubulin (upper panel) or fibrillarin (lower panel). Scale bar, 10 µm. (B) Western-blot analysis of endogenous HCA66 distribution in HeLa cell subcellular fractions. The different lanes correspond to the total extract (Tot.) or the cytoplasmic (Cyto.), nuclear (Nuclei), nucleoplasmic (Np.), soluble nucleolar (Sol. No.) or insoluble nucleolar (Insol. No.) fractions. HDAC2 and nucleolin are markers of the nucleoplasmic and nucleolar material, respectively. (C) Sucrose gradient analysis of ribosomal subunit levels in HeLa cells treated with HCA66 siRNAs. Cytoplasmic extracts prepared from HeLa cells treated with HCA66 or scrambled siRNAs were centrifuged trough 4.5–45% sucrose gradients. Fractions were collected and the absorbance at 254 nm was measured during collection.

Chrystelle Bonnart, et al. Nucleic Acids Res. 2012 July;40(13):6270-6289.
6.
Figure 6.

Figure 6. From: Mammalian HCA66 protein is required for both ribosome synthesis and centriole duplication.

Some molecular mechanisms underlying the function of HCA66/Utp6p in ribosome synthesis are conserved in yeast and mammals. (A) Schematic representation of Utp6p (440 amino acids), HCA66 (597 amino acids) and the different modified versions of HCA66 bearing single (A99E, H137A, E153A, R170A) or double (K102A + D106A) amino acid substitutions within the HAT repeats. The conserved N-terminal domain is colored in green, the HAT motifs in red. (B) Northern-blot analysis showing the accumulation levels of some pre-rRNAs (indicated on the left) in HeLa cells transfected with siRNAs targeting the 3′-UTR of HCA66 mRNA, either alone or in combination with vectors expressing EGFP only or wild-type and modified versions of EGFP-HCA66. The 18S-E/30S ratios, averaged from three experiments, are reported on the histogram presented on the right. (C) Immunoprecipitation experiment using anti-FLAG antibodies and HeLa cells co-expressing FLAG-WDR36 (+) or FLAG alone (−) and wild-type (WT) or altered version (A99E or K102A + D106A) of EGFP-HCA66. A fraction of the total extracts used for the experiments (T) and of the immunoprecipitated material (IP) was analyzed by western blot.

Chrystelle Bonnart, et al. Nucleic Acids Res. 2012 July;40(13):6270-6289.
7.
Figure 7.

Figure 7. From: Mammalian HCA66 protein is required for both ribosome synthesis and centriole duplication.

Defects in centrosome function induced by the expression of a dominant negative version of HCA66 do not impair ribosome synthesis in HeLa cells. (A) FACS analysis showing the cell cycle profiles of HeLa cell cultures expressing EGFP, EGFP-HCA66, EGFP-HCA661–86 or mock-transfected 48 h after transfection. Total cells (adherent and floating cells) were analyzed in each case. The arrowheads below each graph indicate the positions of the G1 and G2/M peaks. Arrows indicate the presence of a sub-G1 peak, prominent in cells expressing EGFP-HCA661–86. (B) Proportion of dead cells in cultures expressing EGFP, EGFP-HCA66, EGFP-HCA661–86 or mock-transfected 24, 48 or 72 h after electroporation. The histogram represents the percentage of trypan blue positive (dead) cells in each population, considering total cells. (C) Agarose gel electrophoresis of genomic DNAs extracted from HeLa cells expressing EGFP, EGFP-HCA66, EGFP-HCA661–86 or mock-transfected, 24, 48 or 72 h after electroporation. Genomic DNAs have been extracted from total cells. Molecular weight ladder: SmartLadder (Eurogentec). (D) Northern-blot analysis using probe a (Figure 2B and Supplementary Table S1) showing the accumulation levels of some pre-rRNAs in HeLa cells expressing EGFP, EGFP-HCA66, EGFP-HCA661–86 or mock-transfected, 24, 48 or 72 h after electroporation. RNAs were extracted from total cells.

Chrystelle Bonnart, et al. Nucleic Acids Res. 2012 July;40(13):6270-6289.

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