A Role for PML3 in Centrosome Duplication and Genome Stability Zhi-Xiang Xu, Wen-Xin Zou, Pei Lin, Kun-Sang Chang  Molecular Cell  Volume 17, Issue 5, Pages 721-732 (March 2005) DOI: 10.1016/j.molcel.2005.02.014 Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 1 A Role for PML in Centrosome Function (A) PML deficiency induced centrosome amplification. IF staining was performed with the PML+/+ and PML−/− MEFs using the antibody against Aurora A. Fluorescence images were recorded with a Kodak digital imaging system mounted on top of a Leica fluorescence microscope. (B) Percentage of centrosome amplification in early-passage (passage 5) MEFs from PML+/+ and PML−/− mice. Centrosomes were immunostained with a monoclonal antibody against Aurora A as described above. The results are shown as average ± SEM from three experiments. 200 cells were counted in each experiment. (C) Colocalization of PML and the centrosome proteins Aurora A and γ-tubulin. Double-color IF staining was performed with 293T cells using γ-tubulin and Aurora A monoclonal antibodies and a rabbit PML polyclonal antibody. Chromosomal DNA was counterstained with TOPRO. Images were captured with a Zeiss LSM 5 confocal microscope. (D) 293T and CCD-37 cells were repeatedly treated with two pairs of siRNAs as described in the Experimental Procedures. Western blot analysis was performed with total protein isolated from each round of siRNA treatment to examine the effects on PML expression. (E and F) Percentage of 293T (E) and CCD-37 (F) cells with amplified centrosomes after each round of siRNA treatment. Double-color IF staining was performed with a PML polyclonal antibody and γ-tubulin monoclonal antibody. Results represent an average ± SEM of three independent experiments from 200 cells (PML knockdown) counted per group. Molecular Cell 2005 17, 721-732DOI: (10.1016/j.molcel.2005.02.014) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 2 PML3-Specific Localization to the Centrosome (A and B) Proliferating Mo59K cells were fixed, and double-color IF staining was performed with PML isoform-specific polyclonal antibodies and a monoclonal antibody against the centrosome protein Aurora A or γ-tubulin. Chromosomal DNA was counterstained with DAPI. Images were captured as described for Figure 1. (C) Localization of PML3 to the centrosome during various cell cycle phases. MO59K cells were fixed, and double-color IF staining was performed with the PML3-specific polyclonal antibody and γ-tubulin monoclonal antibody. Chromosomal DNA was counterstained with TOPRO. Images of cells at various cell cycle phases were captured with a Zeiss LSM 5 confocal microscope. (D) Detection of PML3 in the centrosome fraction by discontinuous sucrose gradient centrifugation. Protein isolated from each gradient fraction within the 40%–70% range was subjected to Western blot analysis with PML isoform-specific antibodies and a γ-tubulin antibody. Molecular Cell 2005 17, 721-732DOI: (10.1016/j.molcel.2005.02.014) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 3 PML3-Specific Knockdown by siRNA Induces Centrosome Amplification (A) Specific knockdown expression of different PML isoforms by siRNA. The specific siRNAs against PML isoforms and a mock control were designed and transfected into proliferating 293T and CCD-37 cells as described in the Experimental Procedures. At 72 hr after transfection, total proteins were isolated for Western blot analysis with the PML isoform-specific antipeptide antibodies. (B) Cells treated with PML isoform-specific siRNAs were fixed, and IF staining was performed with a γ-tubulin monoclonal antibody. (C) PML3 knockdown by siRNA induced centrosome amplification in 293T and CCD-37 cells. The fold increase in the number of 293T cells with centrosome amplification was determined by counting an average of 200 cells (PML knockdown) per group. Values are mean ± SEM of two independent experiments. (D) The effects of reexpression of different PML isoforms by transient transfection on centrosome amplification in PML−/− MEFs. MEFs were transiently transfected with the expression plasmids pcDNA3/PML1, pcDNA3/PML2, pcDNA3/PML3, pcDNA3/PML4, and pcDNA3/PML5 by using FuGENE 6. At 72 hr after transfection, cells were fixed, and double-color IF staining was performed with a γ-tubulin monoclonal antibody and PML isoform-specific polyclonal antibodies. The percentage of cells with centrosome amplification was determined by counting an average of 200 positively transfected cells. The results represent an average ± SEM of three independent experiments. Total proteins in each group were isolated, and Western blot analysis was performed with the PML antibody against the GST-PML fusion protein (Xu et al., 2003). The filter was reprobed with an α-tubulin antibody to serve as a loading control. Molecular Cell 2005 17, 721-732DOI: (10.1016/j.molcel.2005.02.014) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 4 PML3 Is Located at the Pole of the Mitotic Spindle (A) Proliferating Mo59K and U937 cells were fixed and coimmunostained with anti-PML3 (red) and anti-α-tubulin (green) antibodies. Cells were treated with ice-cold stripping buffer or incubated with nocodazole for 24 hr to disrupt the microtubules. Colocalization of PML3 and α-tubulin was determined by double-color IF staining with an α-tubulin monoclonal antibody and PML3 polyclonal antibody. (B) The aberrant centrosomes in PML-deficient cells colocalized with active sites of microtubule nucleation. Double-color IF staining was performed with anti-α-tubulin monoclonal and anti-γ-tubulin polyclonal antibodies in PML+/+ and PML−/− MEFs. (C) Percentage of PML+/+ and PML−/− MEFs containing the aberrant multipolar spindles. The results represent the average percentage ± SEM determined in three independent experiments. In each experiment, at least 200 dividing cells were counted. Molecular Cell 2005 17, 721-732DOI: (10.1016/j.molcel.2005.02.014) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 5 Colocalization of PML3 and the Centrosomes in APL Blasts and in the APL-Derived NB4 Cells APL bone morrow samples and NB4 cells were immobilized on slides by cytocentrifugation. Double-color IF staining was performed with a PML isoform-specific polyclonal antibody and the γ-tubulin monoclonal antibody. The images were captured as described in Figure 1A. Molecular Cell 2005 17, 721-732DOI: (10.1016/j.molcel.2005.02.014) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 6 Deregulation of the Centrosome Duplication Cycle in PML-Deficient Cells Leads to Centrosome Amplification (A) PML+/+ and PML−/− MEFs were treated with 2 mM hydroxyurea. Cells were harvested at various time points as indicated and analyzed to determine the number of centrosomes by staining with a γ-tubulin antibody. The results represent an average ± SEM of three independent experiments. (For all the graphs presented in this section, we counted 200 cells per group.) (B) To determine whether the amplified centrosomes consisted of a pair of centrioles, double-color IF staining was performed with polyclonal centrin-1 and monoclonal α-tubulin antibodies at 48 hr after hydroxyurea treatment. Colocalization of centrin-1 and α-tubulin was shown in the amplified centrosome (bottom). (C and D) 293T (C) and CCD-37 (D) cells were treated with PML1- and PML3-specific siRNAs as described for Figure 4. The percentage of cells (PML1 or PML3 knockdown) with more than two centrosomes after treatment with or without 2 mM hydroxyurea (HOU) for 48 hr was determined. The results represent an average ± SEM of three independent experiments. Molecular Cell 2005 17, 721-732DOI: (10.1016/j.molcel.2005.02.014) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 7 PML3 Physically Interacts with Aurora A, Inhibits Its Kinase Activity, and Affects Cdk2/Cyclin Activity (A) Coimmunoprecipitation (CoIP) was performed by using total protein isolated from U2OS cells with PML isoform-specific antibodies. Western blotting (WB) was performed with an Aurora A antibody. (B) CoIP was performed by using an Aurora A antibody, and WB was carried out with different PML isoform-specific antibodies. (C) The effects of various PML isoforms on Aurora A expression and Thr288 phosphorylation. PML expression plasmids were transiently transfected into U2OS cells. Western blot analysis was performed by using the Aurora A or Aurora A Thr288 phosphospecific antibody. The Western blot was reprobed with a PML polyclonal antibody, which recognized all of the PML isoforms. (D) Subcellular localization of Cdk2, cyclin E, and p21 in PML+/+ and PML−/− MEFs. Equal quantities of protein fractions (30 µg) from PML+/+ and PML−/− MEFs isolated from cytoplasm (C), the nucleus (N), and total proteins (T) were analyzed for the expression of Cdk2, cyclin E, and p21 with WB. The same filter was reprobed with an anti-α-tubulin antibody to serve as a cytoplasmic protein loading control and anti-histone H3 antibody as a nuclear protein loading control. (E) Association of cyclin E and Cdk2 in normal and PML−/− MEFs. A coimmunoprecipitation assay was performed by using a protein fraction (100 µg) isolated from C, N, and T with a Cdk2-specific antibody. WB analysis of the precipitated proteins was performed by using cyclin E, Cdk2, and p21 antibodies. (F) Cdk2 kinase activity in PML−/− and normal MEFs. A coimmunoprecipitation assay was performed with a rabbit polyclonal anti-Cdk2 antibody. The immunoprecipitated proteins in protein A-Sepharose beads were used for kinase activity analysis with histone H1 as a substrate. Cdk2/cyclin E kinase was used as a positive control. (G) Inhibition of PML3 expression by a specific siRNA. PML3-specific knockdown by siRNA was performed as described in Figure 3. (H) The effects of PML3 knockdown on Cdk2 activity. The Cdk2 activity in cells treated with a PML3 and control siRNA was determined as described for (F). Molecular Cell 2005 17, 721-732DOI: (10.1016/j.molcel.2005.02.014) Copyright © 2005 Elsevier Inc. Terms and Conditions