Journal: Molecular Biology of the Cell

Article Title: CEP135 isoform dysregulation promotes centrosome amplification in breast cancer cells

doi: 10.1091/mbc.E18-10-0674

Figure Lengend Snippet: Centriole overduplication in breast cancer cells that have CA. (A) Nonamplified and amplified centrioles have a full complement of representative centriolar proteins. SAS-6 (left, grayscale), CPAP (middle, grayscale) and CEP135 (right, grayscale) are labeled relative to CEP192 (red) and centrin (green) and imaged using structured illumination microscopy (SIM). (B) New centriole assembly at nonamplified and amplified centrioles. Left panel, centrioles labeled for SAS-6 (grayscale), CEP192 (red), and centrin (green). Right panel, schematic of G1-, early and late S phase, and amplified centrioles. Arrows denote multiple new, SAS-6–positive procentrioles. (C) Percentage of the total cell population that have more than two new (SAS-6–positive) daughter centrioles. (D) Number of new centrioles (SAS-6 positive) assembled in cells that have amplified centrosomes. (E) Multiple and single daughter centrioles form from mother centrioles. Top panel, schematic of multiple and single daughter centrioles forming from mother centrioles. Bottom panel, representative images of centrioles labeled for SAS-6 (grayscale), CEP192 (red), and centrin (green) in S-phase MDA-231 cells that have overduplicated centrioles. Arrows denote multiple daughter centriole assembly events at a single mother centriole. (F) Relative frequency of duplication of multiple daughter centrioles compared with a single daughter centriole from a single mother centriole. (C, D) Statistical tests compare to MCF10A cells. Mean ± SEM. Fisher’s exact test and Mann–Whitney U test. ***, p < 0.005 and ****, p < 0.0005. Scale bars, 1 μm.

Article Snippet: Antibodies used for immunofluorescence are α-centrin (1:2000; 20H5; Abcam), α-γ-tubulin (1:1000; DQ-19; Sigma), α-CEP135 full (1:5000; generous gift from T. K. Tang, Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan), α-CEP135 mini (1:2000; Dahl et al. , 2015 ), α-SAS-6 (1:2000; Bethyl; A301-802A), α-α-tubulin (1:500; DM1A; Sigma), α-centromere derived from human calcinosis, Raynaud’s phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia (CREST) patient serum (1:2000; generous gift from J. DeLuca, Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO), α-CPAP (1:350; Proteintech CENPJ 11517-1-AP), and α-CEP192 (1:2000; generous gift from A. Holland, Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD).

Techniques: Amplification, Labeling, Microscopy, MANN-WHITNEY

Journal: Molecular Biology of the Cell

Article Title: CEP135 isoform dysregulation promotes centrosome amplification in breast cancer cells

doi: 10.1091/mbc.E18-10-0674

Figure Lengend Snippet: CEP135 isoform transcript and protein levels are altered in breast cancer cells. (A) Top panel, CEP135 full and CEP135 mini genes. Red and green bars denote coding exons (Ex) of CEP135 full and CEP135 mini , respectively. Blue bars denote noncoding exons. Gray lines denote introns (In). F 1 and R 1 (red) and F 1 and R 2 (green) denote forward and reverse primers for CEP135 full and CEP135 mini , respectively. Black pentagons represent stop codons for translation termination. Bottom panels, CEP135 full , CEP135 mini , and control (GUSB) RT-PCR in breast cancer cells. (B) The relative CEP135 full (red) and CEP135 mini (green) transcript levels and the CEP135 full:mini (black) ratio. (C) The CEP135 full:mini transcript ratio in breast cancer cells. (D) Linear regression fit and Pearson coefficient ( R ) of the CEP135 full:mini transcript ratio relative to the percentage of CA cells in the breast cancer cell populations. (E) CEP135 full (red) and centrin (grayscale) at one centrosome of a G2 phase cell. (F) CEP135 mini (green) and centrin (grayscale) at one centrosome of G2 phase cells. (G) Centrosomal CEP135 full (red) and CEP135 mini (green) protein fluorescence intensities per centrosome of a G2 phase cell and their ratios (black). (B, C, G) Statistical tests compared to MCF10A cells. Mean ± SEM. Student’s t test and Mann–Whitney U test. *, p < 0.05, **, p < 0.01, ***, p < 0.005, and ****, p < 0.0005. Scale bars, 1 μm.

Article Snippet: Antibodies used for immunofluorescence are α-centrin (1:2000; 20H5; Abcam), α-γ-tubulin (1:1000; DQ-19; Sigma), α-CEP135 full (1:5000; generous gift from T. K. Tang, Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan), α-CEP135 mini (1:2000; Dahl et al. , 2015 ), α-SAS-6 (1:2000; Bethyl; A301-802A), α-α-tubulin (1:500; DM1A; Sigma), α-centromere derived from human calcinosis, Raynaud’s phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia (CREST) patient serum (1:2000; generous gift from J. DeLuca, Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO), α-CPAP (1:350; Proteintech CENPJ 11517-1-AP), and α-CEP192 (1:2000; generous gift from A. Holland, Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD).

Techniques: Control, Reverse Transcription Polymerase Chain Reaction, Fluorescence, MANN-WHITNEY

Journal: Molecular Biology of the Cell

Article Title: CEP135 isoform dysregulation promotes centrosome amplification in breast cancer cells

doi: 10.1091/mbc.E18-10-0674

Figure Lengend Snippet: Elevated CEP135 full increases breast cancer cell CA and chromosome missegregation. (A) Top panel, schematic and timeline of exogenous mCh-CEP135 full-Tet expression in MDA-231 cells. Bottom panel, relative centrosomal fluorescence intensity of mCh-CEP135 full in noninduced and induced mCh-CEP135 full-Tet MDA-231 cells. (B) SIM localization of mCh-CEP135 full (red) and CEP192 (green) at centrioles in mCh-CEP135 full-Tet cells. (C) Left panel, amplified centrioles in mCh-CEP135 full-Tet cells. mCh-CEP135 full-Tet (red) cells were colocalized with SAS-6 (green) or CPAP (green). Right panel, mean number of new, daughter centrioles (SAS-6 positive) in noninduced and induced mCh-CEP135 full-Tet CA MDA-231 cells. (D) Centrosomes in noninduced and induced mCh-CEP135 full-Tet cells. mCh-CEP135 full-Tet (red) cells were stained for centrin (grayscale) and γ-tubulin (green). (E) Left panel, percentage of cells with CA in noninduced and induced mCh-CEP135 full-Tet MDA-231 cells. Right panel, number of amplified centrioles (red) and centrosomes (green) in noninduced and tetracycline-induced mCh-CEP135 full-Tet MDA-231 cells displaying CA. (F) Left panels, bipolar and multipolar mitoses in noninduced and tetracycline-induced mCh-CEP135 full-Tet MDA-231 cells stained for PCM (γ-tubulin, green), DNA (Hoechst 33342, blue) and kinetochores (α-CREST, grayscale). Right panel, percentage of mitotic cells with more than two poles in noninduced and induced mCh-CEP135 full-Tet cells. (G) Left panel, anaphase-lagging chromosomes in induced mCh-CEP135 full-Tet (red) MDA-231 cells stained for DNA (Hoechst 33342, blue) and kinetochores (α-CREST, grayscale). Arrow and inset denote a lagging chromosome. Right panel, percentage of late anaphase cells with lagging chromosomes in noninduced and induced mCh-CEP135 full-Tet MDA-231 late mitotic cells. (H) Left panel, micronuclei in induced mCh-CEP135 full-Tet (red) MDA-231 cells stained for DNA (Hoechst 33342, blue) and kinetochores (α-CREST, grayscale). Arrow and inset denote a micronucleus. Right panel, percentage of interphase cells with micronuclei in noninduced and induced mCh-CEP135 full-Tet MDA-231 cells. Mean ± SEM. Fisher’s exact test and Mann–Whitney U test. *, p < 0.05 and ****, p < 0.0005. Scale bars, 1 μm.

Article Snippet: Antibodies used for immunofluorescence are α-centrin (1:2000; 20H5; Abcam), α-γ-tubulin (1:1000; DQ-19; Sigma), α-CEP135 full (1:5000; generous gift from T. K. Tang, Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan), α-CEP135 mini (1:2000; Dahl et al. , 2015 ), α-SAS-6 (1:2000; Bethyl; A301-802A), α-α-tubulin (1:500; DM1A; Sigma), α-centromere derived from human calcinosis, Raynaud’s phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia (CREST) patient serum (1:2000; generous gift from J. DeLuca, Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO), α-CPAP (1:350; Proteintech CENPJ 11517-1-AP), and α-CEP192 (1:2000; generous gift from A. Holland, Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD).

Techniques: Expressing, Fluorescence, Amplification, Staining, MANN-WHITNEY

Journal: Molecular Biology of the Cell

Article Title: CEP135 isoform dysregulation promotes centrosome amplification in breast cancer cells

doi: 10.1091/mbc.E18-10-0674

Figure Lengend Snippet: Elevated CEP135 mini is sufficient to decrease CA and alters chromosome segregation. (A) Top panel, schematic and timeline of tetracycline induction in GFP-CEP135 mini-Tet MDA-231 cells. Bottom panel, relative centrosome fluorescence intensity of GFP-CEP135 mini in noninduced and induced GFP-CEP135 mini-Tet cells. (B) Nonamplified and underduplicated centrosomes in noninduced and induced GFP-CEP135 mini-Tet cells. GFP-CEP135 mini-Tet (green) cells stained for centrioles (centrin, grayscale) and PCM (γ tubulin, red). (C) Percentage of cells with underduplicated (gray), normal (black), and amplified (red) centrosomes in noninduced and induced GFP-CEP135 mini-Tet cells. (D) Frequency of centrioles (red) and centrosomes (green) in the total population of noninduced and induced GFP-CEP135 mini-Tet MDA-231 cells. (E) Left panel, nonamplified centrosomes in noninduced and induced GFP-CEP135 mini-Tet cells. GFP-CEP135 mini-Tet (green) cells stained for centrioles (centrin, grayscale) and PCM (γ tubulin, red). Right panel, relative fluorescence intensity of γ-tubulin in noninduced and tetracycline-induced GFP-CEP135 mini-Tet cells. (F) Left panels, bipolar, multipolar, and apolar mitoses in noninduced and induced GFP-CEP135 mini-Tet MDA-231 cells stained for PCM (γ-tubulin, red), DNA (Hoechst 33342, blue), and kinetochores (α-CREST, grayscale). Right panels, percentage of multipolar and apolar mitoses in noninduced and induced GFP-CEP135 mini-Tet MDA-231 cells. (G) Left panel, anaphase-lagging chromosomes in induced GFP-CEP135 mini-Tet (green) MDA-231 cells stained for PCM (γ-tubulin, red), DNA (Hoechst 33342, blue), and kinetochores (α-CREST, grayscale). Arrow and inset denote a lagging chromosome. Right panel, percentage of late anaphase cells with lagging chromosomes in noninduced and induced GFP-CEP135 mini-Tet MDA-231 cells. (H) Left panel, micronuclei in induced GFP-CEP135 mini-Tet MDA-231 cells stained for DNA (Hoechst 33342, blue) and kinetochores (α-CREST, grayscale). Arrow and inset denote a micronucleus. Right panel, percentage of interphase cells with micronuclei in noninduced and induced GFP-CEP135 mini-Tet MDA-231 cells. Mean ± SEM. Fisher’s exact test and Mann–Whitney U test. *, p < 0.05, ***, p < 0.005, and ****, p < 0.0005. Scale bars, 1 μm.

Article Snippet: Antibodies used for immunofluorescence are α-centrin (1:2000; 20H5; Abcam), α-γ-tubulin (1:1000; DQ-19; Sigma), α-CEP135 full (1:5000; generous gift from T. K. Tang, Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan), α-CEP135 mini (1:2000; Dahl et al. , 2015 ), α-SAS-6 (1:2000; Bethyl; A301-802A), α-α-tubulin (1:500; DM1A; Sigma), α-centromere derived from human calcinosis, Raynaud’s phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia (CREST) patient serum (1:2000; generous gift from J. DeLuca, Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO), α-CPAP (1:350; Proteintech CENPJ 11517-1-AP), and α-CEP192 (1:2000; generous gift from A. Holland, Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD).

Techniques: Fluorescence, Staining, Amplification, MANN-WHITNEY

Journal: Molecular Biology of the Cell

Article Title: CEP135 isoform dysregulation promotes centrosome amplification in breast cancer cells

doi: 10.1091/mbc.E18-10-0674

Figure Lengend Snippet: CEP135 mini is an alternatively polyadenylated isoform, and mutations near the CEP135 mini poly(A) signal reduce the CEP135 full:mini ratio and centrosome number in breast cancer cells. (A) Top panel, schematic of CEP135 full and CEP135 mini genes. Red and green bars denote coding exons (Ex) of CEP135 full and CEP135 mini , respectively. Blue bars denote noncoding exons. Gray lines denote introns (In). Black pentagons denote translation stop codons. Bottom panel, alternate models for CEP135 mini transcript biogenesis. (B) Top panel, schematic of the CEP135 mini 3′UTR-specific forward primer (F 1 ) and reverse primers (R 1 , R 2 , and R 3 ) used to map the approximate end of the CEP135 mini 3′UTR using 3′ RNA-ligated RACE. Bottom panel, RT-PCR and PCR. (C) Top panel, schematic of control and mutant 3′UTR CEP135 mini transcripts. Red bar denotes the Cas9-mediated insertion site in the 3′UTR. Bottom panel, CEP135 full , CEP135 mini , and GUSB transcript levels in control and 3′UTR mutant MDA-231 cells. (D) CEP135 full , CEP135 mini , and CEP135 full:mini transcript levels in control and 3′UTR mutant MDA-231 cells. (E) Left panels, centrosomal CEP135 mini protein in control and 3′UTR mutant MDA-231 cells stained for CEP135 mini (green) and centrin (grayscale). Right panel, quantification of CEP135 mini and centrin fluorescence intensities in control and 3′UTR mutant MDA-231 cells. (F) Percentage of cells with underduplicated (gray), normal (black), and amplified (red) centrosomes in control and 3′UTR mutant MDA-231 cells. (G) Number of centrioles and centrosomes per cell in control and 3′UTR mutant MDA-231 cells. (H) Normal and underduplicated centrosomes in control and 3′UTR mutant MDA-231 cells stained for centrioles (centrin, red) and γ-tubulin (green). (I) Relative γ-tubulin fluorescence intensity in control and 3′UTR mutant MDA-231 cells. Mean ± SEM. Student’s t test, Fisher’s exact test, and Mann–Whitney U test. *, p < 0.05, **, p < 0.01, and ****, p < 0.0005. Scale bars, 1 μm.

Article Snippet: Antibodies used for immunofluorescence are α-centrin (1:2000; 20H5; Abcam), α-γ-tubulin (1:1000; DQ-19; Sigma), α-CEP135 full (1:5000; generous gift from T. K. Tang, Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan), α-CEP135 mini (1:2000; Dahl et al. , 2015 ), α-SAS-6 (1:2000; Bethyl; A301-802A), α-α-tubulin (1:500; DM1A; Sigma), α-centromere derived from human calcinosis, Raynaud’s phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia (CREST) patient serum (1:2000; generous gift from J. DeLuca, Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO), α-CPAP (1:350; Proteintech CENPJ 11517-1-AP), and α-CEP192 (1:2000; generous gift from A. Holland, Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD).

Techniques: Reverse Transcription Polymerase Chain Reaction, Control, Mutagenesis, Staining, Fluorescence, Amplification, MANN-WHITNEY