Journal: PLoS ONE

Article Title: Genome-Wide Analysis of DNA Methylation Differences in Muscle and Fat from Monozygotic Twins Discordant for Type 2 Diabetes

doi: 10.1371/journal.pone.0051302

Figure Lengend Snippet: Differentially methylated genes.

Article Snippet: KLHL12 , cg04462209 , 321 , 3.7±1.4 , 2.8±1.4 , 0.9 , 0.0017 , 1.0.

Techniques: Methylation

Journal: The Journal of Cell Biology

Article Title: Centrosomal organization of Cep152 provides flexibility in Plk4 and procentriole positioning

doi: 10.1083/jcb.202301092

Figure Lengend Snippet: Spatial organization of proximal centriole proteins in HeLa cells. Logarithmically growing HeLa C1-GFP cells were immunolabeled with antibodies to detect Cep44, Cep192, Cep57, Cep63, or Cep152. Some samples were additionally expanded and labeled for acetylated tubulin. Imaging was done using STED. Rotational images of centrioles in the top view were additionally generated by averaging three images of the same centrosome rotated for 0°, 40°, and 80° (as depicted in ). (A) Examples of fluorescent signals for various proteins. Nt = N-terminus. The outer diameter or lateral dimensions of the signals are indicated. (B) Original and averaged images of expanded centrioles imaged in the top view. (C) Images of centrosomes with duplicated mother centrioles immunolabeled for Cep192, which is localized on mother centrioles and procentrioles (PC). (D) Four Z sections of vertically oriented centrosomes illustrate different levels of Cep192 along the mother centriole. (E and F) Examples of expanded centrosomes with immunolabeled Cep152 using antibodies against the Nt or middle region (Mid; ) and acetylated tubulin. Numbers in F indicate denser portions of the signal. (G) G1 centrosomes immunolabeled for Cep152 Nt. Numbers indicate nine groups of signals, with two adjacent signals within (some indicated by arrows). (H) Organization of Cep152 on centrosomes with various degrees of Cep152 depletion. Cep152 was depleted in HeLa C1-GFP cells, as depicted in the gray box. Cells were immunolabeled for Cep152 and procentriole marker SAS-6. Scale bars: 0.5 µm for STED, 1 µm for twofold expansion + STED, and 2 µm for fourfold expansion + STED.

Article Snippet: To deplete Cep152 and Cep192, G1 HeLa C1-GFP cells were treated with Cep192 siRNA (5′-GCU​AGU​AUG​UCU​GAU​ACU​UGG-3′; Dharmacon), Cep152 siRNA (sc-90225; Santa Cruz), non-targeting siRNA (5′-UGG​UUU​ACA​UGU​CGA​CUA​A-3′; Dharmacon), or control siRNA-A (sc-37007; Santa Cruz) 2 h after shake-off as described above.

Techniques: Immunolabeling, Labeling, Imaging, Generated, Marker

Journal: The Journal of Cell Biology

Article Title: Centrosomal organization of Cep152 provides flexibility in Plk4 and procentriole positioning

doi: 10.1083/jcb.202301092

Figure Lengend Snippet: Spatial arrangement of various centrosomal components. (A–E) Cells were immunolabeled with indicated antibodies and expanded. Some samples were additionally labeled for acetylated tubulin. Centrosomes were imaged using STED. (A) Longitudinally oriented centrosomes immunolabeled for Cep44 and acetylated tubulin. Cep44 localizes to procentrioles from their early stages of formation. (B) STED images of centrioles in top view, co-labeled with Cep63 and Cep57. The two proteins are in proximity, consistent with biochemical analyses showing that they interact. (C) Image of longitudinally imaged centriole immunolabeled for Cep63 signals, showing the distribution of Cep63 in a linear fashion, consistent with its ninefold radial distribution around mother centrioles. (D) Image of a centriole tilted toward the imaging plane and immunolabeled for Cep152 and acetylated tubulin to illustrate the three-dimensional arrangement of the Cep152 signal. (E) Images of Cep63 and Cep152 from U2OS cells show a similar arrangement as found in HeLa cells. Scale bars: 0.5 µm for STED, 1 µm for twofold expansion + STED, and 2 µm for fourfold expansion + STED.

Article Snippet: To deplete Cep152 and Cep192, G1 HeLa C1-GFP cells were treated with Cep192 siRNA (5′-GCU​AGU​AUG​UCU​GAU​ACU​UGG-3′; Dharmacon), Cep152 siRNA (sc-90225; Santa Cruz), non-targeting siRNA (5′-UGG​UUU​ACA​UGU​CGA​CUA​A-3′; Dharmacon), or control siRNA-A (sc-37007; Santa Cruz) 2 h after shake-off as described above.

Techniques: Immunolabeling, Labeling, Imaging

Journal: The Journal of Cell Biology

Article Title: Centrosomal organization of Cep152 provides flexibility in Plk4 and procentriole positioning

doi: 10.1083/jcb.202301092

Figure Lengend Snippet: Centrosomal organization of catalytically uninhibited and inhibited overexpressed Plk4 and centriolar rosettes. (A) Centrosomal organization of overexpressed Plk4 in the absence and in the presence of its catalytic inhibitor CEN. S phase arrested RPE-1 Plk4 cells were treated as depicted in the gray box. Dox was added to induce Plk4 expression, after which CEN was added to inhibit Plk4. Cells were fixed and immunolabeled for Plk4 and SAS-6 and imaged using STED. The centrosomal intensity of Plk4 was measured from STED images and plotted. The plot shows all individual points and average ± SD. n = number of centrosomes; N = number of independent experiments. (B) Three Z sections of centriolar rosettes from Plk4-overexpressing RPE-1 Plk4 cells. The middle Z section is shown in . CP110 labels centriole distal ends and SAS-6 labels procentriole cartwheels. (C) Organization of centriolar rosettes induced by a transient CEN treatment after fixation in methanol (MetOH). S phase arrested cells were treated with CEN for 3 h, after which CEN was washed out to allow the formation of procentrioles around mother centrioles for 3 h. Cells were immunolabeled for Cep152 and SAS-6 and imaged by STED. White lines connect the centers of SAS-6 foci and the physical center of the mother centriole. The angles measured between two adjacent lines are indicated. Scale bars: 0.5 µm.

Article Snippet: To deplete Cep152 and Cep192, G1 HeLa C1-GFP cells were treated with Cep192 siRNA (5′-GCU​AGU​AUG​UCU​GAU​ACU​UGG-3′; Dharmacon), Cep152 siRNA (sc-90225; Santa Cruz), non-targeting siRNA (5′-UGG​UUU​ACA​UGU​CGA​CUA​A-3′; Dharmacon), or control siRNA-A (sc-37007; Santa Cruz) 2 h after shake-off as described above.

Techniques: Expressing, Immunolabeling

Journal: The Journal of Cell Biology

Article Title: Centrosomal organization of Cep152 provides flexibility in Plk4 and procentriole positioning

doi: 10.1083/jcb.202301092

Figure Lengend Snippet: Localization of Plk4 foci and procentrioles in relation to the mother centriole microtubule wall. (A) Images of Plk4 foci around centrioles after 2 h of CEN treatment. Samples were immunolabeled for Plk4, expanded, and postlabeled for acetylated tubulin. White lines connect the centers of Plk4 foci and the physical center of the mother centriole. The angles measured between two adjacent lines are indicated. (B) A schematic depicting two positions Plk4 foci and procentrioles occupy with respect to mother centriole microtubules. O = facing opposite microtubule blades. B = facing opposite the gap between microtubule triplets. Right: Examples of Plk4 foci/procentrioles in the B and O positions. (C) Electron micrographs of duplicated centrioles from RPE-1 C1-GFP cells. One procentriole is in B and another in the O position. (D) Centriolar rosettes after transient CEN treatment. Cells were treated for 3 h, after which CEN was washed out to allow the formation of procentrioles around mother centrioles. Centrin1-GFP marks centrioles. Cep152 labeling was used to confirm the orthogonal orientation of mother centrioles. White lines and angles as in A. (E) Centriolar rosettes from Dox-induced Plk4-overexpressing RPE-1 Plk4 cells. CP110 labels centriole distal ends and SAS-6 procentriole cartwheels. White lines and angles, as in A. Asterix marks overlapping procentrioles formed at different lateral highs. (F) Electron micrograph of a centriolar rosette from Plk4-overexpressing RPE-1 Plk4 cells. (G) Quantification of procentrioles’ position around mother centriole. (H) Quantification of angles between adjacent Plk4 foci and procentrioles from various experiments. The angles were determined as indicated in A. Cep44 and acetylated tubulin signals exhibit an average angular distance of 40°, consistent with their symmetrical ninefold pattern, and were used to validate the quantification approach. (I) Plk4 and procentrioles at different lateral positions along the mother centriole longitudinal axis obtained by CEN treatment (STED images) or Dox-induced Plk4 overexpression (electron micrograph). Scale bars: 0.5 µm for STED, 2 µm for fourfold expansion + STED, and 0.4 µm for electron micrographs.

Article Snippet: To deplete Cep152 and Cep192, G1 HeLa C1-GFP cells were treated with Cep192 siRNA (5′-GCU​AGU​AUG​UCU​GAU​ACU​UGG-3′; Dharmacon), Cep152 siRNA (sc-90225; Santa Cruz), non-targeting siRNA (5′-UGG​UUU​ACA​UGU​CGA​CUA​A-3′; Dharmacon), or control siRNA-A (sc-37007; Santa Cruz) 2 h after shake-off as described above.

Techniques: Immunolabeling, Labeling, Over Expression

Journal: The Journal of Cell Biology

Article Title: Centrosomal organization of Cep152 provides flexibility in Plk4 and procentriole positioning

doi: 10.1083/jcb.202301092

Figure Lengend Snippet: The mutual position of Plk4 and a set of proximal centrosomal proteins in relation to mother centriole microtubules. Logarithmically growing HeLa C1-GFP cells were immunolabeled with antibodies to detect Plk4, Cep44, Cep192, Cep57, Cep63, or Cep152. Samples were expanded fourfold and immunolabeled for acetylated tubulin. Centrioles positioned vertically to the imaging plane were imaged using STED. Rotational images of individual proteins and acetylated tubulin were generated as described in and aligned in different combinations, as described in the text and in Materials and methods. Centriole microtubule blades were used as a reference during alignment. Proteins were assigned different colors for easier visualization. (A) Alignment of proximal centrosomal components from G1 centrosomes, characterized by lower amounts of Cep192, Cep63, and Cep152 in comparison to S phase cells. (B) Alignment of proximal centrosomal components from S phase centrosomes. (C) Alignment of centrosomal Plk4 signal with Cep152 and mother centriole microtubules. Plk4 signals are from G1 and S phase centrosomes and from S phase centrosomes treated with Plk4 inhibitor CEN. Scale bars: 2 µm in A and B, and 0.5 µm in B inserts and C.

Article Snippet: To deplete Cep152 and Cep192, G1 HeLa C1-GFP cells were treated with Cep192 siRNA (5′-GCU​AGU​AUG​UCU​GAU​ACU​UGG-3′; Dharmacon), Cep152 siRNA (sc-90225; Santa Cruz), non-targeting siRNA (5′-UGG​UUU​ACA​UGU​CGA​CUA​A-3′; Dharmacon), or control siRNA-A (sc-37007; Santa Cruz) 2 h after shake-off as described above.

Techniques: Immunolabeling, Imaging, Generated, Comparison

Journal: The Journal of Cell Biology

Article Title: Centrosomal organization of Cep152 provides flexibility in Plk4 and procentriole positioning

doi: 10.1083/jcb.202301092

Figure Lengend Snippet: Asymmetrical distribution of Cep152 in RPE-1 cells and its relationship with Cep192. Logarithmically growing RPE-1 C1-GFP cells were immunolabeled for Cep44, Cep192, Cep57, Cep63, or Cep152. Cep152 was detected using an antibody against its N-terminus (Nt) or middle portion (Mid; ). Some samples were additionally expanded twofold and labeled for acetylated tubulin. (A) The discontinuous appearance of Cep152 signals with gaps (arrows) extending over a variable number of centriole microtubule blades. Centrioles are shown in the top view. (B) The discontinuous appearance of Cep57 and Cep63 showing gaps (arrows). In contrast, Cep44 and Cep192 show continuous ring-like distribution. (C) Cep57, Cep63, and Cep152 signal in relation to Centrin1-GFP signal marking centrioles. Regardless of the gaps in Cep57, Cep63, and Cep152 signals, mother centrioles duplicate and regularly form only one daughter centriole. (D) Quantification of centrosomes with gaps in the signal of the indicated protein around the mother centriole. Centrioles imaged in vertical or near vertical orientation were quantified. (E and F) RPE-1 Plk4 cells were treated with Dox for 18 h to induce overexpression of FLAG-Plk4 and formation of centriolar rosettes, and cells were immunolabeled for indicated proteins. (E) Centrosomes with centriolar rosettes to illustrate the absence of procentrioles at regions lacking Cep57, Cep63, and Cep152. Cep192 forms a complete ring around mother centrioles. Right: The absence of Plk4 signal on the regions of the centrosome lacking Cep152. (F) Quantification of centrosomes in RPE-1 Plk4 cells exhibiting gaps in the signals of Cep192, Cep57, Cep63, and Cep152. Centrioles imaged in vertical or near vertical orientation were quantified. (G) Experimental strategy used in H–K (for more details, please see Materials and methods). (H) The effect of Cep192 or Cep152 depletion on the assembly of centriolar rosettes. Cells were depleted for Cep192 or Cep152, and centriolar rosettes were induced by transient treatment with CEN and Cep192 and Cep152 were immunolabeled. Centrin1-GFP marks centrioles. (I) Quantification of cells from H containing centriolar rosettes, duplicated centrioles, or at least one unduplicated centriole. 200 cells were counted per condition in each experiment. (J) The effect of Cep192 or Cep152 depletion on Cep152 and Cep192 centrosomal levels. Intensities of Cep192 and Cep152 were determined from widefield recordings and plotted for individual centrosomes. (K) The effect of Cep192 depletion on centrosomal Plk4 levels. Plk4 and Cep192 were labeled and their centrosomal intensity was quantified from widefiled images. Red lines: average intensity. (L) Localization of Cep57, Cep192, Cep63, Cep152, and SAS-6 on de novo–formed centrioles in S phase RPE-1 Plk4 cells treated with Dox for 18 h. The wider end of the SAS-6 signal distinguishes the centriole’s proximal (p) end from the distal (d) end. Cep192 associates with the centrioles laterally, Cep63 and Cep152 associate with the centriole’s proximal ends, while Cep57 does not associate with de novo formed centrioles. Scale bars: 0.5 µm for STED, 1 µm for twofold expansion/STED, and 2 µm for widefield.

Article Snippet: To deplete Cep152 and Cep192, G1 HeLa C1-GFP cells were treated with Cep192 siRNA (5′-GCU​AGU​AUG​UCU​GAU​ACU​UGG-3′; Dharmacon), Cep152 siRNA (sc-90225; Santa Cruz), non-targeting siRNA (5′-UGG​UUU​ACA​UGU​CGA​CUA​A-3′; Dharmacon), or control siRNA-A (sc-37007; Santa Cruz) 2 h after shake-off as described above.

Techniques: Immunolabeling, Labeling, Over Expression