cep192  (Bethyl)

Journal: bioRxiv

Article Title: Bora, CEP192 and Cenexin activate different Plk1 pools and regulate distinct cell and centrosome cycle transitions

doi: 10.1101/2025.09.30.679461

Figure Lengend Snippet: (A-H) Representative time stamp images and quantifications from FRAP movies from G2 phase Plk1-EGFP tagged hTERT-RPE1 cells treated with non-targeting siRNA control (A; quantification of recovery in (B)) , Cep192 siRNA (C quantification of recovery in (D)) , Cenexin siRNA (E quantification of recovery in (F)) and siCep192 + siCenexin (G quantification of recovery in (H)) . (I) Box and Whisker plot for measurement of mobile Plk1 pool on centrosomes under indicated conditions (N=3 independent experiments; n= 43, 44, 43 and 45 for siControl, siCep192, siCenexin and siCep192 + siCenexin, respectively.) (J) Box and Whisker plot for measurement of mobile Plk1 pool on centrosomes after Plk1 inhibition derived from N=3 independent experiments comprising n= 43, and 53 individual cells for DMSO and Plk1i, respectively. (K) Representative immunofluorescence images of hTERT-RPE1: Plk1-EGFP cells treated with indicated siRNAs and stained for Pericentrin (Red) and EGFP (Green). (L) Box and Whisker plot for measurement of Plk1 levels on centrosomes during G2 phase under the conditions indicated in (K) generated from N=5 independent experiments comprising n= 287: siControl, 279: siBora, 276: siCep192 and 287: siCenexin cells. Data presented as the entire range with values between first and third quartiles in boxes. ‘ + ’ sign in each column represent the mean value (p values for individual comparisons indicated on graphs: Kruskal-Wallis test). Scale bars Images: 5 µm and zoomed insets: 0.5 µm.

Article Snippet: The following antibodies were used in this study: Mouse anti-α-tubulin (Geneva antibody facility: AA345-M2a; 1:250: ExM) , Mouse anti-β-tubulin (Geneva antibody facility: AA344-M2a; 1:250: ExM) , Mouse anti-α-tubulin (Clone: DM1α, Sigma Aldrich, T9026, 1:5000: Western Blotting), Bora (gift from Erich Nigg, 1:1000 Western blotting)), CEP192 (Bethyl: A302-324A, 1:1000 Western blotting), Cenexin (abcam: ab43840, 1:1000 Western blotting), Aurora A (BD Biosciences: 610939, 1:1000 Western blotting), Plk1 (abcam: ab17057, 1:1000 Western blotting), Rabbit polyclonal anti-Pericentrin (abcam: ab4448; 1:250: ExM, 1:2000: IF), Mouse anti-Cyclin A2 (Clone: E32.1, abcam: ab38; 1:1000: Western Blotting), Chicken anti-GFP (Thermo Fisher Scientific: A10262; 1:2000 IF) and Mouse monoclonal anti-Separase (abcam: ab16170; 1:500 Western blotting).

Techniques: Control, Whisker Assay, Inhibition, Derivative Assay, Immunofluorescence, Staining, Generated

Journal: bioRxiv

Article Title: Bora, CEP192 and Cenexin activate different Plk1 pools and regulate distinct cell and centrosome cycle transitions

doi: 10.1101/2025.09.30.679461

Figure Lengend Snippet: (A) Live-cell time-lapse images of hTERT-RPE1: mRuby-PCNA cells treated with indicated siRNA combinations as they progress through S phase. (B) Box and whisker plot for measurement of the duration of S phase in cells after depletion of proteins indicated in (A) . Data derived from N=3 independent experiments comprising n= 45: siBora + Cep192, 52: siCep192 + siCenexin, 57: siBora + siCenexin and 51: siBora + siCep192 + siCenexin cells. (C) Live-cell time-lapse images of hTERT-RPE1: mRuby-PCNA cells treated with indicated siRNA combinations as they progress through G2 phase. (D) Box and whisker plot for measurement of the duration of S phase in cells after depletion of proteins indicated in (A) . Data derived from N=3 independent experiments comprising n= 82: siBora + Cep192, 56: siCep192 + siCenexin, 64: siBora + siCenexin and 71: siBora + siCep192 + siCenexin cells. Data presented as the entire range with values between first and third quartiles in boxes. ‘ + ’ sign in each column represent the mean value (p values for individual comparisons indicated on graphs: Kruskal-Wallis test). Scale bars: 5 µm.

Article Snippet: The following antibodies were used in this study: Mouse anti-α-tubulin (Geneva antibody facility: AA345-M2a; 1:250: ExM) , Mouse anti-β-tubulin (Geneva antibody facility: AA344-M2a; 1:250: ExM) , Mouse anti-α-tubulin (Clone: DM1α, Sigma Aldrich, T9026, 1:5000: Western Blotting), Bora (gift from Erich Nigg, 1:1000 Western blotting)), CEP192 (Bethyl: A302-324A, 1:1000 Western blotting), Cenexin (abcam: ab43840, 1:1000 Western blotting), Aurora A (BD Biosciences: 610939, 1:1000 Western blotting), Plk1 (abcam: ab17057, 1:1000 Western blotting), Rabbit polyclonal anti-Pericentrin (abcam: ab4448; 1:250: ExM, 1:2000: IF), Mouse anti-Cyclin A2 (Clone: E32.1, abcam: ab38; 1:1000: Western Blotting), Chicken anti-GFP (Thermo Fisher Scientific: A10262; 1:2000 IF) and Mouse monoclonal anti-Separase (abcam: ab16170; 1:500 Western blotting).

Techniques: Whisker Assay, Derivative Assay

Journal: bioRxiv

Article Title: Bora, CEP192 and Cenexin activate different Plk1 pools and regulate distinct cell and centrosome cycle transitions

doi: 10.1101/2025.09.30.679461

Figure Lengend Snippet: (A-C) Flow cytometry-based quantification of cell cycle profile of hTERT-RPE1 (WT), hTERT-RPE1: Usp28 - / - and hTERT-RPE1: Usp28 - / - 0:0 cells revealing percentage of cells in G1 (A) , S (B) and G2 (C) phases derived from N=4 independent experiments with each treatment comprising around 30,000 cells. (D) Flow cytometry-based quantification of cell cycle profile or RPE1 cells treated with Control vs Cep192 siRNA showing percentage of cells in G1, S and G2 phases obtained from N=5 independent experiments with each treatment comprising of 30,000 cells. (E) Representative images of S phase hTERT-RPE1 cells expressing cellular Plk1 activity sensor and Cyclin A2-mScarlet treated with indicated siRNAs or inhibitors. (F) Box and Whisker plot for FRET measurement-based quantification of cellular Plk1 activity calculated from S phase cells represented in (E) derived from N=4 independent experiments comprising n= 203: siControl, 204: siBora, 215: siCep192, 219: siCenexin, 215: Plk1i cells. The leftmost G2 Phase – siControl bar shown is replica of siControl data shown in for side-by-side comparison purposes. G) Representative images of S phase hTERT-RPE1 cells expressing centrosomal Plk1 activity sensor and Cyclin A2-mScarlet treated with indicated siRNAs or inhibitors. (H) Box and Whisker plot for FRET measurement-based quantification of cellular Plk1 activity calculated from S phase cells represented in (G) derived from N=3 independent experiments comprising n= 163: siControl, 187: siBora, 187: siCep192, 184: siCenexin, 178: Plk1i cells. The leftmost G2 Phase – siControl bar shown is replica of siControl data shown in for side-by-side comparison purposes. Data presented as the entire range with values between first and third quartiles in boxes. ‘ + ’ sign in each column represent the mean value (p values for individual comparisons indicated on graphs: Kruskal-Wallis test). Scale bars, Images: 5 µm and zoomed insets: 0.5 µm.

Article Snippet: The following antibodies were used in this study: Mouse anti-α-tubulin (Geneva antibody facility: AA345-M2a; 1:250: ExM) , Mouse anti-β-tubulin (Geneva antibody facility: AA344-M2a; 1:250: ExM) , Mouse anti-α-tubulin (Clone: DM1α, Sigma Aldrich, T9026, 1:5000: Western Blotting), Bora (gift from Erich Nigg, 1:1000 Western blotting)), CEP192 (Bethyl: A302-324A, 1:1000 Western blotting), Cenexin (abcam: ab43840, 1:1000 Western blotting), Aurora A (BD Biosciences: 610939, 1:1000 Western blotting), Plk1 (abcam: ab17057, 1:1000 Western blotting), Rabbit polyclonal anti-Pericentrin (abcam: ab4448; 1:250: ExM, 1:2000: IF), Mouse anti-Cyclin A2 (Clone: E32.1, abcam: ab38; 1:1000: Western Blotting), Chicken anti-GFP (Thermo Fisher Scientific: A10262; 1:2000 IF) and Mouse monoclonal anti-Separase (abcam: ab16170; 1:500 Western blotting).

Techniques: Flow Cytometry, Derivative Assay, Control, Expressing, Activity Assay, Whisker Assay, Comparison

Journal: Nature Communications

Article Title: The DNA replication machinery transmits dual signals to prevent unscheduled licensing and execution of centrosome duplication

doi: 10.1038/s41467-025-63002-3

Figure Lengend Snippet: a List of genes involved in microcephalic primordial dwarfism (MPD) based on previous studies . The genes involved in MPD are mostly classified as centrosomal proteins, DNA replication components, or a regulator for DNA damage response. b The number of Cep192 foci upon siRNA treatment against the indicated proteins. c , Histograms represent the frequency of interphase cells with >2 Cep192 foci observed in ( b ). n = 3 independent experiments, 50 cells for each. d Schematic of the DONSON gene, indicating mutations observed in cells from MPD patient 2 (P2) . The genomic structure is based on the longest ORF, containing ten coding exons (white rectangles) (NCBI NM_017613.3 ). e The number of Cep192 foci in hTERT-immortalized fibroblasts derived from the MPD patient 2. Fibroblasts were infected with pMSCV-empty-vector or pMSCV-DONSON and immunostained with antibodies against Cep192 (red). Arrowheads indicate centrosomes. f Histograms represent the frequency of interphase cells with the indicated phenotypes observed in ( e ). n = 3 independent experiments, 50 cells for each. g The number of centrin and Cep192 foci in G1 or S/G2 phase upon DONSON or Cdc6 depletion. HeLa cells were treated with siRNA treatment against the indicated proteins for 48 h and 10 μM EdU for 30 min. CENP-F and EdU double-negative cells were classified as G1 phase cells, and others as S or G2 phase cells. Arrowheads indicate centrioles. h Histograms represent the frequency of cells with the indicated phenotype observed in ( g ). n = 3 independent experiments, 30 cells for each. i Time-lapse observation of centriole disengagement in DONSON-depleted cells. HeLa GFP-centrin-1 cells were visualized every 20 min for 48 h after 24 h treatment with siRNA and 3 h treatment with 100 nM SiR-DNA. Arrowheads indicate engaged centriole pairs, and left–right arrows indicate precociously disengaged centrioles. j Cumulative scatterplot indicates the duration from nuclear envelope breakdown (NEBD) to centriole disengagement observed in ( i ). k Schematic model of the phenotypes of DONSON or Cdc6 depletion. All scale bars, 5 μm. Values are mean percentages ± s.d. Tukey’s multiple comparison test was used in c , and a two-tailed, unpaired Student’s t-test was used in ( f ) to obtain P -value. Throughout all the figures in this study, the single-channel images were generated by displaying only the signal from one channel extracted directly from the original merged representative image, without any adjustment to intensity or contrast. The insets represent magnified views of regions taken directly from either the merged image or the corresponding single-channel image, also without any further modification. The insets are displayed in the following order from top to bottom: merged, green, red, and cyan. All source data are provided as a Source Data file.

Article Snippet: The following primary antibodies were used in this study: rabbit antibodies against Cep192 (Bethyl Laboratories, A302–324A, IF 1:1000), C-Nap1 (Proteintech, 14498-1-AP, IF 1:1000), CP110 (Proteintech, 12780-1-AP, IF 1:300), Cdt1 (Abcam, ab202067, IF 1:500), Cdc6 (Abcam, ab109315, WB 1:100), RFP-tag (MBP, PM005, IF 1:1000), Cep57 (GeneTex, GTX115931, IF 1:1000), Cep57L1 (Proteintech, 24957-1-AP, IF 1:500), γ-Tubulin (Merck, T5192, IF 1:1000), PCNT (Abcam, ab4448, IF 1:2000), Cep152 (Bethyl Laboratories, A302–480A, IF 1:1000), Cep97 (Novus Biologicals, NPB1-83591 IF 1:500), CENP-F (Bethyl Laboratories, A301–617 A IF 1:1000), Phospho-Chk1 (S296) (Abcam, ab79758, WB 1:100), CyclinB1 (Cell Signaling Technology, 4138, IF 1:200, WB 1:1000), BLM (Abcam, ab2179, IF 1:250), ATR (Bethyl Laboratories, A300-137A, WB 1:500), ETAA1 (Abcam, ab192402, WB 1:100), TopBP1 (Abcam, ab2402, WB 1:1000), mNeonGreen-tag (Cell Signaling Technology, 53061, WB 1:100), p97 (VCP; GeneTex, GTX101089, IF 1:1000), Cdc45 (Cell Signaling Technology, 11881, IF 1:200, WB 1:500), LRR1 (Atlas antibodies, HPA069364, IF 1:200, WB 1:1000) and MCM3 (Cell Signaling Technology, 4012, WB 1:1000), Emi1 (Proteintech, 10872-1-AP, WB 1:1000); mouse antibodies against FLAG-tag (Merck, F1804, IF 1:1000, WB 1:1000), centrin-2 (Merck, 20H5, IF 1:1000), PCNA (Santa Cruz Biotechnology, sc-56, IF 1:1000, WB 1:500), CENP-F (Mitosin; BD Biosciences Pharmingen, 610768, IF 1:1000), Acetylated-Tubulin (Abcam, Ab179484 , IF 1:300), Cdc6 (Santa Cruz Biotechnology, sc-13136, IF 1:200), HSP90 (BD Transduction Laboratories, 610418, WB 1:5000), Cdh1 (Fzr1; Abcam, ab89535, IF 1:300), HsSAS-6 (Santa Cruz Biotechnology, sc-81431, IF 1:1000), EB1 (BD Transduction Laboratories, 610534, IF 1:500), Phospho-Plk1 (T210) (Abcam, ab39068, IF 1:300, WB 1:200), Chk1 (Santa Cruz Biotechnology, sc-8408, WB 1:1000), α-Tubulin (Merck, DM1A, IF 1:1000, WB 1:5000), HA-tag (BioLegend, 901501, WB 1:1000), mNeonGreen-tag (Proteintech, 32F6, IF 1:500), Cyclin-E1 (Cell Signaling Technology, 4129, WB 1:1000), Cyclin-A2 (Cell Signaling Technology, 4656, WB 1:1000), Histone H3 (Santa Cruz Biotechnology, sc-517576, WB 1:5000), LaminB1 (Santa Cruz Biotechnology, sc-374015, WB 1:100) and γ-Tubulin-Alexa Fluor 647 (Abcam, ab191114, IF 1:500); rat antibodies against centrin-2 (BioLegend, 698602, IF 1:500) and PCNA (Abcam, ab252848, IF 1:2000).

Techniques: Derivative Assay, Infection, Plasmid Preparation, Comparison, Two Tailed Test, Generated, Modification

Journal: Nature Communications

Article Title: The DNA replication machinery transmits dual signals to prevent unscheduled licensing and execution of centrosome duplication

doi: 10.1038/s41467-025-63002-3

Figure Lengend Snippet: a Time-lapse observation of mitotic cell division in DONSON-depleted cells. HeLa GFP-centrin-1 cells were visualized every 20 min for 48 h after 24 h treatment with siRNA and 3 h treatment with 100 nM SiR-DNA. Left–right arrows indicate precociously disengaged centrioles. b Histograms represent the frequency of cells in mitosis with the indicated phenotypes observed in ( a ). n = 3 independent experiments, 30 cells for each. c Mitotic cell division of hTERT-immortalized fibroblasts derived from the patients with mutations in DONSON . Fibroblasts were infected with pMSCV-empty-vector or pMSCV-DONSON and immunostained with antibodies against Cep192 (red). d , f Inducible expression of ETAA1 upon DONSON depletion. HeLa-Tet3G mNG-ETAA1 cells were treated with siControl or siDONSON following Dox treatment for 24 h. e , g Histograms represent the frequency of cells in mitosis with the indicated phenotypes observed in ( d ) and ( f ), respectively. n = 3 independent experiments, 30 cells for each. h A speculative model showing how DNA replication machinery exerts direct control over the licensing and execution of centrosome duplication. All scale bars, 5 μm. Values are mean percentages ± s.d. Two-tailed, unpaired Student’s t-test was used in ( b ), ( e ), and ( g ) to obtain the P -value. Source data are provided as a Source Data file.

Article Snippet: The following primary antibodies were used in this study: rabbit antibodies against Cep192 (Bethyl Laboratories, A302–324A, IF 1:1000), C-Nap1 (Proteintech, 14498-1-AP, IF 1:1000), CP110 (Proteintech, 12780-1-AP, IF 1:300), Cdt1 (Abcam, ab202067, IF 1:500), Cdc6 (Abcam, ab109315, WB 1:100), RFP-tag (MBP, PM005, IF 1:1000), Cep57 (GeneTex, GTX115931, IF 1:1000), Cep57L1 (Proteintech, 24957-1-AP, IF 1:500), γ-Tubulin (Merck, T5192, IF 1:1000), PCNT (Abcam, ab4448, IF 1:2000), Cep152 (Bethyl Laboratories, A302–480A, IF 1:1000), Cep97 (Novus Biologicals, NPB1-83591 IF 1:500), CENP-F (Bethyl Laboratories, A301–617 A IF 1:1000), Phospho-Chk1 (S296) (Abcam, ab79758, WB 1:100), CyclinB1 (Cell Signaling Technology, 4138, IF 1:200, WB 1:1000), BLM (Abcam, ab2179, IF 1:250), ATR (Bethyl Laboratories, A300-137A, WB 1:500), ETAA1 (Abcam, ab192402, WB 1:100), TopBP1 (Abcam, ab2402, WB 1:1000), mNeonGreen-tag (Cell Signaling Technology, 53061, WB 1:100), p97 (VCP; GeneTex, GTX101089, IF 1:1000), Cdc45 (Cell Signaling Technology, 11881, IF 1:200, WB 1:500), LRR1 (Atlas antibodies, HPA069364, IF 1:200, WB 1:1000) and MCM3 (Cell Signaling Technology, 4012, WB 1:1000), Emi1 (Proteintech, 10872-1-AP, WB 1:1000); mouse antibodies against FLAG-tag (Merck, F1804, IF 1:1000, WB 1:1000), centrin-2 (Merck, 20H5, IF 1:1000), PCNA (Santa Cruz Biotechnology, sc-56, IF 1:1000, WB 1:500), CENP-F (Mitosin; BD Biosciences Pharmingen, 610768, IF 1:1000), Acetylated-Tubulin (Abcam, Ab179484 , IF 1:300), Cdc6 (Santa Cruz Biotechnology, sc-13136, IF 1:200), HSP90 (BD Transduction Laboratories, 610418, WB 1:5000), Cdh1 (Fzr1; Abcam, ab89535, IF 1:300), HsSAS-6 (Santa Cruz Biotechnology, sc-81431, IF 1:1000), EB1 (BD Transduction Laboratories, 610534, IF 1:500), Phospho-Plk1 (T210) (Abcam, ab39068, IF 1:300, WB 1:200), Chk1 (Santa Cruz Biotechnology, sc-8408, WB 1:1000), α-Tubulin (Merck, DM1A, IF 1:1000, WB 1:5000), HA-tag (BioLegend, 901501, WB 1:1000), mNeonGreen-tag (Proteintech, 32F6, IF 1:500), Cyclin-E1 (Cell Signaling Technology, 4129, WB 1:1000), Cyclin-A2 (Cell Signaling Technology, 4656, WB 1:1000), Histone H3 (Santa Cruz Biotechnology, sc-517576, WB 1:5000), LaminB1 (Santa Cruz Biotechnology, sc-374015, WB 1:100) and γ-Tubulin-Alexa Fluor 647 (Abcam, ab191114, IF 1:500); rat antibodies against centrin-2 (BioLegend, 698602, IF 1:500) and PCNA (Abcam, ab252848, IF 1:2000).

Techniques: Derivative Assay, Infection, Plasmid Preparation, Expressing, Control, Two Tailed Test