Journal: Nature Communications

Article Title: Selective autophagy maintains centrosome integrity and accurate mitosis by turnover of centriolar satellites

doi: 10.1038/s41467-019-12094-9

Figure Lengend Snippet: Interphase centrosomes display compositional changes and accumulation of abnormal CS upon autophagy deficiency. a U2OS cells transfected with control, ULK1, ATG7 or ATG5 siRNAs, stained for Pericentrin and Hoechst33342. b , c Quantification of ( a ). Columns represent mean fluorescence intensity ( b ) or mean centrosome area ( c ) ±SD, n = 3 of >60 cells. * P ≤ 0.05, ** P ≤ 0.01, **** P ≤ 0.0001. Unpaired Student’s t -test, two-tailed. d Centrin distribution related to NEDD1-stained centrosomes in U2OS cells treated with control, ULK1 or ATG7 siRNAs. Arrows indicate examples of extra-centrosomal centrin foci. e Quantification of ( d ). Columns represent the mean frequency of acentrosomal centrin foci ±SD, n = 3 of > 100 cells. * P ≤ 0.05. Unpaired Student’s t -test, two-tailed. f Colocalization between centrin and PCM1 in U2OS cells transfected with control or ATG7 siRNAs. g PCM1 distribution in U2OS cells treated with control, ULK1 or ATG7 siRNAs. Arrows indicate string-like PCM1. h String-like distribution of PCM1 in ATG7-depleted cells. i Quantification of integrated density of experiments represented in ( g ). Columns represent the mean ± SD, n = 3 of >50 cells. * P ≤ 0.05, ** P ≤ 0.01. Unpaired Student’s t -test, two-tailed. j Quantification of CS characteristics of experiments represented in ( g ). Columns represent the mean ± SD, n = 3 of >50 cells. ns P > 0.05, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001. Unpaired Student’s t -test, two-tailed. Scale bars, 10 µm. Source data are provided as a Source Data file

Article Snippet: Primary antibodies were as follows; ATG5 Cell Signaling Technology, 12994S, WB 1:1000), ATG7 (Cell Signaling Technology, 8558S, WB 1:1000), β-actin (Novus Biologicals, NB600-501, WB 1:5000), β-tubulin (Sigma-Aldrich, T4026, IF 1:200), centrin (a gift from I. Cheeseman , Whitehead Institute for Biomedical, Research, Cambridge, USA, IF 1:1000), CEP63 (Proteintech, 16268-1-AP, IF 1:200), CEP131 (Bethyl, A301-425A, WB 1:1000; Abcam, ab84864, IF 1:300; Abcam, ab99379, WB 1:1000, IF 1:500), CEP290 (Abcam, ab84870, IF 1:200, WB 1:1000), CPAP (Proteintech, 11517-1-AP, IF 1:200), DIC 1/2 (Santa Cruz Biotechnology, sc-13524, WB 1:2000), EG5 (BD Transduction Laboratories, 611186, WB 1:2000), GABARAP (Abgent, AP1821a, WB 1:1000; Cell Signaling Technology, 13733S, WB 1:1000, IF 1:100), GABARAPL2 (Abcam, ab126607, WB 1:1000, IF 1:100), GAPDH (Merck-Millipore, CB1001, 1:20,000), GFP (Santa Cruz Biotechnology, sc-8334, WB 1:1000), γ-tubulin (Sigma-Aldrich, T3559, IF 1:200; Sigma-Aldrich, T5326, IF 1:250), HA (Sigma-Aldrich, H3663, WB 1:1000, IF 1:200), Histone H3 (Abcam, ab201456, WB 1:2000), LAMP2 (Abcam, ab25631, WB 1:2000; Developmental Studies Hybridoma Bank University of Iowa, H4B4, IF 1:100), LC3B (Cell Signaling Technology, 3868S, WB 1:1000, IF 1:200), LC3B (Cell Signaling Technology, 2775S, WB 1:1000), MIB1 (Novus Biologicals, NBP1-95846, WB 1:1000), NEDD1 (Santa Cruz Biotechnology, sc-100961, IF 1:200), NuMA (Abcam, ab36999, IF 1:200), OFD1 (Sigma-Aldrich, HPA031103, IF 1:200, WB 1:1000), p62/SQSTM1 (MBL International, PM045, WB 1:2000), p150Glued (BD Biosciences, 610473, WB 1:2000), PCM1 (Bethyl Laboratories, A301-150A, WB 1:1000, IF 1:500, EM 1:20; Santa Cruz Biotechnology, sc-398365, IF 1:200; Santa Cruz Biotechnology, sc-50164, IF 1:200), Pericentrin (Abcam, ab4448, WB 1:1000, IF 1:200), SSX2IP (Sigma-Aldrich, HPA027306, IF 1:200, WB 1:1000), ULK1 (Cell Signaling Technology, 8054, WB 1:1000), VDAC (Cell Signaling Technology, 12454 S, WB 1:1000), vinculin (Sigma-Aldrich, V4505, WB 1:2000).

Techniques: Transfection, Control, Staining, Fluorescence, Two Tailed Test

Journal: eLife

Article Title: Self-assembly of pericentriolar material in interphase cells lacking centrioles

doi: 10.7554/eLife.77892

Figure Lengend Snippet:

Article Snippet: Antibody , anti-NEDD1 (rabbit polyclonal) , Rockland , Rockland Cat# 109–401 C38S, RRID: AB_10893219 , (1:1000) for WB.

Techniques: Sequencing, Luciferase, Imaging, Software, Recombinant, Plasmid Preparation, Knock-Out, Mutagenesis

Journal: Cell cycle (Georgetown, Tex.)

Article Title: Involvement of centrosome amplification in radiation-induced mitotic catastrophe.

doi: 10.4161/cc.6.3.3834

Figure Lengend Snippet: Fig. 1. GFP-centrin-1 as a marker of IR- induced centrosome amplification. (A) Human U2OS cells expressing GFP-centrin-1 (green) and H2B-RFP (red) were fixed in methanol and stained with the following antibodies to centrosomal or centrosome-associated proteins, Aurora A, γ-tubulin, CEP170, Ninein and Nedd1 (blue, Alexa 350). Scale bar, 10 mm. U2OS cells as described in (A), before (B) and after (C) treatment with 10 Gy IR.

Article Snippet: Cells were fixed in either 4% PFA or methanol as described in ref. 22 and stained using the following primary antibodies: γ-tubulin (T3559, Sigma), ninein (AM), CEP170 (GG), Nedd1 (AM), Aurora A (35C1, Abcam), a-tubulin (B512, Sigma), TPX-2 (BL1679, Bethyl Labs), BubR1 (8G1, AbCam) and Mad2 (Abcam).

Techniques: Marker, Amplification, Expressing, Staining

Journal: The EMBO Journal

Article Title: Glutamylation of centrosomes ensures their function by recruiting microtubule nucleation factors

doi: 10.1038/s44318-025-00435-y

Figure Lengend Snippet: ( A – C ) NIH3T3 cells co-transfected with Ce3-FRB-CEP170C and either CCP5CD-Neon-FKBP or CCP5CDDM-Neon-FKBP were treated with DMSO (0.1%) or rapamycin (Rapa, 100 nM) for 1 h. Following treatment, cells were fixed and immunostained for γ-tubulin ( A ), CEP192 ( B ), and NEDD1 ( C ), respectively. The right panels show magnified images of the areas outlined by dashed squares. Scale bar, 10 μm. ( D – F ) Quantification of the normalized fluorescence intensity of γ-tubulin ( D ), CEP192 ( E ), and NEDD1 ( F ) in cells from ( A ), ( B ), and ( C ), respectively. Data represent the mean ± SEM. n (Neon-FKBP, CCP5CD-Neon-FKBP, and CCP5CDDM-Neon-FKBP) = 87, 142, and 113 cells in ( D ); 117, 174, and 159 cells in ( E ); 153, 229, and 192 cells in ( F ), respectively, from three to four independent experiments. ( G , H ) NIH3T3 cells were co-transfected with Ce3-FRB-CEP170C and CCP5CD-Neon-FKBP or with Ce3-FRB-CEP170C-NEDD1 and CCP5CDDM-Neon-FKBP. In ( G ), transfected cells were treated with 100 nM rapamycin (Rapa) for 1 h and then immunostained for γ-tubulin. In ( H ), transfected cells were incubated on ice with nocodazole (3.3 μM) for 40 min to depolymerize microtubules. Following depolymerization, cells were washed with DMEM for 1 min, allowed to recover for 3 min at RT, and then immunostained with anti-α-tubulin antibody. Hollow arrowheads indicate regrown microtubules, as shown by α-tubulin-labeled filaments extending from the centrosomal region in ( H ). Scale bar, 2 μm. ( I , J ) Quantification of the normalized intensity of γ-tubulin in ( I ) and area of centrosome-derived microtubules in ( J ) are shown. Data represent the mean ± SEM. n = 212 and 222 cells in CCP5CD and CCP5CDDM groups, respectively, for ( I ); n = 158 and 149 cells in CCP5CD and CCP5CDDM groups, respectively, for ( J ); three independent experiments. ( K ) Schematic of the co-sedimentation experiment protocol. Purified glutamylated or unmodified microtubules were generated and incubated with NIH3T3 cell lysates. The lysate proteins co-sedimented with microtubules (MTs) were collected and subjected to immunoblotting. ( L ) Immunoblot analysis of cell lysates collected using the protocol in ( K ), probing for NEDD1, glutamylated tubulin, and α-tubulin. ( M ) Quantification of NEDD1 levels co-sedimented with unmodified or glutamylated microtubules. Data represent the mean ± SEM from three independent experiments. ( N ) 3D-SIM images of tubulin glutamylation (Glu-tub) and NEDD1 in COS7 cells. The distribution profiles of tubulin glutamylation and NEDD1 are shown from both top view and side view of centrosomes. Scale bar, 1 µm. ( O ) Charge potential analysis of the centrosome-binding domain of wild-type (WT) NEDD1 and the negatively charged NEDD1 mutant (Neg). ( P ) COS7 cells transfected with WT NEDD1 or Neg NEDD1 were immunostained for pericentrin (red). The right panels show magnified images of the areas outlined by dashed squares. Scale bar, 10 µm. ( Q , R ) Quantification of the percentage of cells with centrosomal or cytosolic localization of NEDD1 ( Q ) and the centrosome-to-cytosol intensity ratio of NEDD1 ( R ). Data represent the mean ± SEM in ( Q ) and mean ± SD (red) in (R). n = 200 and 204 cells in the WT and Neg NEDD1 groups, respectively; Three independent experiments. Student’s t tests were performed, and P values are indicated. Noted: Single focal plane of Ce3-FRB-CEP170C images may not always display a two-centriole pattern. .

Article Snippet: A recent study found that PLK4-mediated phosphorylation of serine 325 in NEDD1 promotes its binding to nascent centrioles (Chi et al, ).

Techniques: Transfection, Fluorescence, Incubation, Labeling, Derivative Assay, Sedimentation, Purification, Generated, Western Blot, Binding Assay, Mutagenesis

Journal: The EMBO Journal

Article Title: Glutamylation of centrosomes ensures their function by recruiting microtubule nucleation factors

doi: 10.1038/s44318-025-00435-y

Figure Lengend Snippet: ( A , B ) COS7 cells co-transfected with the indicated constructs were treated with DMSO (0.1%) or Rapa (100 nM rapamycin) for 1 h. Following treatment, cells were immunostained with antibodies against NEDD1 ( A ) and γ-tubulin ( B ), respectively. Scale bar, 10 µm. ( C , D ) Quantification of the normalized intensity of NEDD1 ( C ) in cells from ( A ) and γ-tubulin ( D ) in cells from ( B ). Data represent as mean ± SEM. n (from left to right) = 62, 51, 84, 101, 67, 55, 53, and 54 cells in ( C ); 143, 152, 134, 135, 37, 36, 85, and 69 cells in ( D ); 3–6 independent experiments. Students’ t tests were performed, and P values are indicated.

Article Snippet: A recent study found that PLK4-mediated phosphorylation of serine 325 in NEDD1 promotes its binding to nascent centrioles (Chi et al, ).

Techniques: Transfection, Construct