Journal: Communications biology

Article Title: The centrosomal protein 131 participates in the regulation of mitochondrial apoptosis.

doi: 10.1038/s42003-023-05676-3

Figure Lengend Snippet: Fig. 1 Characterization of CEP131 knockout Jurkat cells. a Schematic representation of the CEP131 gene, with positioning of the CRISPR sequence guide (blue) in exon 3. Genomic sequences in wild-type (WT) and two bi-allelic clones (KO#1 and KO#2; mutations in red) are shown. b The RNA expression level of CEP131 was assessed by qPCR in WT, CEP131KO#1, and KO#2 cells (mean ± SEM, n = 3 biological replicates, fold change using ACTB and HPRT1 as housekeeping genes for normalization, one-way ANOVA, ****p < 0.0001). c, d Lysates from WT, CEP131 KO#1, and KO#2 Jurkat cells were prepared and analyzed by immunoblotting with antibodies specific to the indicated proteins. e Representative immunofluorescence images of PCM1 (green) and γ-tubulin (red) in WT, CEP131 KO#1, and KO#2 Jurkat cells. Nuclei were counterstained with 4’−6-diamidino-2- phenylindole (DAPI). Scale bars, 10 µm. f, g Radial profile analysis of PCM1 in a 29 µm circle whose center is defined by γ-tubulin, in WT, CEP131 KO#1, and KO#2 Jurkat cells (mean ± SEM of n = 10 cells/condition). h Cell lysates from WT, CEP131 KO#1, and KO#2 Jurkat were immunoprecipitated (IP) with antibodies against PCM1 or with non- relevant Ig (negative control). Samples were then analyzed by immunoblotting as indicated. Inputs are the total lysates collected before the IP. i WT, CEP131 KO#1, and KO #2 Jurkat cells were treated with 10 µM nocodazole for 1 h, prior to washing. Microtubule regrowth was assessed after 5 min by confocal microscopy analysis of α-tubulin. Representative images of three independent experiments (left) (scale bars, 10 µm), and quantification of α-tubulin intensity of one representative experiment (right) (n = 70 cells/condition; one-way ANOVA, ****p < 0.0001). Data information: (c, d, h) GAPDH served as a loading control. Molecular weight markers (Mr) are shown. Data are representative of three independent experiments.

Article Snippet: The following antibodies were used: CEP72 (A301-297A, Bethyl), CEP131 (A301-415A, Bethyl), CEP290 (IHC-00365-2, Bethyl), cytochrome-c (SC-13561, Santa Cruz), MIB1 (M5948, Sigma-Aldrich), PCM1 (5213 S, Cell Signaling), α-Tubulin (66031-1-1 g, Proteintech), γ-Tubulin (T6557, Sigma-Aldrich).

Techniques: Knock-Out, CRISPR, Sequencing, Genomic Sequencing, Clone Assay, RNA Expression, Western Blot, Immunoprecipitation, Negative Control, Confocal Microscopy, Control, Molecular Weight

Journal: Communications biology

Article Title: The centrosomal protein 131 participates in the regulation of mitochondrial apoptosis.

doi: 10.1038/s42003-023-05676-3

Figure Lengend Snippet: Fig. 3 CEP131 participates in mitochondrial dynamics. a Flow cytometry analysis of the mitochondrial mass using MitoTracker Green in wild-type (WT), CEP131 KO#1, and KO#2 Jurkat cells (mean ± SEM, n = 7 biological replicates, one-way ANOVA, ns: non-significant). b Expression of the mitochondrial DNA (mtDNA) normalized to the nuclear DNA (nDNA) assessed by qPCR. For mtDNA and nDNA, the expression of three genes were measured and the mean of three ratios mtDNA/nDNA is presented for n = 3 biological replicates (one-way ANOVA, ns: non-significant). c Flow cytometry analysis of the mitochondrial transmembrane potential using TMRM (tetramethylrhodamine) in WT, CEP131 KO#1, and KO#2 Jurkat cells (mean ± SEM, n = 7 biological replicates, one-way ANOVA, *p < 0.05, ****p < 0.0001). d Schematic overview of the workflow used to measure mitochondrial volumes. e TOM20 staining analysis by structure illumination microscopy (SIM) in WT, CEP131 KO#1, and #2 cells. Top panel, max intensity projection of z-stacked images are shown. Bottom panel, each detected object is represented by one color as explained in (d). Representative images of three independent experiments are shown (scale bars, 10 µm). f Cumulative plots of mitochondrial volume distribution in WT and CEP131 knockout cells derived from (e) (three cells/conditions). Data are representative of three independent experiments. g Cell lysates from WT, CEP131 KO#1, and KO#2 cells stimulated with 1 µM antimycin and 1 µM oligomycin for the indicated times were prepared and analyzed by immunoblotting with specific antibodies. Black arrowhead, ubiquitinated MFN1. GAPDH served as a loading control. Molecular weight markers (Mr) are shown. Data are representative of three independent experiments.

Article Snippet: The following antibodies were used: CEP72 (A301-297A, Bethyl), CEP131 (A301-415A, Bethyl), CEP290 (IHC-00365-2, Bethyl), cytochrome-c (SC-13561, Santa Cruz), MIB1 (M5948, Sigma-Aldrich), PCM1 (5213 S, Cell Signaling), α-Tubulin (66031-1-1 g, Proteintech), γ-Tubulin (T6557, Sigma-Aldrich).

Techniques: Flow Cytometry, Expressing, Staining, Microscopy, Knock-Out, Derivative Assay, Western Blot, Control, Molecular Weight

Journal: Communications biology

Article Title: The centrosomal protein 131 participates in the regulation of mitochondrial apoptosis.

doi: 10.1038/s42003-023-05676-3

Figure Lengend Snippet: Fig. 1 Characterization of CEP131 knockout Jurkat cells. a Schematic representation of the CEP131 gene, with positioning of the CRISPR sequence guide (blue) in exon 3. Genomic sequences in wild-type (WT) and two bi-allelic clones (KO#1 and KO#2; mutations in red) are shown. b The RNA expression level of CEP131 was assessed by qPCR in WT, CEP131KO#1, and KO#2 cells (mean ± SEM, n = 3 biological replicates, fold change using ACTB and HPRT1 as housekeeping genes for normalization, one-way ANOVA, ****p < 0.0001). c, d Lysates from WT, CEP131 KO#1, and KO#2 Jurkat cells were prepared and analyzed by immunoblotting with antibodies specific to the indicated proteins. e Representative immunofluorescence images of PCM1 (green) and γ-tubulin (red) in WT, CEP131 KO#1, and KO#2 Jurkat cells. Nuclei were counterstained with 4’−6-diamidino-2- phenylindole (DAPI). Scale bars, 10 µm. f, g Radial profile analysis of PCM1 in a 29 µm circle whose center is defined by γ-tubulin, in WT, CEP131 KO#1, and KO#2 Jurkat cells (mean ± SEM of n = 10 cells/condition). h Cell lysates from WT, CEP131 KO#1, and KO#2 Jurkat were immunoprecipitated (IP) with antibodies against PCM1 or with non- relevant Ig (negative control). Samples were then analyzed by immunoblotting as indicated. Inputs are the total lysates collected before the IP. i WT, CEP131 KO#1, and KO #2 Jurkat cells were treated with 10 µM nocodazole for 1 h, prior to washing. Microtubule regrowth was assessed after 5 min by confocal microscopy analysis of α-tubulin. Representative images of three independent experiments (left) (scale bars, 10 µm), and quantification of α-tubulin intensity of one representative experiment (right) (n = 70 cells/condition; one-way ANOVA, ****p < 0.0001). Data information: (c, d, h) GAPDH served as a loading control. Molecular weight markers (Mr) are shown. Data are representative of three independent experiments.

Article Snippet: The following antibodies were used: CEP72 (A301-297A, Bethyl), CEP131 (A301-415A, Bethyl), CEP290 (IHC-00365-2, Bethyl), cytochrome-c (SC-13561, Santa Cruz), MIB1 (M5948, Sigma-Aldrich), PCM1 (5213 S, Cell Signaling), α-Tubulin (66031-1-1 g, Proteintech), γ-Tubulin (T6557, Sigma-Aldrich).

Techniques: Knock-Out, CRISPR, Sequencing, Genomic Sequencing, Clone Assay, RNA Expression, Western Blot, Immunoprecipitation, Negative Control, Confocal Microscopy, Control, Molecular Weight

Journal: bioRxiv

Article Title: BioE3 enables the identification of bona fide targets of E3 ligases

doi: 10.1101/2023.08.18.553883

Figure Lengend Snippet: (a) Western blot of BioE3 experiment performed on HEK293FT stable cell line expressing TRIPZ-bio GEF Ubnc and transfected with EFS-BirA-MIB1 WT or BirA-MIB1 CA . Specific biotinylation of MIB1 targets was observed at different biotin timings. Molecular weight markers are shown to the left of the blots in kDa. (b) Confocal microscopy of BioE3 experiment performed on U2OS stable cell line expressing TRIPZ-bio GEF Ubnc transfected with EFS-BirA-MIB1 WT or BirA-MIB1 CA . Colocalization of streptavidin (Strep, magenta), BirA-MIB1 (green) and Centrin-2 (CETN2, blue) was observed at the centrosomes and selected for digital zooming (yellow dotted-line squares). Black and white panels show the green, magenta and blue channels individually. Scale bar: 8 µm. (c) Volcano plot of LC-MS analysis comparing streptavidin pull-downs of BioE3 experiments performed on HEK293FT stable cell line expressing TRIPZ-bio GEF Ubnc and transfected with EFS-BirA-MIB1 WT or BirA-MIB1 CA (3 biological replicates per condition). Proteins significantly enriched (Log2 MIB1 WT / MIB1 CA > 0 and p -value < 0.05) were considered as MIB1 targets. Statistical analyses were performed by two-sided Student’s t -test. Data are provided as Supplementary Data 3. (d) Western blot validations of centrosomal MIB1 targets identified in (c) : PCM1, USP9X and CEP131. IN: input; St PD: streptavidin pull-down. Molecular weight markers are shown to the left of the blots in kDa. (e) Gene ontology analysis of the MIB1 targets defined in (c) . Depicted biological processes and cellular components were significantly enriched. Dotted line represents the threshold of the p -value (0.05). Data are provided as Supplementary Data 4. (a-d) Data are representative of 3 independent transfection experiments with similar

Article Snippet: Primary antibodies used as follows: Cell Signaling Technology: anti-biotin-HRP (1/1000; Cat#7075S), anti-alpha-Actinin (1/5000; Cat#6487S), anti-PCM1 (1/1000; Cat#5213S); SinoBiological: anti-BirA (1/1000; Cat#11582-T16); Proteintech: anti-USP9X (1/1000; Cat#55054-1-AP), anti-CEP131 (1/1000; Cat#25735-1-AP), anti-SUMO2/3 (1/1000; Cat#67154-1-Ig), anti-GAPDH (1/5000; Cat#60004-1-Ig), anti-PML (1/1000; Cat#21041-1-AP), anti-ROCK1 (1/1000; Cat#21850-1-AP), anti-GIGYF2 (1/1000; Cat#24790-1-AP), anti-CLINT1 (1/1000; Cat#10470-1-AP), anti-ARFGAP1 (1/1000; Cat#13571-1-AP); GenScript: anti-AviTag (1/1000; Cat#A00674); Sigma-Aldrich: anti-CCT8 (1/1000; Cat#HPA021051), anti-TP53BP2 (1/1000; Cat#HPA021603); anti-ubiquitin (1/5; ZTA10; generated at IFOM ); Jackson ImmunoResearch: anti-Mouse-HRP (1/5000; Cat#115-035-062), anti-Rabbit-HRP (1/5000; Cat#111-035-045).

Techniques: Western Blot, Stable Transfection, Expressing, Transfection, Molecular Weight, Confocal Microscopy, Liquid Chromatography with Mass Spectroscopy