Journal: Cell

Article Title: Time-series reconstruction of the molecular architecture of human centriole assembly

doi: 10.1016/j.cell.2024.03.025

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-CEP152 , Thermo Fisher Scientific , Cat# A302-479A, RRID: AB_1966085.

Techniques: Recombinant, Software

Journal: PLoS ONE

Article Title: Cep63 and Cep152 Cooperate to Ensure Centriole Duplication

doi: 10.1371/journal.pone.0069986

Figure Lengend Snippet: (A) Diagram of Cep152 full length and truncation proteins used in the following experiments. Numbers indicate amino acid positions. (B) Cep63 interacts with the C-terminal half of Cep152. MBP or MBP-Cep63 pull down experiments were carried out after incubation in lysates of 293 HEK cells expressing GFP-Cep152 full length (FL), N-terminal half (1–803), or C-terminal half (804–1654), and Cep152 proteins were detected by Western blotting with anti-Cep152 antibodies (Cep152 9AP). Input shows 10% of cell lysate used for pull down experiments. (C) The C-terminal half of Cep152 is required for its centrosomal localisation. 293 HEK cells expressing the GFP-Cep152 proteins used in (B) were stained with DAPI (blue) and anti-Centrin 3 antibodies (red), GFP direct fluorescence is shown in green. Lower panels show magnification of one centrosome (boxed region). Scale bars 5 µm.

Article Snippet: Commercial anti-α-tubulin mouse (Sigma-Aldrich), Centrin 2 rabbit (Santa Cruz), Centrin 3 mouse (Abcam), Cep63 rabbit (Millipore), Cep152 rabbit (no. 479, Bethyl), Cep192 (Bethyl), Flag M2 mouse (Sigma-Aldrich), γ-tubulin mouse (Sigma-Aldrich), Ninein rabbit (Abcam), Pericentrin rabbit (Covance), HsSAS-6 mouse (Santa Cruz) and PCNA mouse (PC10, Santa Cruz) antibodies were used for immunofluorescence and/or Western blotting.

Techniques: Incubation, Expressing, Western Blot, Staining, Fluorescence

Journal: PLoS ONE

Article Title: Cep63 and Cep152 Cooperate to Ensure Centriole Duplication

doi: 10.1371/journal.pone.0069986

Figure Lengend Snippet: (A) Diagram of Cep63 full length and truncation proteins used in the following experiments. Numbers indicate amino acid positions. (B) Cep63 C-terminus interacts with Cep152. Expression of Flag-Cep63 (FL), truncation proteins, or Flag-empty vector control (e) was induced in 293 FlpIn TREX cell lines by incubation with 2 µg/ml doxycycline for 72 hours, then proteins were immunoprecipitated using anti-Flag resin. Western blots show endogenous Cep152, detected by anti-Cep152 (Bethyl), and Cep63 truncations detected by anti-Cep63 (Millipore). Inputs are 5% of the lysate used for Flag IP. Red arrowhead points to Cep63 truncation 1–135 present in the Flag IP. (C-F) U2OS cells were transfected with YFP-Cep63 (FL), truncation proteins, or YFP-empty vector (e) for 48 hours. (C) Cells were stained with anti-Cep152 (red) and γ-tubulin (blue) antibodies; YFP-tagged proteins were detected by direct fluorescence (green). Scale bar 1 µm. (D) Whole cell lysates from (C) were analysed by Western blot with anti-GFP antibodies to visualise YFP-tagged proteins. (E) The localisation of YFP-Cep63 proteins to the centrosome was scored in 3 independent experiments, n >10. (F) Overexpression of Cep63 425–541 and 136–541 deplete Cep152 from the centrosome. The ratio of Cep152 to γ-tubulin fluorescence intensities at the centrosome was measured for multiple cells from the experiment shown in (C), n >10.

Article Snippet: Commercial anti-α-tubulin mouse (Sigma-Aldrich), Centrin 2 rabbit (Santa Cruz), Centrin 3 mouse (Abcam), Cep63 rabbit (Millipore), Cep152 rabbit (no. 479, Bethyl), Cep192 (Bethyl), Flag M2 mouse (Sigma-Aldrich), γ-tubulin mouse (Sigma-Aldrich), Ninein rabbit (Abcam), Pericentrin rabbit (Covance), HsSAS-6 mouse (Santa Cruz) and PCNA mouse (PC10, Santa Cruz) antibodies were used for immunofluorescence and/or Western blotting.

Techniques: Expressing, Plasmid Preparation, Incubation, Immunoprecipitation, Western Blot, Transfection, Staining, Fluorescence, Over Expression

Journal: PLoS ONE

Article Title: Cep63 and Cep152 Cooperate to Ensure Centriole Duplication

doi: 10.1371/journal.pone.0069986

Figure Lengend Snippet: (A-D) Control RNAi or RNAi of Cep63, Cep152, or Cep192, was carried out for 4 days in U2OS cells, followed by immunofluorescence on replicate samples, with anti- γ-tubulin and anti-Cep63 (A), Cep152 (B), or Cep192 (C) antibodies. Fluorescence intensity of Cep63, Cep152, Cep192, and γ-tubulin at the centrosome were measured (graphs A-D). All intensity measurements were normalised to the mean of the control population and p values are indicated above (students’ t-test). (E-G) Images of γ-tubulin and Cep63, Cep152, or Cep192 immunofluorescence at the centrosome from the experiment shown in A-D. Scale bar 1 µm. (H) Western blots of whole cell lysates from U2OS cells used in experiments (A-G) using anti-Cep152 (Bethyl) and α-tubulin antibodies.

Article Snippet: Commercial anti-α-tubulin mouse (Sigma-Aldrich), Centrin 2 rabbit (Santa Cruz), Centrin 3 mouse (Abcam), Cep63 rabbit (Millipore), Cep152 rabbit (no. 479, Bethyl), Cep192 (Bethyl), Flag M2 mouse (Sigma-Aldrich), γ-tubulin mouse (Sigma-Aldrich), Ninein rabbit (Abcam), Pericentrin rabbit (Covance), HsSAS-6 mouse (Santa Cruz) and PCNA mouse (PC10, Santa Cruz) antibodies were used for immunofluorescence and/or Western blotting.

Techniques: Immunofluorescence, Fluorescence, Western Blot

Journal: PLoS ONE

Article Title: Cep63 and Cep152 Cooperate to Ensure Centriole Duplication

doi: 10.1371/journal.pone.0069986

Figure Lengend Snippet: (A) Cep63 is present at the PCM before HsSAS-6 recruitment. Telophase, G1 phase, S or G2 phase and mitotic HeLa cells, as indicated, were stained with anti-centrin 2 (red), Cep63 (green), and HsSAS-6 (blue) antibodies. Centrosomes from each cell are shown. Scale bar 1 µm. (B) HsSAS-6 foci were counted in U2OS cells with nuclear PCNA foci (a marker of DNA replication) after 96 hours RNAi as indicated, n >150, 3 experiments. P values from a students’ t-test are indicated on the graph. (C) U2OS cells were stained with anti-Cyclin A and HsSAS-6 antibodies after control, Cep63, or Cep152 RNAi. Cyclin A status, negative (-, early G1 phase), dull (+, G1-S), or bright (++, S-G2), and the number of HsSAS-6 foci (0,1,2,>2) were scored in asynchronous populations in three independent experiments, n >150.

Article Snippet: Commercial anti-α-tubulin mouse (Sigma-Aldrich), Centrin 2 rabbit (Santa Cruz), Centrin 3 mouse (Abcam), Cep63 rabbit (Millipore), Cep152 rabbit (no. 479, Bethyl), Cep192 (Bethyl), Flag M2 mouse (Sigma-Aldrich), γ-tubulin mouse (Sigma-Aldrich), Ninein rabbit (Abcam), Pericentrin rabbit (Covance), HsSAS-6 mouse (Santa Cruz) and PCNA mouse (PC10, Santa Cruz) antibodies were used for immunofluorescence and/or Western blotting.

Techniques: Staining, Marker

Journal: Cell reports

Article Title: Bimodal Binding of STIL to Plk4 Controls Proper Centriole Copy Number.

doi: 10.1016/j.celrep.2018.05.030

Figure Lengend Snippet: Figure 1. The Autophosphorylation of Plk4 Is Critical for Limiting a Single Site for Procentriole Assembly (A) U2OS cells stained with indicated antibodies were observed using super resolution microscopy. White broken line, outline of the Nuclei. Insets, magnified views around the centrosome. A red arrowhead, an intense focus in a Plk4 ring of the mother centriole. Scale bar, 0.5 (inset) and 5 mm (whole nuclei). Note that we judged centrioles to be ‘‘before’’ or ‘‘after’’ duplication based on the staining patterns of Cep152 and Cep192. (B) (Top) Top view of the centriole duplication process and distribution of Plk4 (blue) and Cep152 (red). A blue arrowhead, an intense focus of the Plk4 ring. (Middle) How to quantify the signal intensities of Plk4 along with the mother centriole wall. (Bottom) Representative panels of centriolar Plk4 and Cep152. Scale bar, 0.5 mm. See also STAR Methods. (C) Signal intensity of the Plk4 around the mother centriole wall according to the method in (B). n = 18 or n = 13 for before or after duplication respectively. The values are ±SEM.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit polyclonal anti-STIL Bethyl laboratories Cat#A302-441A Rabbit polyclonal anti-STIL Abcam Cat#ab89314 Rabbit polyclonal anti-Cep152 Bethyl laboratories Cat#A302-479A Rabbit polyclonal anti-Cep152 Sonnen et al., 2013 N/A Rabbit polyclonal anti-phospho-S305 Plk4 Sillibourne et al., 2010 N/A Rabbit polyclonal anti-Cep192 Bethyl laboratories Cat#A302-324A Rabbit polyclonal anti-HA Abcam Cat#ab9110 Mouse monoclonal anti-centrin-2 Millipore Cat#20H5 Mouse monoclonal anti-centrin-HsSAS-6 Santa Cruz Bio-technology Cat#sc-81431 Mouse monoclonal anti-Plk4 Merck Millipore clone 6H5, Cat#MABC544 Mouse monoclonal anti-Flag Sigma Cat#F1804 Mouse monoclonal anti-HA Covance Cat#MMS-101P Mouse monoclonal anti-a-tubulin Sigma Cat#DM1A Mouse monoclonal anti-Myc Santa Cruz Biotechnology Cat#9E10 Alexa Fluor 488 goat anti-mouse IgG (H+L) Molecular probe Cat#11001 Alexa Fluor 568 goat anti-mouse IgG (H+L) Molecular probe Cat#11011 Goat polyclonal antibodies-HRP against mouse IgG Promega Cat#W402B Goat polyclonal antibodies-HRP against rabbit IgG Promega Cat#W401B Bacterial and Virus Strains DH5a E. coli Strain used for cloning Takara Cat#9057 BL21 gold E. coli Strain used for protein purification Agilent Technology Cat#230130 Chemicals, Peptides, and Recombinant Proteins aphidicolin Sigma A0781 centrinone Wong et al., 2015 N/A Flag antibody-conjugated M2 agarose Sigma Cat#A2220 SimplyBlue Safe Invitrogen Cat#LC6060 Experimental Models: Cell Lines Human: U2OS ECACC Cat#92022711 Human: HEK293T ECACC Cat#12022001 Oligonucleotides Stealth siRNA targeting sequence: 30UTR of HsSAS-6 (50-GAGCUGUUAAAGACUGGAUACUUUA-30) Life Technologies N/A Negative control Low GC Duplex #2 for siRNA treatment Life Technologies Cat#12935110 siRNA targeting sequence: 30UTR of Plk4 (50-CTCCTTTCAGACATATAAG-30) Sigma Genosys N/A siRNA targeting sequence: 30UTR of STIL (50-GTTTAAGGGAAAAGTTATT-30) JBios N/A siRNA targeting sequence: ORF of Cep152 (50-GCGGATCCAACTGGAAATCTA-30) JBios N/A Silencer select siRNA targeting sequence: ORF of STIL Life Technologies Cat#s12863 Negative control for siRNA treatment Life Technologies Cat#4390843 Recombinant DNA pcDNA3-Plk4-Flag full-length and Ddegron constructs Yamashita et al., 2001 N/A pcDNA14-Plk4-3Flag full-length and mutant constructs This study N/A (Continued on next page) e1 Cell Reports 23, 3160–3169.e1–e4, June 12, 2018

Techniques: Staining, Super-Resolution Microscopy

Journal: Cell reports

Article Title: Bimodal Binding of STIL to Plk4 Controls Proper Centriole Copy Number.

doi: 10.1016/j.celrep.2018.05.030

Figure Lengend Snippet: Figure 2. The C-Terminal Region of STIL, Rather Than the Coiled Coil, Is Essential for Stimulating Plk4 Kinase Activity (A) HEK293T cells expressing Plk4-3Flag together with HA, HA-STIL, HsSAS-6-GFP, or HA-Cep152 were analyzed by western blotting using indicated anti- bodies. Tubulin was used for loading control. The values at the bottom indicate the average ratios (relative to the HA co-expression) of Plk4-3Flag protein level from two independent experiments. (legend continued on next page)

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit polyclonal anti-STIL Bethyl laboratories Cat#A302-441A Rabbit polyclonal anti-STIL Abcam Cat#ab89314 Rabbit polyclonal anti-Cep152 Bethyl laboratories Cat#A302-479A Rabbit polyclonal anti-Cep152 Sonnen et al., 2013 N/A Rabbit polyclonal anti-phospho-S305 Plk4 Sillibourne et al., 2010 N/A Rabbit polyclonal anti-Cep192 Bethyl laboratories Cat#A302-324A Rabbit polyclonal anti-HA Abcam Cat#ab9110 Mouse monoclonal anti-centrin-2 Millipore Cat#20H5 Mouse monoclonal anti-centrin-HsSAS-6 Santa Cruz Bio-technology Cat#sc-81431 Mouse monoclonal anti-Plk4 Merck Millipore clone 6H5, Cat#MABC544 Mouse monoclonal anti-Flag Sigma Cat#F1804 Mouse monoclonal anti-HA Covance Cat#MMS-101P Mouse monoclonal anti-a-tubulin Sigma Cat#DM1A Mouse monoclonal anti-Myc Santa Cruz Biotechnology Cat#9E10 Alexa Fluor 488 goat anti-mouse IgG (H+L) Molecular probe Cat#11001 Alexa Fluor 568 goat anti-mouse IgG (H+L) Molecular probe Cat#11011 Goat polyclonal antibodies-HRP against mouse IgG Promega Cat#W402B Goat polyclonal antibodies-HRP against rabbit IgG Promega Cat#W401B Bacterial and Virus Strains DH5a E. coli Strain used for cloning Takara Cat#9057 BL21 gold E. coli Strain used for protein purification Agilent Technology Cat#230130 Chemicals, Peptides, and Recombinant Proteins aphidicolin Sigma A0781 centrinone Wong et al., 2015 N/A Flag antibody-conjugated M2 agarose Sigma Cat#A2220 SimplyBlue Safe Invitrogen Cat#LC6060 Experimental Models: Cell Lines Human: U2OS ECACC Cat#92022711 Human: HEK293T ECACC Cat#12022001 Oligonucleotides Stealth siRNA targeting sequence: 30UTR of HsSAS-6 (50-GAGCUGUUAAAGACUGGAUACUUUA-30) Life Technologies N/A Negative control Low GC Duplex #2 for siRNA treatment Life Technologies Cat#12935110 siRNA targeting sequence: 30UTR of Plk4 (50-CTCCTTTCAGACATATAAG-30) Sigma Genosys N/A siRNA targeting sequence: 30UTR of STIL (50-GTTTAAGGGAAAAGTTATT-30) JBios N/A siRNA targeting sequence: ORF of Cep152 (50-GCGGATCCAACTGGAAATCTA-30) JBios N/A Silencer select siRNA targeting sequence: ORF of STIL Life Technologies Cat#s12863 Negative control for siRNA treatment Life Technologies Cat#4390843 Recombinant DNA pcDNA3-Plk4-Flag full-length and Ddegron constructs Yamashita et al., 2001 N/A pcDNA14-Plk4-3Flag full-length and mutant constructs This study N/A (Continued on next page) e1 Cell Reports 23, 3160–3169.e1–e4, June 12, 2018

Techniques: Activity Assay, Expressing, Western Blot, Control

Journal: Cell reports

Article Title: Bimodal Binding of STIL to Plk4 Controls Proper Centriole Copy Number.

doi: 10.1016/j.celrep.2018.05.030

Figure Lengend Snippet: Figure 3. Direct Interaction between the STIL STAN and the First Linker Region of Plk4 (A and B) GST pull-down assay was performed using recombinant GST-Plk4 FL wild-type (WT) or kinase-dead (KD) with HA-STIL[N3] (A) or HA-STIL [C] (B). Precipitant materials were analyzed by western blotting using indicated antibodies. (C) Diagrams of the Plk4 and STIL proteins. Each fragment used for the GST pull-down assay is shown in below. The minimum regions of Plk4 and STIL for their interaction are shown in red. The position of ND is shown as a red line in the degron motif. (D and E) GST pull-down assay was performed using recombinant GST-Plk4 FL WT, kinase-dead, or fragments (L1PB3 and PB1-3 in D, L1 in E) with HA-STIL[C]. Precipitant materials were analyzed by western blotting using HA antibodies or by blue staining (BS). (F) GST pull-down assay was performed using recombinant GST-Plk4[L1PB3] with STIL frag- ments (C, CS, and CSC). Precipitant materials were analyzed by western blotting using STIL antibody or by BS. (G) (Top) Schematic diagram of the experimental procedure. HEK293T cells expressing HA-STIL[C] alone or together with Plk4ND-3Flag WT or KD were treated with 10 mM MG132 for 6 hr. (Bottom) The lysates were subjected to co-immunoprecip- itation assay and analyzed by western blotting using indicated antibodies. See also Figure S4.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit polyclonal anti-STIL Bethyl laboratories Cat#A302-441A Rabbit polyclonal anti-STIL Abcam Cat#ab89314 Rabbit polyclonal anti-Cep152 Bethyl laboratories Cat#A302-479A Rabbit polyclonal anti-Cep152 Sonnen et al., 2013 N/A Rabbit polyclonal anti-phospho-S305 Plk4 Sillibourne et al., 2010 N/A Rabbit polyclonal anti-Cep192 Bethyl laboratories Cat#A302-324A Rabbit polyclonal anti-HA Abcam Cat#ab9110 Mouse monoclonal anti-centrin-2 Millipore Cat#20H5 Mouse monoclonal anti-centrin-HsSAS-6 Santa Cruz Bio-technology Cat#sc-81431 Mouse monoclonal anti-Plk4 Merck Millipore clone 6H5, Cat#MABC544 Mouse monoclonal anti-Flag Sigma Cat#F1804 Mouse monoclonal anti-HA Covance Cat#MMS-101P Mouse monoclonal anti-a-tubulin Sigma Cat#DM1A Mouse monoclonal anti-Myc Santa Cruz Biotechnology Cat#9E10 Alexa Fluor 488 goat anti-mouse IgG (H+L) Molecular probe Cat#11001 Alexa Fluor 568 goat anti-mouse IgG (H+L) Molecular probe Cat#11011 Goat polyclonal antibodies-HRP against mouse IgG Promega Cat#W402B Goat polyclonal antibodies-HRP against rabbit IgG Promega Cat#W401B Bacterial and Virus Strains DH5a E. coli Strain used for cloning Takara Cat#9057 BL21 gold E. coli Strain used for protein purification Agilent Technology Cat#230130 Chemicals, Peptides, and Recombinant Proteins aphidicolin Sigma A0781 centrinone Wong et al., 2015 N/A Flag antibody-conjugated M2 agarose Sigma Cat#A2220 SimplyBlue Safe Invitrogen Cat#LC6060 Experimental Models: Cell Lines Human: U2OS ECACC Cat#92022711 Human: HEK293T ECACC Cat#12022001 Oligonucleotides Stealth siRNA targeting sequence: 30UTR of HsSAS-6 (50-GAGCUGUUAAAGACUGGAUACUUUA-30) Life Technologies N/A Negative control Low GC Duplex #2 for siRNA treatment Life Technologies Cat#12935110 siRNA targeting sequence: 30UTR of Plk4 (50-CTCCTTTCAGACATATAAG-30) Sigma Genosys N/A siRNA targeting sequence: 30UTR of STIL (50-GTTTAAGGGAAAAGTTATT-30) JBios N/A siRNA targeting sequence: ORF of Cep152 (50-GCGGATCCAACTGGAAATCTA-30) JBios N/A Silencer select siRNA targeting sequence: ORF of STIL Life Technologies Cat#s12863 Negative control for siRNA treatment Life Technologies Cat#4390843 Recombinant DNA pcDNA3-Plk4-Flag full-length and Ddegron constructs Yamashita et al., 2001 N/A pcDNA14-Plk4-3Flag full-length and mutant constructs This study N/A (Continued on next page) e1 Cell Reports 23, 3160–3169.e1–e4, June 12, 2018

Techniques: Pull Down Assay, Recombinant, Western Blot, Staining, Expressing

Journal: Cell reports

Article Title: Bimodal Binding of STIL to Plk4 Controls Proper Centriole Copy Number.

doi: 10.1016/j.celrep.2018.05.030

Figure Lengend Snippet: Figure 4. The Conserved TIM Domain of STIL Limits the Localization of Plk4 at Centrioles and Procentriole Number (A) Diagrams of STIL domains and mutants used in this figure. Replacements of amino acids were shown in red. (B) Alignment of the TIM domain within human, mouse, Xenopus and zebrafish STIL, and Drosophila Ana2. Identical residues are colored in yellow; similar residues are in gray. Asterisks: the residues identical in all aligned sequences; colons: conserved substitutions; periods: semi-conserved substitutions. Red asterisks indicate the residues, which are changed in the LR-AC mutant. (C) HEK293T cells expressing Plk4ND-3Flag together with HA vector, HA-STIL FL, DTIM (aaD1268–1287), LR-AC (L1280A, R1281C), or 5A (S1061A, S1116A, S1181A, T1238A, and T1250A) were analyzed by western blotting using anti-Flag, STIL, or tubulin antibodies. The histograms depict the average ratios (relative to the HA co-expression) of phosphorylated Plk4ND (p-Plk4ND) to the total Plk4ND protein level. The values are mean ±SEM from three independent experiments. *p < 0.05, **p < 0.01, n.s., not significant relative to the HA co-expression. (D) In vitro kinase assay was performed using recombinant GST-Plk4[KL1] and HA-STIL[C], [CLRAC], [CDTIM], and [TIM: aa 1268–1287]. The incorporation of [g-32P] ATP to the substrates and the loaded proteins were analyzed in the same way as in Figure 2G. Protein amount of TIM fragment was detected by western blotting using anti-STIL antibody. (E and F) U2OS cells were treated with control siRNA (siCnt) or siRNA targeting 30 UTR of endogenous STIL (siSTIL), followed by transfection with an HA vector (HA), HA-STIL full-length WT, DTIM, or LR-AC mutant. The cells were immunostained with antibodies against HA and Plk4 (E) or centrin (F). (E) Signal intensities of centriolar localization of endogenous Plk4 and HA-STIL proteins were shown in the box-and-whisker plot (N R 33). (F) Quantification of fraction of centrosomes with the indicated number of daughter centrioles from three independent experiments (N R 30, the values are mean ± SEM). *p < 0.05, **p < 0.01, n.s., not significant. Scale bar, 0.5 mm. See also Figure S4.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit polyclonal anti-STIL Bethyl laboratories Cat#A302-441A Rabbit polyclonal anti-STIL Abcam Cat#ab89314 Rabbit polyclonal anti-Cep152 Bethyl laboratories Cat#A302-479A Rabbit polyclonal anti-Cep152 Sonnen et al., 2013 N/A Rabbit polyclonal anti-phospho-S305 Plk4 Sillibourne et al., 2010 N/A Rabbit polyclonal anti-Cep192 Bethyl laboratories Cat#A302-324A Rabbit polyclonal anti-HA Abcam Cat#ab9110 Mouse monoclonal anti-centrin-2 Millipore Cat#20H5 Mouse monoclonal anti-centrin-HsSAS-6 Santa Cruz Bio-technology Cat#sc-81431 Mouse monoclonal anti-Plk4 Merck Millipore clone 6H5, Cat#MABC544 Mouse monoclonal anti-Flag Sigma Cat#F1804 Mouse monoclonal anti-HA Covance Cat#MMS-101P Mouse monoclonal anti-a-tubulin Sigma Cat#DM1A Mouse monoclonal anti-Myc Santa Cruz Biotechnology Cat#9E10 Alexa Fluor 488 goat anti-mouse IgG (H+L) Molecular probe Cat#11001 Alexa Fluor 568 goat anti-mouse IgG (H+L) Molecular probe Cat#11011 Goat polyclonal antibodies-HRP against mouse IgG Promega Cat#W402B Goat polyclonal antibodies-HRP against rabbit IgG Promega Cat#W401B Bacterial and Virus Strains DH5a E. coli Strain used for cloning Takara Cat#9057 BL21 gold E. coli Strain used for protein purification Agilent Technology Cat#230130 Chemicals, Peptides, and Recombinant Proteins aphidicolin Sigma A0781 centrinone Wong et al., 2015 N/A Flag antibody-conjugated M2 agarose Sigma Cat#A2220 SimplyBlue Safe Invitrogen Cat#LC6060 Experimental Models: Cell Lines Human: U2OS ECACC Cat#92022711 Human: HEK293T ECACC Cat#12022001 Oligonucleotides Stealth siRNA targeting sequence: 30UTR of HsSAS-6 (50-GAGCUGUUAAAGACUGGAUACUUUA-30) Life Technologies N/A Negative control Low GC Duplex #2 for siRNA treatment Life Technologies Cat#12935110 siRNA targeting sequence: 30UTR of Plk4 (50-CTCCTTTCAGACATATAAG-30) Sigma Genosys N/A siRNA targeting sequence: 30UTR of STIL (50-GTTTAAGGGAAAAGTTATT-30) JBios N/A siRNA targeting sequence: ORF of Cep152 (50-GCGGATCCAACTGGAAATCTA-30) JBios N/A Silencer select siRNA targeting sequence: ORF of STIL Life Technologies Cat#s12863 Negative control for siRNA treatment Life Technologies Cat#4390843 Recombinant DNA pcDNA3-Plk4-Flag full-length and Ddegron constructs Yamashita et al., 2001 N/A pcDNA14-Plk4-3Flag full-length and mutant constructs This study N/A (Continued on next page) e1 Cell Reports 23, 3160–3169.e1–e4, June 12, 2018

Techniques: Mutagenesis, Expressing, Plasmid Preparation, Western Blot, In Vitro, Kinase Assay, Recombinant, Control, Transfection, Whisker Assay