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