Valentina Quarantotti et al The EMBO Journal Expanded View Figures Figure EV1. The effect of combined nocodazole A PCM1 locus and cytochalasin-B treatment on the distribution of endogenously labelled PCM1-GFP in DT40 cells. 32 33 3435 UTR A Diagram showing the GFP construct used to target the chicken PCM1 locus at the C-terminus on both alleles, by homologous recombination. Highlighted the Sal1 and BamH1 sites used for 2.8 kb 0.7 kb ~ 3 kb 3.3 kb restriction digestion to clone the LA (Left Arm) LA GFP Blasti/Puro/His RA and the RA (Right Arm) and to replace the resistance cassette. Clones were screened for Sal1BamH1 BamH1 Sal1 antibiotic resistance genes blasticidin (Blasti), puromycin (Puro) or Histidinol (His). LoxP sites B flanking the resistance cassette are represented by red triangles. The dashed lines indicate the sites of recombination and integration in the GFP γ-tubulin GFP, γ-tubulin, DNA PCM1 locus. Confirmation of targeting was carried out by Western blotting, as shown in Fig 1B–D. B Representative immunofluorescence images of DMSO cell lines with genotypes as indicated, treated with both nocodazole (2 lg/ml) and cytochalasin-B (1 lg/ml). DMSO-treated cells were used as a control (DMSO, upper panels). 2 PCM1-GFP Treatments were carried out for h, and cells were co-stained with antibodies against GFP WT (green) and c-tubulin (red). DNA is in blue. Nocodazole + Images correspond to maximum intensity Cytochalasin-B projections of confocal micrographs. Asterisks mark cells with dispersed satellites. Note that drug treatment leads to an increase in large and a decrease in small satellite granules in all three genotypes, but the effects are more prominent in acentriolar than in WT cells. Scale bars: 5 lm. DMSO * * PCM1-GFP STIL-KO Nocodazole + Cytochalasin-B DMSO * PCM1-GFP CEP152-KO Nocodazole + Cytochalasin-B ª 2019 The Authors The EMBO Journal e101082 | 2019 EV1 The EMBO Journal Valentina Quarantotti et al Figure EV2. Comparisons of CS-WT with published datasets. ▸ A Venn diagram showing the number of proteins identified in three datasets: CS-WT, PCM1-BioID (Gupta et al, 2015) and PCM1-FLAG IP (Gupta et al, 2015). Note that this and all subsequent analyses were performed on human orthologues of the chicken proteins from CS-WT. B Venn diagrams showing the number of published CS components (illustrated in the table in C) detected in each of the indicated datasets. C Table depicts previously reported CS components and their detection in the indicated datasets. D Venn diagram showing the number of centrosomal proteins based on the human centrosome proteome dataset (Jakobsen et al, 2011) detected in each of the indicated datasets. Note that the majority of the proteins detected in both CS-WT and PCM1-BioID are centrosomal proteins (33/43). E Venn diagram showing the overlap between proteins identified in CS-WT and in a functional screen for positive regulators of Hedgehog signalling (Breslow et al, 2018). EV2 The EMBO Journal e101082 | 2019 ª 2019 The Authors Valentina Quarantotti et al The EMBO Journal AB C Published CS components Number of published CS components BBS4 FOPNL CCDC112 PCM1-BioID in indicated datasets CS-WT (Gupta et al., 2015) CDK1 HAUS6 LRRC49 PCM1-BioID CS-WT (Gupta et al., 2015) CEP95 WDR67 ODF2L CEP290 PCM1 TEX9 176 29 84 # 11 9 5 DZIP1 CCDC14 CCDC11 14 4 15 HOOK3 CEP90 CCDC13 8 # 0 5 NIN CEP131 CCDC113 15 PCNT KIAA0753 CEP126 0 PRKACB MIB1 CETN3** PCM1-FLAG IP SDCCAG8 OFD1 DISC1 (Gupta et al., 2015) PCM1-FLAG IP SPAG5 SSX2IP HAP1 (Gupta et al., 2015) C2CD3 CCNB2* HTT # D CCDC18 CEP72 KIZUNA CCDC66 NPHP1* Number of centrosomal proteins CSPP1** in indicated datasets CEP63 MED4 PAR6a PCM1-BioID CEP89 WRAP73* TCTN3 CS-WT (Gupta et al., 2015) FOP C11ORF49 VPS4 Present exclusively in CS-WT 48 22 16 Present in CS-WT and PCM1-BioID Present in all datasets 11 Present in PCM1-BioID and PCM1-FLAG IP 1 7 Present exclusively in PCM1-BioID Absent from all datasets 1 *Absent from CS-WT, but present in CS-STIL and/or CS-CEP152 **Detected in Filtered Data from WTPCM1-GFP cells PCM1-FLAG IP and present in CS-STIL and CS-CEP152 (Gupta et al., 2015) #Not in Gallus gallus genome E Positive regulators of Hedgehog signalling CS-WT (Breslow et al., 2018) 193 30 442 Centrosome/transition zone components Other proteins BBS7 FOPNL ATP5B EDC4 C2CD3 KIAA0753 BBS2/4/9 HSP90B1 CBY1 OFD1 CAPZB KIF7 CEP44 PPP2R3 CUL3 LONP1 CEP76 SLMAP DNAJC13 SETDB1 CEP90 TEDC1 CEP120 TTC8/BBS8 CEP295 TUBD1 FAM92A FOP Figure EV2. ª 2019 The Authors The EMBO Journal e101082 | 2019 EV3 The EMBO Journal Valentina Quarantotti et al Figure EV3. Localisation of centriolar satellite candidates in Jurkat cells. ▸ A Representative immunofluorescence images of Jurkat cells co-stained with antibodies against selected new CS candidates (green) and PCM1 (red). SSX2IP, a known CS component, is shown as positive control. The framed panel at the bottom illustrates the relative distributions of the centrosomal marker c-tubulin (red) and the CS protein PCM1 (green) in Jurkat cells. DNA is in blue. Images correspond to maximum intensity projections of confocal micrographs. High magnification images are included to aid visualisation of framed areas. Scale bars: 5 lm. B Representative immunofluorescence images of Jurkat cells mock-treated with DMSO or incubated with nocodazole (2 lg/ml) to depolymerise microtubules. Cells were co-stained with antibodies against PCM1 (green), c-tubulin (red) and a-tubulin (blue). Nocodazole reduces PCM1 signal in the pericentrosomal region. High magnification images are included to aid visualisation of framed areas and correspond to framed areas. Scale bar: 5 lm. C Knock-down efficiency of siRNAs assessed by qPCR. The relative expression of each candidate upon siRNA treatment was assessed by qPCR and shown relative to cells treated with a control siRNA. Each datapoint represents a biological replicate. Note that T3JAMsi1 enhances rather than reduces mRNA expression. Bar graphs show mean Æ SE. EV4 The EMBO Journal e101082 | 2019 ª 2019 The Authors Valentina Quarantotti et al The EMBO Journal A B γ-tubulin PCM1 PCM1, γ-tubulin, DNA DMSO Nocodazole PCM1 PCM1 CS candidate PCM1 CS candidate, PCM1, DNA SSX2IP γ-tubulin γ-tubulin BICD2 CEP63 α-tubulin α-tubulin CEP170 CEP215 -tubulin α CETN2 , -tubulin γ , CP110 PCM1 PCM1 SPICE1 PCM1 γ-tubulin WDR90 C BICD2 CCDC77 CEP170 HERC2 MYCBP2 T3JAM TRIM37 TRIM41 WDR37 3.0 2.5 2.0 1.5 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 i i i i i i2 s s 1 2 1 i1 i2 s 1 s 1 2 s l s si s s si si2 l si si1 ol o rol ol tr tr trol si 37 37 si3 tro 41 41 si m m m dr37 Bicd2Bicd2 si1 3jam i i Control si Con Con ContHerc2Herc2 si Con ControlT3jam siT ContrTr Trim37Tr ConTrimTri ControlW siWdr37 si2 Ccdc77Ccdc77 si1 si2 Cep170Cep170 si1 si2 MycBP2MycBP2 si si2 Figure EV3. ª 2019 The Authors The EMBO Journal e101082 | 2019 EV5 The EMBO Journal Valentina Quarantotti et al Figure EV4. SILAC-based quantitative MS analyses of whole-cell and centriolar satellite proteomes of WTPCM1-GFP and STIL-KOPCM1-GFP cell lines. ▸ A Reproducibility of SILAC-WCP experiments. B, C Reproducibility of SILAC-CS experiments. Scatterplots (in red) showing the reproducibility and quantile–quantile plots (in blue) showing similar ratio distribution between the replicates, for the GFP pull-downs (B) and the IgG CT pull-downs (C). Protein ratios of the reverse experiment have been inverted. D Venn diagram showing the overlap between the proteins down-regulated in SILAC-CS-STIL and the centrosome proteome dataset (Jakobsen et al, 2011). E Venn diagram showing the overlap between the proteins up-regulated in SILAC-CS-STIL and the centrosome proteome dataset (Jakobsen et al, 2011). Note that centrosomal proteins are under-represented among the up-regulated proteins. EV6 The EMBO Journal e101082 | 2019 ª 2019 The Authors Valentina Quarantotti et al The EMBO Journal A WCP B PCM1-GFP PCM1-GFP Log2 FC, WT /STIL KO -6 -2 2 6 -6 -2 2 6 -6 -2 2 6 SILAC-CS: GFP pull-downs PCM1-GFP PCM1-GFP 6 Log FC, STIL-KO /WT STIL(H)/ 2 WT(L) 2 (Forward) -6 -4 -2 0 2 4 -2 replicate #1 Q−Q plot Q−Q plot Q−Q plot Q−Q plot Q−Q plot STIL(L)/ -6 WT(H) STIL(H)/ (Forward) WT(L) replicate #1 Q−Q plot Q−Q plot (Forward) replicate #2 Q−Q plot Q−Q plot Q−Q plot Q−Q plot STIL(L)/ -6 -2 2 6 WT(H) STIL(H)/ (Forward) WT(L) replicate #2 Q−Q plot (Forward) -6 -4 -2 0 2 4 replicate #3 -2 2 6 Q−Q plot Q−Q plot Q−Q plot STIL(H)/ -6 WT(L) STIL(H)/ (Reverse) WT(L) replicate #1 (Forward) -6 -4 -2 0 2 4 -6 -4 -2 0 2 4 -6 -4 -2 0 2 4 -6 -4 -2 0 2 4 replicate #4 Q−Q plot Q−Q plot -6 -2 2 6 STIL(L)/ WT(H) (Reverse) -2 2 6 C replicate #1 Q−Q plot -6 STIL(L)/ SILAC-CS: IgG pull-downs WT(H) Log FC, STIL-KOPCM1-GFP/WTPCM1-GFP (Reverse) 2 replicate #2 -6 -4 -2 0 2 4 -6 -2 2 6 -6 -2 2 6 -6 -2 2 6 -6 -2 2 6 STIL(L)/ WT(H) (Forward) -2 0 2 4 replicate #1 -4 D Q−Q plot Q−Q plot Proteins down in STIL Centrosome proteome STIL(L)/ (SILAC-CS) (Jakobsen et al., 2011) WT(H) (Forward) -4 -2 0 2 4 replicate #2 Q−Q plot -6 1829 136 STIL(H)/ WT(L) (Reverse) replicate #1 -4 -2 0 2 4 -6 -6 -6 -4 -2 0 2 4 -6 -4 -2 0 2 4 E Proteins up in STIL Centrosome proteome (SILAC-CS) (Jakobsen et al., 2011) 214 161 Figure EV4.