Figure S1 (Kim et al) Introduction into the Saccharomyces cerevisiae reporter strain AH109 (Trp-/Leu-/His-/Ade-) pGBKT7-hsRad21 human fetal kidney cDNA library in the pACT2 vector containing the GAL4 activation domain + Grown at 30 for 3 to 5 days on SD agar plates (Trp-/Leu-/His-/Ade-) in the presence of X--Gal Cloning & sequencing MPSSPLRVAVVCSSNQNRSMEAHNILSKRGFSVRSFGTGTHVKLPGPAPD KPNVYDFKTTYDQMYNDLLRKDKELYTQNGILHMLDRNKRIKPRPERFQNC KDLFDLILTCEERVYDQVVEDLNSREQETCQPVHVVNVDIQDNHEEATLGAF LICELCQCIQHTEDMENEIDELLQEFEEKSGRTFLHTVCFY A shSsu72 #2 shLuc Myc-Ssu72 Endo Ssu72 Myc- Ssu72 Ssu72 (monoclonal) C Figure S2 (Kim et al) shLuc shSsu72 #1 shSsu72 #2 Endo Ssu72 Ssu72 (polyclonal) D B shLuc shSsu72 #1 shSsu72 #2 Ssu72 (monoclonal) (KDa) Endo Ssu E DAPI Ssu72 Rad21 Merge shLuc shSsu72#2 Ssu72 (monoclonal) shSsu72#1 A Figure S3 (Kim et al) InterProPrometaMetaAnaTelo DAPI Ssu72 Rad21 Merge B DAPI Ssu72 Rad21 Merge InterProPrometaMetaAnaTelo Pre-extraction Figure S4 (Kim et al) A B H2B-GFP Rad21-RFP Phase contrast Merge HeLa -Rad21-RFP + pMyc HeLa -Rad21-RFP + pMyc-Ssu72 H2B-GFP SA2-RFP HeLa -SA2-RFP + shLuc Phase contrast Merge HeLa -SA2-RFP + shSsu72 (live cell imaging) Figure S5 (Kim et al) HeLa-SA2-RFP + pHA-Ssu72 SA2-RFP H2B-CFP HeLa-SA2-RFP + pHA 2 m4 m5 m6 m 0 m MBP-GFP Bleaching NEBD SA2-RFP H2B-CFP MBP-GFP Bleaching NEBD 2 m4 m6 m8 m0 m10 m16 m Asy (h) HeLa -Con HeLa -HA-Ssu72 Asy (h) Thy-DB/Release HA (Ssu72) Plk1 Cyclin B1 Securin Actin Asy (h) Asy ControlHA-Ssu72 Thy-DB / Release A B Figure S6 (Kim et al) C 01:02:50 Control NEBD 00:00:0000:08:5800:26:5600:47:5300:53:5100:56:5100:59:51 00:00:0200:09:0000:26:5800:47:5500:53:5400:56:5400:59:5301:02:52 HA-Ssu72 NEBD H2B- GFP 00:00:0000:10:3000:26:5400:42:5200:55:4901:20:4201:45:5001:57:5202:15:4502:29:3703:10:3003:39:4703:42:4703:45:47 00:00:0200:10:3200:26:5600:42:5400:55:5101:20:4401:45:5201:57:5502:15:4702:29:3903:10:3203:39:5003:42:4903:45:49 D Mitotic duration (min) 142 min 62 min ControlHA-Ssu72 E Time duration (NEBD to anaphase onset) Asy (h) 2n 4n 2n 4n Asy (h) shLuc shSsu72 Figure S7 (Kim et al) Human Drosophila Human Drosophila Human Drosophila Human Drosophila A B C D Cys13 Arg19 Asn15 Asn17 Asp143 Met16 D-box LxCxE Polo-box binding PPase E GST-Ssu72 WT GST-Ssu72 C12S GST-Ssu72 1-12 Phosphatase Activity (pNPP at 405 nm) Protein concentration ( g) GST-Ssu72 GST-Ssu72 + Na 3 VO (M) Na 3 VO 4 concentration GST-Ssu72 WT GSTSsu72 1-12 GST GST-Ssu72 C12S FG Figure S8 (Kim et al) PPase LxCxE D-box Polo-box binding SA2 4A 1232 T1079A T1112AT1118AT1151A X X X X 1 A Figure S9 (Kim et al) B shSsu72 + Myc-SA2 4A shSsu72 + Myc-SA2 WTshSsu72 shLuc C shSsu72 + Myc-SA2 4A shSsu72 + Myc-SA2 WTshSsu72 shLuc Myc (SA2) Ssu72 Actin Gene5 primer3 primer Smc1att cag agc cga gag agg gatct tgg cga ttt cat tct gc Smc3tgg agc gct gga aaa ata tgcct ggg gaa gtg atc caa gt Rad21caa tgc caa cca tga ctg atcgg tgt aag aca gcg tgt aaa SA2tca ccg gcc agt agc agt agccc aca tgc tat cca caa gg GAPDHggc atg gac tgt ggt cat gagtgc acc acc aac tgc tta gc A B Smc1 Smc3 Rad21 Con Ssu72 SA2 Marker * * * * 100 bp 200 bp Figure S10 (Kim et al) C