Pei et al. Supplementary Figure S1

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1 Pei et al. Supplementary Figure S1 C H-CUL9: Myc-ROC1: U2OS/pcDN3 U2OS/H-CUL9 U2OS/ + H-CUL9 IP: -H -myc input -H -myc H-CUL9 Myc-ROC1 -H -H -H -H H-CUL9: wt RR myc-roc1: IP: -H E1 + Ubc P-Ub + TP P-(Ub)n 32 P-Ub Nucleus (75%) Nuc. & cyto. (25%) Cytoplasm (0%) Nucleus (75%) Nucleus & Cytoplasm (20%) localization Cytoplasm (5%) Supplementary Figure S1. CUL9 binds to ROC1, localizes in the cytoplasm, but does not sequesters to the cytoplasm.. CUL9 binds with ROC1. H- CUL9 was co-transfected 293T cells with ROC1 and its association was examined by reciprocal IP-Western.. CUL9 immunocomplexes contain ubiquitin ligase activity. H-tagged wild-type or mutant CUL9 with the two C-terminal RING fingers deleted ( RR) were transiently transfected into 293T cells, either alone or with ROC1. H immunocomplexes were incubated in vitro with E1, E2 (UbcH5) and 32 P-labeled ubiquitin in the presence of TP. Self-ubiquitinylation ligase activity (E2 activation) was assayed by the synthesis of polyubiquitin chain. C. CUL9 does not sequester in the cytoplasm. Subcellular localization of endogenous or ectopically expressed in the absence or presence of ectopic expression of CUL9 was analyzed by indirect immunofluorescence in U2OS cells. Subcellular localization of was categorized into three groups and the percentage of each group was calculated from microscopic examination of approximately 300 cells.

2 m Locus Pei et al. Supplementary Figure S2 X N TG X N X E1 E2 E3 E4 E7E8 E9 Targeting Construct Targeted llele Conditional llele Deleted llele TK NEO E1 E2 E3 E4 E7 E8 E9 X N N X N X NEO Probe a b c E1 Flp E9 X N TG X N X X N E1 E1 a d N E8 c X Cre e E9 FLP site E9 Cre site C D E mt WT mt WT kb 0.5 kb 1.4 kb 0.16 kb Cre-treated 3D 3H 1F GPDH F f/f / 1 2 G - DPI / f/f H f/f / f/f / UV 1 h 2 h 2 1 h 2 h 2 UV 2 h 2 h ax MDM2 p21 Supplementary Figure S2. Generation and characterization of mutant mouse and ES cells, Schematic presentation of targeting strategy for generating null ES cells kb fragment of mouse genomic DN, spanning from the promoter region to exon 9, was amplified by PCR from mouse ES cell genomic DN and verified by DN sequencing. Lox P sites were inserted at the 5 and 3 ends of the region to be deleted. To select for homologous recombination, a neomycin resistance gene, flanked by frt

3 sites, was inserted immediately upstream of the 5 lox P deletion site, and a thymidine kinase negative selection marker was inserted upstream of exon 1., Isolation of NeoFlox/+ heterozygous ES clones. The linearized targeting construct was electroporated into ES cells, G418 and ganciclovir doubly-resistant clones were screened for homologous recombination by Southern blotting. Genomic DN from ES cells was digested by Xho1 and hybridized with the probe. C, Isolation of Flox/+ ( F/+ ) heterozygous ES clones. NeoFlox/+ ES cells were transfected with an expression vector encoding Flp recombinase, neo-removed F/+ heterozygous ES clones were screened and isolated by PCR with the primers a and b. te that 0.5 kb-fragment shows the DN amplified from neo-removed F allele, while 1.7 kb-fragment shows the DN amplified from Neoflox allele. D, Generation of F/NeoFlox and -/- ES clones. F/+ ES cells were retargeted with the same targeting construct, followed by another round of G418 and ganciclovir double-selection and screening for homologous recombination by PCR and Southern blotting. F/NeoFlox ES clones were expanded and transfected with CMV-Cre vector or infected with retroviral Cre. Deletion of exons 2-7 from both alleles was confirmed by genomic PCR using primers a and c. te that 0.16 kb-fragment shows the DN amplified from neo-removed and exons 2-7 deleted allele, while 1.4 kb-fragment shows the DN amplified from neo-unremoved but exons 2-7 deleted Neo allele. E, Reduced or loss of gene expression was determined by RT-PCR. RN was isolated from F/F ES cells and analyzed by RT-PCR with primers d and e. te that Cre-mediated deletion was incomplete in Clone3H, resulting in a reduction, but not complete loss, of expression. F, protein level is unchanged in the absence of. Western blot analysis of steady state levels in total protein lysates of F/F and -/- ES cells using antibodies as indicated. G, Loss of in ES cells did not change localization. Subcellular localization of endogenous in F/F and -/- ES was analyzed by indirect immunofluorescence. H, Cell extracts were prepared from F/F and -/- ES cells at the different time points after exposure to 50 J/m 2 UV irradiation. The expression of individual proteins was determined by Western blot.

4 Pei et al. Supplementary Figure S3 a Pituitary tumor (T516, -/-, 26 M) b Sarcoma (T505, -/-, 25 M) c Lymphoma (T449, -/-, 21 M) d Lung denoma (T584, -/-, 16 M) Liver e Hepatocelluar tumor (T450, -/-, 21 M) f Lung adenoma (T342, +/-, 12 M) g Insulinoma (T333, +/-, 13 M) h Pituitary tumor (T451, +/-, 21M)

5 Supplementary Figure S3. Representative tumors developed in mutant mice. Insets in a and b showing the gross picture of the tumors, in c showing the liver periportal invasion of lymphoma, and in g and h showing the lower magnification picture of the tumors. M, month.

6 Pei et al. Supplementary Figure S4 Lung Liver Pancrease Pre- lymphoma lymphoma +/+ ;E -Myc + IgM 3% 83% 53% 42% one marrow +/- ;E -Myc + 11% 79% 83% 1% Spleen 220 Supplementary Figure S4. deficiency accelerates E -Myc-induced lymphomagenesis.. H.E. staining of representative lymphoma developed in 3 month old mice, illustrating lymphoma invasion into lung, liver and pancreases in a +/- ;E -Myc+ mouse.. FCS analysis of representative pre- and lymphomas. one marrow or spleen cells prepared from two representative mice at two months of age were stained with 220 and IgM. The expression of 220+IgM low and 220+IgM high confirmed the origin of tumors as pre- lymphoma and lymphoma, respectively.

7 Pei et al. Supplementary Figure S5 Genotype (Mouse ID) WT (755) WT (756) WT (774) +/- (757) +/- (758) +/- (760) +/- (761) +/- (770) +/- (773) -/- (762) -/- (768) -/- (769) -/- (771) -/- (772) -/- (759) Lung Liver tumor Lymphoma Tumor 1 Yes (1) Yes (2) Yes (1) Yes (2) Yes (2) 2 Yes (4) Yes (2) Yes (3) Carcinoma 3 Yes 4 Yes 4 +/- Lung b1 -/- Lung b2 -/- Lung b3 -/- Lung b4 100 µm 100 µm 100 µm 25 µm -/- Liver b5 -/- Liver b6 -/- Liver b7 -/- Liver b8 Lym 1 Tumor numbers are shown in the parenthesis. 2 Lung adenocarcinoma with bronchus metastasis. 3 Multiple hepatocellular carcinomas in liver. 4 Lymphoma with multiple organ infiltration. HCC Lym 400 µm 25 µm 400 µm 25 µm Supplementary Figure S5. mutant mice are susceptible to carcinogen.. DEN-induced-tumor incidences in the mice of different genotypes. ll mice were treated with DEN as described in the Methods, and dissected at 11 months of age. Numbers of lung tumor were counted by H.E. staining-confirmed lung tumors.. Representative tumors developed in +/- (b1) and -/- (b2-b8) mice. te the bronchus metastasis in b3 was derived from primary lung adenocarcinoma in b2 and enlarged in b4 (indicated by red arrows). Hepatocellular carcinoma (HCC) in b5 (black arrow) that is enlarged in b6 is mixed with surrounding lymphoma (Lym). Multiple liver infiltrations by lymphoma (b7) are indicated by arrows and enlarged in b8.

8 Pei et al. Supplementary Figure S h 2 h 2 WT rdu -/ DN area Thymus +/+ -/- Supplementary Figure S6. Disruption of attenuated apoptosis, but did not significantly affect the cell cycle progression of ES cells.. f/f and -/- ES cells were exposed to 50 J/m 2 UV irradiation, pulse labeled with 10 M rdu for 1 hour, and harvested at indicated time after UV and stained with anti-rdu-fitc and PI. The distribution of cells in different phases of the cell cycle is determined by flow cytometry and shown in the table. The quantification of four different cell populations, sub-g1, G1, S and G2/M, are presented in Fig. 4D.. Littermate mice at 5 weeks of age were irradiated (10 Gy), 9 hours later, thymus was isolated, fixed and stained with TUNEL.

9 Pei et al. Supplementary Figure S7 +/- +/+ -/- wt mt -/- +/+ +/- +/+ ; +/+ -/- ; +/+ -/- ; -/- +/+ ; -/- UV Ser18-Phos- p21 ax ctin C Relative Ser18- Relative /+ ; +/+ -/- ; +/+ -/- ; -/- +/+ ; -/- Relative ax Relative p

10 Supplementary Figure S7.. Characterization of mt MEFs. Left panel, genomic DN isolated from littermate MEFs were digested with Nco1 and hybridized with a probe. Right panel, cell lysates prepared from MEFs were analyzed by western blot., C. Deletion of reduces the accumulation of and targets genes. Total cell lystes were prepared from MEF cells of different genotypes 2ours afrer UV irradiation (200 J/m 2 ) and separated by SDS-PGE, followed by direct immunoblotting with indicated antibodies (). Protein levels were quantified using NIH Image (version 1.33U) and relative protein levels were cauclauted after normalizing againt actin (C).

11 Pei et al. Supplementary Figure S8 Embryo (E13.5) +/+ -/- -/- +/+ +/+ -/- +/+ -/- MEF UV 24h WT -/- -/- WI-38/UV rain Thymus Spleen Testis Muscle rain Thymus Spleen Testis Muscle rain Thymus Spleen Testis Muscle E6 Mock p21 ax Supplementary Figure S8. Deletion of does not significantly affect expression.. Whole cell lysates were prepared from E13.5 mouse embryos (top panel) or UVirradiated MEFs (lower panel) of indicated genotype and separated by SDS-PGE, followed by western blotting using indicated antibodies.. Whole cell lysates from different tissues or organs of mice of indicated genotype at 7-8 week of age and separated by SDS-PGE, followed by western blotting using indicated antibodies. Human WI-38 cells transfected with or without E6 were UV irradiated were included as a positive control for and p21 protein expression. Loss of in embryo slightly decreases protein level and deletion of in adult tissue does not affect expression.