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1 Supporting Information Bommi-Reddy et al /pnas Fig. S1. Reintroducing wild-type pvhl in VHL / cells does not affect cell growth in vitro.(a) Immunoblot analysis of 786-O and RCC4 VHL / renal carcinoma cells infected with a retrovirus encoding HA-tagged wild-type pvhl or the backbone vector. Note that RCC4 produces both HIF1 and HIF2, whereas 786-O cells produce only HIF2.(Band D) 786-O (B) and RCC4 (D) VHL / cells infected as in (A) were plated at 1,000 cells per well in a 96-well plate in the presence of DMEM and 10% FBS. Cell proliferation was monitored by XTT assay. Results represent an average of three different experiments in triplicate. Error bars 1 SEM. (C) 786-O VHL / cells infected as in (A) were plated at a density of cells per 10-cm plate and incubated overnight. Cells then were trypsinized 24, 48, or 72 h after plating, washed, and fixed in 70% ethanol. Fixed cells were suspended in PBS containing 5 g/ml propidium iodide and analyzed on a fluorescence-activated cell sorter (FACScan, Becton Dickinson) using CellQuest software. 1of12
2 Fig. S2. Reproducibility of alamarblue fluorescence values in independent screens using 786-O cells. Shown are mean alamarblue values (n 2) for each of two independent screens (screen 1 and screen 2) done in duplicate along with correlation coefficients. 2of12
3 Fig. S3. VHL status influences response to shrna-mediated kinase inhibition. (A) Raw alamarblue fluorescence values for RCC4 cells infected with lentiviruses encoding the indicated shrnas. Open and filled bars depict values for RCC4 cells infected with a retrovirus encoding HA-pVHL or with the empty vector, respectively. 3of12
4 Fig. S4. Kinase shrnas that inhibit RCC4 VHL / cells over a range of viral titers. Viability of RCC4 cells grown in 96-well plates and infected with indicated amounts of lentiviruses encoding shrnas directed against CDK6, MET, ormap2k1. Cell number was assayed using alamarblue 5 days after infection and normalized to scrambled hairpin control. Filled circles and squares depict values for RCC4 cells infected with a retrovirus encoding HA-pVHL or with the empty vector, respectively. Error bars 1 SEM. 4of12
5 Fig. S5. Preferential inhibition of 786-O VHL / cells with kinase sirnas. (A) Immunoblot analysis of 786-O cells 48 h after transfection with the indicated sirnas. scr scrambled sirna. Cells were infected previously with a retrovirus encoding HA-tagged wild-type pvhl (VHL) or empty vector (vector). (B) Percent loss of viability (%LOV) 72 h after transfection with the indicated sirnas. Cells were transfected in 96-well plates, and viability was determined in triplicate using XTT. Error bars 1 SEM. 5of12
6 Fig. S6. A CDK4/6 inhibitor preferentially inhibits VHL / RCC4 cells. Percent loss of viability (%LOV) of RCC4 cells infected with a retrovirus encoding HA-pVHL (circles) or empty vector (squares) and then treated with Cdk4/6 inhibitor (Calbiochem catalog no ) at indicated concentrations for 48 h in 96-well plates. Viable cell number was determined in triplicate by XTT assay and normalized to DMSO-treated controls. Error bar 1 SEM. 6of12
7 Fig. S7. Inhibition of renal carcinoma cell viability by a MEK inhibitor. (A) Percent loss of viability (%LOV) of 786-O cells infected with a retrovirus encoding HA-pVHL (circles) or empty vector (squares) and then treated with the MEK inhibitor CI-1040 at indicated concentrations for 48 h in 96-well plates. Viable cell number was determined in triplicate by XTT assay and normalized to DMSO-treated controls. (B) Immunoblot analysis of 786-O derivatives treated with indicated concentrations of CI-1040 overnight. Note that marked killing of 786-O cells occurs at drug concentrations that are higher than required for MEK inhibition. This observation raises the possibility that killing in A is the result, at least in part, of off-target effects. 7of12
8 Table S1. List of 88 kinases AAK1 CLIK1L MELK PLK1 ADCK4 CLK3 MET RNASEL ALK DDR2 MISR2 ROS AMPKa1 EEF2K MST2 RSK2 ANPb EPHB1 MTOR RSK3 BRD2 EPHB4 MYO3B SGK2 BTK FER MYT1 SgK495 BUBR1 FGFR3 NEK7 SLK CaMK1G FYN NLK SRPK2 CAMK2A HER3 PAK3 STK33 CamK4 HER4 PAK6 SURTK106 CAMKK1 HIPK2 PBK TAK1 camlck HUNK PCTAIRE1 TLK1 CDK3 IRR PDGFRa TNK1 CDK4 JNK2 PDGFRb TRAD CDK6 JNK3 PDHK2 TRRAP CDK7 KHS1 PEK TSSK2 CDK8 LATS2 PITSLRE TYRO3 CDK9 LKB1 PKD2 ULK4 CDK10 MAP2K1 PKD3 VACAMKL CK1e MAP3K8 PKG1 VRK3 CK2a2 MAPAPK3 PKN1 ZC1/HGK 8of12
9 Table S2. Raw alamarblue values for 786-O cells Gene TRC number Average vector Average VHL Rank SGK TYRO MELK MYO3B PKN NLK CK1e DDR MAP3K PKD CDK MST CDK FYN MYT STK HIPK BRD SRPK CDK CDK JNK EPHB JNK ROS CaMK1G PAK camlck LKB PLK CamK PEK PKD PITSLRE PITSLRE NEK AAK ULK TAK IRR SgK SLK EPHB TRAD PKG KHS PDGRFb SURTK FGFR TRRAP MISR CK1e LATS CAMKK PDHK ADCK CAMK2A PBK TSSK PCTAIRE VRK CLK PAK HER Gene TRC number Average vector Average VHL Rank RNASEL CK2a ZC1/HGK MAPAPK JNK MET RSK SLK TLK CDK CDK HER MAP2K FER CLIK1L HUNK VACAMKL BTK BUBR HER TNK CK1e ALK ALK CDK AMPKa CDK PDGFRa EEF2K JNK CDK PKD JNK SCRAM GFP RSK PAK of12
10 Table S3. Raw alamarblue values for RCC4 cells Gene TRCN Average vector Average VHL Rank HER FYN TYRO MELK MISR SGK PKD DDR CDK KHS SLK PITSLRE JNK EPHB MAP3K CK1e PKN CDK RNASEL VRK PEK NEK EPHB LKB PAK FGFR JNK MET BTK TSSK IRR ALK PITSLRE SURTK MST SRPK MYT MYO3B HER HER RSK LATS ROS TRRAP TAK AAK PLK MAP2K BRD CAMKK CDK CaMK1G NLK ULK CDK TLK PDGRFb CAMK2A PDHK TNK ADCK CK1e SLK PAK Gene TRCN Average vector Average VHL Rank HIPK JNK ALK PBK PCTAIRE ZC1/HGK FER STK CLIK1L CK1e MAPAPK CDK TRAD PKD CK2a PDGFRa PKG PKD BUBR SCRAM VACAMKL CDK EEF2K CDK CamK HUNK CLK JNK camlck CDK SgK AMPKa PAK CDK GFP JNK RSK of 12
11 Table S4. Differential loss of viability in 786-O cells Gene/TRC number Differential vector-vhl Rank PAK RSK HUNK HER PDGRFb HER camlck ALK EPHB GFP JNK BRD MAP3K MST CDK NLK JNK MYO3B TAK MELK STK ADCK HIPK CDK PITSLRE PKN SGK MISR ULK LKB PKD DDR PAK NEK CamK SCRAM PAK CK1e LATS PBK TYRO ALK JNK ROS SLK CDK EPHB CK1e PEK CDK TSSK MAPAPK PLK BUBR FYN TRRAP FER SRPK PITSLRE PCTAIRE SURTK CDK PKG ZC1/HGK Gene/TRC number Differential vector-vhl Rank PKD MYT BTK FGFR PDGFRa CLIK1L AAK KHS TNK PDHK CAMK2A PKD SgK SLK CK2a AMPKa CaMK1G CDK TLK RNASEL JNK IRR RSK JNK CLK EEF2K CDK VACAMKL CDK VRK CDK HER CAMKK MET CK1e TRAD MAP2K of 12
12 Table S5. Differential loss of viability in RCC4 cells Gene/TRC number Differential vector-vhl Rank JNK RSK CDK STK CDK PKD CLK ROS BRD camlck JNK MST PKG CamK CDK MYO3B SgK CDK TAK BUBR PDGRFb JNK CK2a PITSLRE NLK DDR CAMK2A SURTK HUNK FER CLIK1L ADCK EEF2K TSSK MELK CDK PKD EPHB CK1e CDK EPHB AMPKa CDK GFP CaMK1G CDK JNK MAPAPK PKN ULK CK1e AAK ALK SCRAM PAK JNK TRAD NEK CK1e SGK TYRO MAP3K VACAMKL BTK Gene/TRC number Differential vector-vhl Rank FGFR PAK FYN SLK PDGFRa PCTAIRE VRK PKD PBK KHS HER ZC1/HGK HER PEK RNASEL TLK TNK CAMKK TRRAP PITSLRE RSK PAK MAP2K MISR ALK MYT LATS LKB IRR HIPK MET SLK PLK SRPK CDK PDHK HER of 12
2. Appendix Tables legend General Legend applicable for Table S1 to S4 (Page 10)
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