UAGACAUUGGGUUCGCCGUTT N A br siCul CCCUUGGAGAAAGACUUUAUA N A
UAGACAUUGGGUUCGCCGUTT
N/A
siCul2
CCCUUGGAGAAAGACUUUAUA
N/A
siCul4B
AAGGTGCGAGAAGATGTA
N/A
siCul3-1
AAGGUGCGAGAAGAUGUATT
N/A
siCul3-2
AACAACUUUCUUCAAACGCUA
N/A
siCul3-3
AACAACACUUGGCAAGGAGAC
N/A
siSPOP-1
CAACUAUCAUGCUUCGGAU
N/A
siSPOP-2
GGUAAAGGUUCCUGAGUGC
N/A
siSPOP-3
AAAUGGUGUUUGCGAGUAA
N/A
siSPOP-4
UAGAACUUUUAAUGACUUCAC
N/A
siBRAF-1
GGUGUGGAAUAUCAAACAATT
N/A
siBRAF-2
GGCUCACUAACUAACGUGATT
N/A
siBRAF-3
AAACGUUUUUCGUACAAAGUU
N/A
siBRAF-4
AUUCAUACAGAACAAUUCCAA
(Continued on next page)
Continued
REAGENT or RESOURCE
SOURCE
IDENTIFIER
Recombinant DNA
pcDNA3.1-HA-NANOG
In our lab
N/A
pcDNA3.1-Flag-dnCul1
In our lab
N/A
pcDNA3.1-Flag-dnCul2
In our lab
N/A
pcDNA3.1-Flag-dnCul3
In our lab
N/A
pcDNA3.1-Flag-dnCul4A
In our lab
N/A
pcDNA3.1-Flag-dnCul4B
In our lab
N/A
pcDNA3.1-Flag-GFP
In our lab
N/A
pcDNA3.1-HA-SPOP
In our lab
N/A
pcDNA3.1-Flag-NANOG
In our lab
N/A
pcDNA3.1-His-ubiquitin
In our lab
N/A
pcDNA3.1-Flag-SPOP
In our lab
N/A
pcDNA3.1-Flag-SPOPDBTB
In our lab
N/A
pcDNA3.1-Flag-SPOPDMATH
In our lab
N/A
pcDNA3.1-HA-SPOPDBTB
In our lab
N/A
pcDNA3.1-HA-SPOPDMATH
In our lab
N/A
Lenti-pWPI-Flag-SPOP
In our lab
N/A
Lenti-pWPI-Flag-SPOP-F133L
In our lab
N/A
Lenti-pWPI-Flag-SPOP-Y87C
In our lab
N/A
Lenti-pWPI-Flag-NANOG
In our lab
N/A
pcDNA3.1-HA-NANOG-DPDSST
In our lab
N/A
pcDNA3.1-HA-NANOG-3A
In our lab
N/A
pcDNA3.1-Flag-NANOG-DPDSST
In our lab
N/A
pGEX-4T-2-GST-SPOP
In our lab
N/A
pcDNA3.1-Flag-NANOG-S65A
In our lab
N/A
pcDNA3.1-Flag-NANOG-P66A
In our lab
N/A
pcDNA3.1-Flag-NANOG-D67A
In our lab
N/A
pcDNA3.1-Flag-NANOG-S68A
In our lab
N/A
pcDNA3.1-Flag-NANOG-S69A
In our lab
N/A
pcDNA3.1-Flag-NANOG-T70A
In our lab
N/A
pcDNA3.1-Flag-NANOG-S71A
In our lab
N/A
pcDNA3.1-Flag-NANOG-P72A
In our lab
N/A
pcDNA3.1-Flag-NANOG-S68Y
In our lab
N/A
Lenti-pWPI-Flag-NANOG-S68Y
In our lab
N/A
pcDNA3.1-Flag-NANOG-S68D
In our lab
N/A
NANOG reporter plasmid
Dr. Duanqing Pei, Guangzhou Institutes cck-8 Biomedicine
N/A
and Health
(Continued on next page)
Continued
REAGENT or RESOURCE
SOURCE
IDENTIFIER
pcDNA3.1-HA-AKT
In our lab
N/A
pcDNA3.1-HA-ARAF
In our lab
N/A
pcDNA3.1-HA-BRAF
In our lab
N/A
pcDNA3.1-HA-CRAF
In our lab
N/A
pcDNA3.1-Flag-BRAF
In our lab
N/A
pET28a-His-NANOG
In our lab
N/A
Retro-Migr-Venus-Mir30-shSPOP
In our lab
N/A
Retro-Migr-Venus-Mir30-shNANOG
In our lab
N/A
pGEX-4T-2-GST-SPOP-DBTB
In our lab
N/A
pGEX-4T-2-GST-SPOP-DMATH
In our lab
N/A
pcDNA3.1-Flag-KCTD2
In our lab
N/A
pcDNA3.1-Flag-KCTD5
In our lab
N/A
pcDNA3.1-Flag-BACURD2
In our lab
N/A
pcDNA3.1-Flag-KLHL9
In our lab
N/A
pcDNA3.1-Flag-KLHL13
In our lab
N/A
pcDNA3.1-Flag-KLHL15
In our lab
N/A
pcDNA3.1-Flag-KLHL17
In our lab
N/A
pcDNA3.1-Flag-KLHL22
In our lab
N/A
pcDNA3.1-Flag-KLHL24
In our lab
N/A
Lenti-pWPI-Flag-NANOG-DPDSST
In our lab
CONTACT FOR REAGENT AND RESOURCE SHARING
Further information and requests for reagents may be directed to and will be fulfilled by the Lead Contact, Ping Wang ([email protected]
EXPERIMENTAL MODEL AND SUBJECT DETAILS
Cell Culture
PC3 (male), 22RV1 (male), LNCaP (male) cells were cultured in RPMI 1640 medium supplemented with 10% FBS. DU145 (male), SPOP WT and KO DU145 (male), AMPKa1/a2 WT and KO MEFs (a kind gift from Dr.Jia Li, National Center for Drug Screening, China) and human embryonic kidney 293T (from 
Cell Bank, Chinese Academy of Sciences) cells were cultured in DMEM medium supple-mented with 10% heat-inactivated fetal bovine serum. E14 mESCs (a kind gift from Dr.Xin Xie, National Center for Drug Screening, China) were maintained on feeder-free gelatin-coated plastics cultured by mES medium (DMEM with 15% FBS (Gibco 10099), 2mM GlutaMAX, 0.1 mM non-essential amino acids (NEAA), 0.1 mM b-mercaptoethanol, 100 U/ml penicillin and 100 mg/ml streptomycin) supplemented with 1000 U/ml LIF and passaged every 3 days. Cells were cultured at 37 C supplied with 5% CO2. Transfection was performed by using calcium phosphate-DNA coprecipitation for HEK293T cells and using Lipofectamine 2000 for DU145, LNCaP, PC3 and E14 cells.
In Vivo Xenograft Assay
All treatments were administered according to the guidelines of Institution Animal Care and Use Committee and all the protocols were approved by East China Normal University and Tongji University. Mice were caged in the groups of five in a laminar airflow cabinet under specific pathogen-free conditions, fed with sterilized food and water, and kept on a 12 hr light/dark cycle. Serial limiting dilution DU145 (AR-) or 22RV1 (AR+) cells were admixed with Matrigel and injected into the backside of male 6-week-old BALB/cA nude mice. The animals were checked two to three times per week. Mean of primary, secondary and tertiary tumor volumes at 5 weeks.