Estrogen Receptor alpha siRNA (m)

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	The D3 mESCs were cultured on a feeder layer of mitomycin-treated MEFs in a traditional complete medium (KS/LIF medium) containing knockout Dulbecco’s modified Eagle’s medium (DMEM/F-12) supplemented with 15% knockout serum replacement, 0.1 mM non-essential amino acids, 1% penicillin/streptomycin, 1 mM L-glutamine, 0.1 mM β-mercaptoethanol (Invitrogen) and 1000 U/ml mouse LIF (Millipore). 2i/LIF condition medium was made from the traditional complete medium by using 3 μM CHIR99021 (Stemgent) and 1 μM PD0325901 (Stemgent) to substitute knockout serum replacement. Prior to experiments, cells were passaged by incubation in 0.25% trypsin/EDTA for 5 min at 37 °C and then plated onto gelatin-coated dishes without a feeder layer for 24 h. Icaritin was purchased from Sigma. All reagents were purchased from Invitrogen unless otherwise specified.mESCs were transiently mESCs were transiently transfected with siRNAs specific for knockdown of Rbl2/p130, Cdx2 or ERα gene (Santa Cruz). Cells were plated on gelatin coated plates for 24 h. Transfection was preceded by using lipofectamine RNAiMAX reagent according to manufacturer’s instructions (Invitrogen). Control siRNA transfection was performed in parallel. Total RNA and protein was extracted at 48 h after transfection.with siRNAs specific for knockdown of Rbl2/p130, Cdx2 or ERα gene (Santa Cruz). Cells were plated on gelatin coated plates for 24 h. Transfection was preceded by using lipofectamine RNAiMAX reagent according to manufacturer’s instructions (Invitrogen). Control siRNA transfection was performed in parallel. Total RNA and protein was extracted at 48 h after transfection.

Protocol tips

The D3 mESCs were cultured on a feeder layer of mitomycin-treated MEFs in a traditional complete medium (KS/LIF medium) containing knockout Dulbecco’s modified Eagle’s medium (DMEM/F-12) supplemented with 15% knockout serum replacement, 0.1 mM non-essential amino acids, 1% penicillin/streptomycin, 1 mM L-glutamine, 0.1 mM β-mercaptoethanol (Invitrogen) and 1000 U/ml mouse LIF (Millipore). 2i/LIF condition medium was made from the traditional complete medium by using 3 μM CHIR99021 (Stemgent) and 1 μM PD0325901 (Stemgent) to substitute knockout serum replacement. Prior to experiments, cells were passaged by incubation in 0.25% trypsin/EDTA for 5 min at 37 °C and then plated onto gelatin-coated dishes without a feeder layer for 24 h. Icaritin was purchased from Sigma. All reagents were purchased from Invitrogen unless otherwise specified.mESCs were transiently mESCs were transiently transfected with siRNAs specific for knockdown of Rbl2/p130, Cdx2 or ERα gene (Santa Cruz). Cells were plated on gelatin coated plates for 24 h. Transfection was preceded by using lipofectamine RNAiMAX reagent according to manufacturer’s instructions (Invitrogen). Control siRNA transfection was performed in parallel. Total RNA and protein was extracted at 48 h after transfection.with siRNAs specific for knockdown of Rbl2/p130, Cdx2 or ERα gene (Santa Cruz). Cells were plated on gelatin coated plates for 24 h. Transfection was preceded by using lipofectamine RNAiMAX reagent according to manufacturer’s instructions (Invitrogen). Control siRNA transfection was performed in parallel. Total RNA and protein was extracted at 48 h after transfection.

Publication protocol

The D3 mESCs were cultured on a feeder layer of mitomycin-treated MEFs in a traditional complete medium (KS/LIF medium) containing knockout Dulbecco’s modified Eagle’s medium (DMEM/F-12) supplemented with 15% knockout serum replacement, 0.1 mM non-essential amino acids, 1% penicillin/streptomycin, 1 mM L-glutamine, 0.1 mM β-mercaptoethanol (Invitrogen) and 1000 U/ml mouse LIF (Millipore). 2i/LIF condition medium was made from the traditional complete medium by using 3 μM CHIR99021 (Stemgent) and 1 μM PD0325901 (Stemgent) to substitute knockout serum replacement. Prior to experiments, cells were passaged by incubation in 0.25% trypsin/EDTA for 5 min at 37 °C and then plated onto gelatin-coated dishes without a feeder layer for 24 h. Icaritin was purchased from Sigma. All reagents were purchased from Invitrogen unless otherwise specified.mESCs were transiently mESCs were transiently transfected with siRNAs specific for knockdown of Rbl2/p130, Cdx2 or ERα gene (Santa Cruz). Cells were plated on gelatin coated plates for 24 h. Transfection was preceded by using lipofectamine RNAiMAX reagent according to manufacturer’s instructions (Invitrogen). Control siRNA transfection was performed in parallel. Total RNA and protein was extracted at 48 h after transfection.with siRNAs specific for knockdown of Rbl2/p130, Cdx2 or ERα gene (Santa Cruz). Cells were plated on gelatin coated plates for 24 h. Transfection was preceded by using lipofectamine RNAiMAX reagent according to manufacturer’s instructions (Invitrogen). Control siRNA transfection was performed in parallel. Total RNA and protein was extracted at 48 h after transfection.

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