Gibco™Ham's F-12 Nutrient Mix

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	supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic/antimycotic (Invitrogen).

Protocol tips
supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic/antimycotic (Invitrogen).

Publication protocol

PC-3 cells obtained from ATCC were cultured in F12 HAM supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic/antimycotic (Invitrogen). The androgen dependent LNCaP-104S cells (a gift from Dr. Shutsung Liao, University of Chicago) were isolated from the parental LNCaP cells and characterized [34]. LNCaP-104S cells were maintained in DMEM containing 10% FBS, 1 nM DHT (Sigma-Aldrich) and 1% antibiotic/antimycotic. C4-2B cells are developed from vertebral metastasis of LNCaP xenografts [35] (a gift from Dr. Leland Chung, Cedars-Sinai Medical Center). C4-2B cells were maintained in RPMI with 10% FBS and 1% antibiotic/antimycotic. All cells are rigorously monitored for mycoplasma contamination using DAPI staining method and authenticated through STR profiling (Additional file 1: Table S1). For inhibition of FGD4 expression, 4 FGD4 siRNAs (FlexiTube Gene Solution, Qiagen) were used for transient transfection using Lipofectamine RNAiMax (ThermoFisher) transfection reagent. Three control siRNAs were used for transient transfection in parallel as the controls. Transfected cells were harvested at 48 h for subsequent experiments. For ectopic expression, DNA of full length coding sequence of FGD4 gene was synthesized (GenScript), cloned into pcDNA 3.1 and pcDNA 3.1-EGFP mammalian expression vectors (FGD4 pcDNA and pcDNA 3.1 FGD4-EGFP) and sequence verified. FGD4 pcDNA, pcDNA 3.1 FGD4-EGFP, pcDNA 3.1 MECP2-EGFP, or the empty vector as the control, was used for transient transfection using Lipofectamine (Invitrogen). Cells were used after 48 h for subsequent experiments.

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