Genjet reagent

Human PDK2 (NM_002611.4) and PDK3 (NM_001142386) cDNAs, cloned in pcDNA3.1+/C-DYK mammalian expression plasmids (C-terminal Flag fusion), were purchased from GeneScript (Piscataway, NJ, USA). pRK5 Flag-PTEN was made by PCR incorporation of an N-terminal Flag sequence to human PTEN (NM_000314) from pRK5 PTEN (37 (link)). Cells were transiently transfected with empty vector, pRK5 Flag-PTEN, pCDNA3.1 PDK2-Flag, or pCDNA3.1 PDK3-Flag using GenJet reagent (SignaGen, Frederick, MD, USA). Cells were lysed in M-PER extraction reagent (ThermoFisher, Waltham, MA, USA) and processed for immunoblot as described (38 (link)). Primary antibody used was mouse anti-Flag (1:500, MAB3118, Sigma Aldrich). Secondary antibody was IRDye 680RD Goat anti-Mouse (LI-COR, Lincoln, NE, USA). Blots were processed with Odyssey CLx Imaging system (LI-COR).

Two hundred and ninety-three cells were seeded (concentration: 5 × 10⁶ cells) in a 10 cm dish and transfected with DNA plasmids (5 μg) using the GenJet™ reagent (SignaGen Laboratories, Frederick, MD, USA). Ishikawa cells, T47D, MCF7, and primary uterine stroma cells were seeded (concentration: 1 × 10⁶ cells) in a 6 cm dish and transfected with double-stranded RNA (50 nM) using the Lipofectamine RNAiMAX reagent (Invitrogen). P62 and KEAP1 small interfering (si)RNAs were obtained from Santa Cruz Biotechnology (Dallas, TX, USA), whereas GPR30 siRNA was from Invitrogen. At 72 h after transfection, the silencing of target genes was confirmed by Western blot.

Simian kidney COS-7 cells (ATCC CRL-1651) were grown at 37° C, 5% CO₂ in DMEM containing high glucose supplemented with 5% heat-inactivated fetal bovine serum (FBS), 1 mM L-glutamine, 100 U/ml penicillin, and 0.1 mg/ml streptomycin. Cells were transfected using GenJet reagent (SignaGen) according to the manufacturer instructions, and processed for analysis 48 h after transfection. The pRK5 PTEN, pRK5 GST-PTEN (N-terminal tagging), pRK5 PTEN-L (Met), and pSG5-AKT1 plasmids have been described [38 (link), 39 (link)]. pRK5 PTEN-L (Leu), pRK5 PTEN-M (Met), pRK5 PTEN-M (Ile), pRK5 PTEN-O (Met), and pRK5 PTEN-O (Leu) were made by PCR oligonucleotide site-directed mutagenesis from pRK5 PTEN-L (Met), as described [38 (link)]. Plasmid pRK5 PTEN-Δ (residues 1-343-Ser) was made by PCR mutagenesis from pRK5 PTEN. The PTEN and GST-PTEN amino acid substitution and truncation variants were made by PCR mutagenesis, and mutations were confirmed by DNA sequencing. Nucleotide and amino acid numbering for PTEN variants correspond to reference sequences from accession numbers NM_000314 and NP_000305, respectively.

DNA transfection of HEK293 cells and STIP1 KO HAP1 cells was performed using the GenJet reagent (SignaGen Laboratories, Frederick, MD, USA; 2 μL for 1μg of DNA). SKOV3 cells were transfected with jetPRIME (Polyplus-transfection SA, Illkirch, France) according to the manufacturer’s instructions.

Cells were used for transfection with plasmids (the manufacturing process has been described previously [50 (link)]) including pEGFP (GFP) and pEGFP-Sp1 (GFP-Sp1) using the GenJet reagent (SignaGen Laboratories, Rockville, MD) and commercial gene-specific SMARTpool short-interfering RNAs (Dharmacon, Lafayette, CO) for HDAC1 (siHDAC1), HDAC2 (siHDAC2), HDAC6 (siHDAC6), Sp1 (siSp1), and nontargeting siRNAs control (siControl) using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer’s protocol. The transfection efficiency was confirmed by western blotting analysis.