Pcdna3.1 myc his a

HOXA9 cDNA was obtained by RT-PCR using primers as follows: forward, 5′- GAA TTC AAT GGC CAC CAC TGG GGC C-3′ and reverse, 5′- TCT AGA CTC GTC TTT TGC TCG GTC-3′. The products were subcloned into pFlag-CMV2 (Sigma-Aldrich, St. Louis, MO, USA) or pcDNA 3.1/myc-His A (Thermo Fisher Scientific, Waltham, MA, USA) by EcoRI and XbaI sites. Nanog-MycDDK was constructed in a pCMV6-Entry expression vector (Origene, Cat. No. RC210243, Rockville, MD, USA). Flag-HOXA9, HOXA9-MycHis, or Nanog-MycDDK was transfected into DLBCL cells (SU-DHL-5 or HT) by a transfection reagent (Lipofectamine 2000, Invitrogen, Grand Island, NY, USA) according to the manufacturer’s instructions. pFlag-CMV2, pcDNA 3.1/myc-His A, or pCMV6-Entry was transfected into cells as a vector control. The vectors are shown in Supplementary Fig. S2, S3.

The 0.8 kb and 4060 bp Mkx promoter sequence were amplified from mouse genomic DNA by PCR using the following primers: Mkxp8K forward primer, 5′- GGGGTACCACTGGAAATGGCTTTATTGTAT-3′; Mkxp8K reverse primer, 5′- CTAGCTAGCGCGGCTGACACTCCTGT-3′; Mkx4K forward primer, 5′- CTAGCTAGCGGATGGAGGCTGGAGAACTTG-3′; and Mkx4K reverse primer, 5′- CCCAAGCTTGCGGCTGACACTCCTGTC-3′. The wild-type Mkx promoter regions were cloned into a pGL3-basic reporter vector using the KpnI and NheI sites for the 855-bp fragment and the XhoI and HindIII sites for the 4060-bp fragment. HEK-293T cells were co-transfected with 100 ng pcDNA3.1(+)/myc-His A (Thermo Fisher Scientific, USA), pCMV5SMAD2-HA or pCMV5B-Flag-SMAD3, 100 ng reporter plasmids and 5 ng pRL-TK plasmid (Promega, USA) using Lipofectamine 2000 (Thermo Fisher Scientific, USA) according to the manufacturer’s instructions. 48 hr after transfection, firefly and Renilla luciferase activities were assayed using the Dual-luciferase reporter assay system (Promega, USA) according to the manufacturer’s protocol. pCMV5 SMAD2-HA was a gift from Joan Massague (Addgene plasmid # 14930) (Hata et al., 1997 (link)). pCMV5B-Flag-SMAD3 was a gift from Jeff Wrana (Addgene plasmid # 11742) (Labbé et al., 1998 (link)). The activity of the firefly luciferase was normalized to Renilla luciferase activity.

Lymph node adenocarcinoma of the prostate (LNCaP; ATCC CRL-1740) and prostate carcinoma derived-3 (PC-3; ATCC CRL-1435) cell lines were cultured in Dulbecco’s modified Eagle medium (DMEM) (Life Technologies) containing 10% (v/v) fetal bovine serum (FBS) (In Vitro Technologies, Noble Park, Vic., Australia) and 0.1% (w/v) penicillin/streptomycin (Pen/Strep) (Life Technologies) at 37 °C supplemented with 5% CO₂. These cell lines were transfected using Lipofectamine (Life Technologies) with an empty pcDNA3.1MycHisA+ (Thermo Fisher Scientific) vector along with versions expressing mouse ADAMTS-15 (wild-type; OriGene) or ADAMTS-15EA (catalytically-inactive, E362A) [17 (link)] and clones selected in the presence of 700 µg/mL Geneticin (Life Technologies).

The HAS3 coding sequence-bearing PCR product spanning from nucleotides 225 to 1886 (Genebank Accession number NM_001199280.1) was cloned into pcDNA3.1(-)/Myc-His A (Invitrogen) and sequence-verified. The Myc/His-tagged HAS3 cDNA fragment was then cloned into pLKO-AS2.zeo lentiviral vector.

Construction of the miR-1225-5p expression plasmid and packaging lentivirus were provided by Genechem Biotechnology (Shanghai, China). Sigma, St Louis, Missouri, USA). After infection, puromycin at 1.5 μg/ml (Sigma) was used to select stably transduced cells over a 10-day period. Stable miR-1225-5p overexpressing clones (MGC803-1225+ or SGC7901-1225+) and empty vector-transfected cells (MGC803-vector or SGC7901-vector) with a low basal level of miR-1225-5p expression were selected for further study. The expression of miR-1225-5p in the stable cell lines was confirmed by quantitative PCR.
The open reading frames (ORFs) of IRS1 were PCR amplified and cloned into mammalian expression vector pcDNA.3.1/myc-His(−)A (Invitrogen) to generate IRS1 expression vectors. The wild-type and mutated 3′-UTRs of the predicted targets ETV1, IRS1, FUS and CSK were amplified and cloned downstream to the luciferase gene in a pmirGLO Dual-Luciferase miRNA Target Expression Vector (Promega, Madison, USA), respectively. Recombinant plasmid transfection was performed with Roche, Basel, Switzerland). The primers used are shown in Supplementary Table S2.