2.2. Plasmids and siRNA

The promoter/regulatory region of the ADTRP gene starting from −789 bp to + 724 bp from the transcriptional start site (TSS) was amplified by PCR analysis using human genomic DNA as the template, the Prime STAR HS DNA polymerase (TaKaRa, Dalian, China) and PCR primers ADTRP-F-KpnI 5′-CGGGGTACCCCTCCTTGTCCAGCCTACAG-3′ and ADTRP-R-XhoI 5′-CCGCTCGAGCCCCTCTTTGA GCTCATCTG-3′. The PCR product was digested with restriction enzymes Kpn I and Xho I (TaKaRa, Dalian, China), and sub-cloned into the multiple cloning site of the pGL3-Basic luciferase vector. This generates a luciferase reporter for the ADTRP promoter, pGL3-Basic-ADTRPp-Luc, in which the ADTRP promoter/regulatory region is inserted upstream of the firefly luciferase coding region.

The half ARE sequence at the position of + 324 bp from the TSS was mutated from TGTTCT to AAAAAT and TAAAAA using site-directed mutagenesis as described elsewhere [21,22], resulting in two mutant reporters pGL3-Basic-ADTRPp-Luc-Mut1 and pGL3-Basic-ADTRPp-Luc-Mut2. The primers for mutagenesis from TGTTCT to AAAAAT included a forward primer 5′-TGCATATACCACTTCCT AAAAATGAGCTGGTATACTTTCC-3′ and a reverse primer 5′-GGAAAGTATACCAGCTCATTT TTAGGAAGTGGTATATGCA-3′ (for pGL3-Basic-ADTRPp-Luc-Mut1). The primers for mutagenesis from TGTTCT to TAAAAA included a forward primer 5′-TGCATATACCACTTCCTTAAAAAGAGCTGGTATACTTTCC-3′ and a reverse primer 5′-GGAAAGTATACCAGTTCTTTTTAAGGAAGTGGTATATGCA-3′ (for pGL3-Basic-ADTRPp-Luc-Mut 2).

A plasmid with the full-length cDNA for the human AR gene, pEZ-M02 AR, was purchased from Gene Copoeia (Guangzhou, China) (human genome build hg19 mRNA: {"type":"entrez-nucleotide","attrs":{"text":"NC_000023.11","term_id":"568815575","term_text":"NC_000023.11"}}NC_000023.11, Entrez GeneID 367, Ensembl: ENSG00000169083). The full length cDNA for AR was amplified by PCR analysis using pEZ-M02 AR as the template and primers AR (920aa) 2763 bp F-BglII: 5′-GGAAGATCTGGATGGAAGTGCAGTTAGGGCTGGG-3’and R-XhoI:5′-CCGCTCGAGTCACTGGGTGTGGAAATAGATGGGCT-3′. The PCR product was digested using restriction enzymes Bgl II and Xho I (TAKARA, Dalian, China) and sub-cloned into the multiple cloning site of eukaryotic tag expression vector pCMV-Myc, generating a mammalian expression plasmid for AR, pCMV-Myc-AR.

The full-length cDNA for PIK3R3 ({"type":"entrez-nucleotide","attrs":{"text":"NC_000001.11","term_id":"568815597","term_text":"NC_000001.11"}}NC_000001.11, Entrez GeneID 8503, Ensembl: ENSG00000117461) was obtained by RT-PCR analysis using RNA samples isolated from HeLa cells. The PCR product was digested with EcoR I and Kpn I (TAKARA, Japan) and sub-cloned into the multiple cloning site of pCDNA3.1(−), resulting in an expression plasmid for PIK3R3, pCDNA3.1(−)-PIK3R3.

The expression plasmid for MIA3/TANGO1, pcDNA3.1(+)-TANGO1-HA, was as described previously [23–28] and kindly provided by Dr. Vivek Malhotra and colleagues.

We purchased siRNAs from Genepharma (Suzhou, China). The sequences for siRNAs are 5′-GGAUCCUCUUUCUCUACAATT-3′ (sense) and 5′-UUGUAGAGAAAGAGGAUCCTT-3′ (antisense) (ADTRP), 5′-GGACUUGCUUUAUGGGAAAdTdT-3′ (sense) and 5′-UUUCCCAUAAAGCAAGUCCdTdT-3′(antisense) (PIK3R3), and 5′-GGUGAAGUCUGAAUGCCAUTT-3′ (sense) and 5′-AUGGCAUUCAGACUUCACCTT-3′(antisense) (MIA3/TANGO1).