Pbasi hu6 neo vector

Human DNMT1-specific shRNA were designed as 81-mers each containing a hairpin-loop (bold font) flanked by siRNAs (underlined) and cloned into the pBAsi-hU6-Neo vector (TAKARA), which uses the U6 RNA polymerase promoter and selective neomycin-resistant gene. The double-stranded oligodeoxyribonucleotides were synthesized as follows: 5′-GATCCGTGAGTGGAAATTAAGACTTTATGTACTGTGAAGCCACAGATGGGTACATAAAGTCTTAATTTCCACTCACTTTTTTA-3′ and 5′-AGCTTAAAAAAGTGAGTGGAAATTAAGACTTTATGTACCCATCTGTGGCTTCACAGTACATAAAGTCTTAATTTCCACTCACG-3′. The oligos consisted of 25 nucleotides (underlined) derived from the DNMT1 gene (Genebank accession number NM_001130823). The DNMT1 target sequences were subjected to NCBI Blast query to confirm the lack of homology to other known genes. The oligonucleotides were annealed and the annealed DNAs were ligated into the BamH I and Hind III sites of the pBAsi-hU6-Neo plasmid to create the new recombinant plasmid pBAsi-hU6-Neo-shDNMT1.

To knockdown endogenous HIF-1α, Nrf2, parkin, and Hsc70, cells were transfected with siRNA against HIF-1α (Cat. No. SI02664053), Nrf2 (SI03246950), Parkin (SI04246550), and Hsc70 (SI02661477) purchased from Qiagen (Hilden, Germany). The knockdown of HIF-2α was performed by siRNA against HIF-2α (Cat. No. AM16708) purchased from Life Technologies (Tokyo, Japan). All siRNAs were transfected using ScreenFect™ A Transfection Reagent (Wako, Osaka, Japan) according to the manufacturer’s instructions. AllStars Negative Control (Cat. No. SI03650318, Qiagen) was used as a control in RNA interference experiments. Regarding the knockdown of Siah2 using shRNA, the target sequence of 5′-ACCCGGAGTGCTTATCTTAAA-3′ was inserted into the pBAsi-hU6 Neo Vector (Takara Bio Inc., Shiga, Japan) according to a previously described procedure^{37 (link)}. The control for the shRNA experiment was the sequence of 5′-CTCGAGTACAACTATAACTCA-3′ against GFP. Transfectants of shRNA were selected using G418.