Sirna duplexes

Expression of PHD was suppressed in 832/13 cells by the introduction of siRNA duplexes (Integrated DNA Technologies, Coralville, IA). Three independent siRNA sequences were designed per PHD isoform (see Table 1). A previously used scrambled siRNA sequence (GAG ACC CUA UCC GUG AUU A) with no known target was used as a control (siCtl) (Pillai et al. 2011). siRNA duplexes were introduced into 832/13 cells at 40–50% confluence using INTERFERin nucleofection (Polyplus Transfection, New York, NY). RNA isolation and real‐time PCR was performed as described earlier (Patterson et al. 2014).

For transient silencing of MMP3, the following 21-nt sequences of small interfering RNA (siRNA) duplexes were synthesized (GenePharma, Shanghai, China) were used: 5’-CAAGAUGAUGUAGAUGGUATT-3’ (sense) and 5’-AAUACCAUCUACAUCAUCUUG-3’ (antisense). In addition, 5’-UUCUCCGAACGUGUCACGUTT-3’ (sense) and 5’-ACGUGACACGUUCGGAGAATT-3’ (antisense) were synthesized as the negative control (NC) siRNA. Forty nM of siRNA duplexes were transfected into cells (2×10⁵/well) using 3 μl of INTERFER in siRNA transfection reagent (Polyplus, NY, CA, USA) in 24-well plates. The efficiency of transient MMP3 silencing was confirmed by Western blot.