Control nontargeting scrambled sirna duplexes

Three different IFNκ siRNA duplexes and control nontargeting scrambled siRNA duplexes were purchased from Life Technologies. The sequence for IFNκ siRNA #1 are as follows: sense: CCCUAUCCCUGGACUGUAAtt and antisense: UUACAGUCCAGGGAUAGGGtg; IFNκ siRNA #2 sense: GAUAGACAAUUUCCUGAAAtt and antisense: UUUCAGGAAAUUGUCUAUCct; IFNκ siRNA #3 sense: CACCUUCAAAUAUUGGAAAtt and antisense: UUUCCAAUAUU UGAAGGUGtg. NHEKs were plated in 24-well plates at 1 × 10⁵ per well the day before transfection. Cells were transfected with siRNA duplexes at a final concentration of 10 nM using lipofectamine 2000 according to the manufacturer's instructions (Invitrogen, Carlsbad, CA). After 24 hours of incubation, the cell culture medium was replaced with EpiLife supplemented either with 0.06 mM CaCl₂ for 24 hours (undifferentiated condition, UD) or with 1.3 mM CaCl₂ for 2 days (differentiated condition, D). HSV-1 at various multiplicity of infection (MOI) was then added to the cells for an additional 24 hours. After incubation with HSV-1, the cells were harvested for RNA extraction, qRT-PCR, and plaque assays.

FOXC1 siRNA duplexes, ZNF750 siRNA duplexes, KLF4 siRNA and control non-targeting scrambled siRNA duplexes were purchased from Life Technologies. The sequence for KLF4 siRNA are: sense: UGACCAGGCACUACCGUAAtt; antisense: UUACGGUAGUGCCUGGUCAtt. The sequence for ZNF750 siRNA are: sense: CCUCAAUGUUGUGAACGGAtt; antisense: UCCGUUCACAACAUUGAGGct. The sequence for FOXC1 siRNA are: sense 1: GAAAGUCGCUUUCUUUUUAtt; antisense: UAAAAAGAAAGCGACUUUCat. Sense 2: ACUCUCCAGUGAACGGGAAtt, antisense: UUCCCGUUCACUGGAGAGUtg; Sense 3: AGAGGAUCGGCUUGAACAAtt; anti-sense: UUGUUCAAGCCGAUCCUCUgt. KCs were plated in 24 well plates at 1x10⁵ per well the day before transfection. Cells were transfected with siRNA duplexes at final concentration of 10 nM using lipofectamine 2000 according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA). After 24 hours incubation, the cells were replaced with EpiLife supplemented either with 0.06mM or 1.3 mM calcium for up to 120 hours. Then the cells were used for total RNA and protein extraction.