Pkcα sirna

PKCα siRNA (Santa Cruz biotechnologies, SC-29449) or Silencer® Negative control siRNA (Thermo, AM4611) were transfected into HepG2 cells using Lipofectamine® RNAiMAX reagent (Invitrogen) following the manufacturer’s protocol. HepG2 cells (5.5 × 10⁵ cells) were seeded for 24 h before siRNA complexes were added. Twenty nM of siRNA were effective for suppressing PKCα expression. At 24 h post-transfection, DENV2 at MOI of 1 was added into the HepG2 cells and the culture was further incubated for 90 min to allow internalization of virus into the cells. Then, the excess virus was removed, and the cells were washed twice with PBS. Finally, DMEM supplement with 2 % FBS was added into the cells, and the cells were further incubated for another 24 h. Cells were collected for detection of intracellular viral RNA by RT-qPCR and viral proteins by immunoblots.

Cells were maintained in DMEM with 10% FBS, 100 units/ml penicillin, and 100 μg/ml streptomycin. For drug treatments, 90% confluent cells were serum-starved for 4 h and then treated with 0.5 μm PMA (Sigma) or SP600125 (Merck Millipore, Billerica, MA). A PKCβ-selective inhibitor (539654, Merck Millipore), anisomycin (Sigma), and TAPI-0 (Merck Millipore) were used for 30 min before drug treatment. Cycloheximide, α-amanitin, and actinomycin D were purchased from Sigma. For overexpression or knockdown of specific proteins, transfection of cDNA or siRNA was performed. When cells reached 30%–40% confluence, cells were transfected with JNK1 siRNA, JNK2 siRNA, PKCα siRNA, PKCβ siRNA, and TACE siRNA (Santa Cruz Biotechnology, Dallas, TX), WT PKCβII cDNA, DN PKCβI (K371R), and DN PKCβII (K371R) cDNA (Addgene, Cambridge, MA) using Lipofectamine 2000 (Invitrogen) for 48–72 h. Control siRNA (catalog no. sc-37007, Santa Cruz Biotechnology), designed not to target any gene, was used as a negative control in knockdown experiments.