Commercially available lentiviral vectors (pLKO.1) expressing short-hairpin RNAs (shRNAs) against FZD9 under the control of the U6 promoter (Thermo Scientific) were engineered to express the Discosoma sp. red fluorescent protein mCherry under the control of the hPGK (human phosphoglycerate kinase) promoter. The following shRNAs against FZD9 and a non-silencing scrambled control shRNA were selected (Thermo Scientific):
For rescue experiments, FZD9 cDNA was amplified from TD NPC cDNA as template by the following primer pair:
The reaction was performed using Phusion High-Fidelity DNA polymerase (New England Biolabs) according to the manufacturer’s protocol. The FZD9 cDNA was cloned into a lentiviral vector driven by the ubiquitin promoter followed by a self-clevage peptide and GFP sequence. The specificity and efficiency of shRNA-control, shRNA-FZD9, and the FZD9-WT constructs were verified by co-transfection into HEK-293 cells. Cell lysates were collected and analyzed by Western blot analysis with anti-FZD9 antibodies (Aviva OAEC02415, 1:1000).
For rescue experiments, FZD9 cDNA was amplified from TD NPC cDNA as template by the following primer pair:
The reaction was performed using Phusion High-Fidelity DNA polymerase (New England Biolabs) according to the manufacturer’s protocol. The FZD9 cDNA was cloned into a lentiviral vector driven by the ubiquitin promoter followed by a self-clevage peptide and GFP sequence. The specificity and efficiency of shRNA-control, shRNA-FZD9, and the FZD9-WT constructs were verified by co-transfection into HEK-293 cells. Cell lysates were collected and analyzed by Western blot analysis with anti-FZD9 antibodies (Aviva OAEC02415, 1:1000).