Inducible stable shRNA transfection

Doxycycline inducible shRNA expression vectors of CNR1 and CNR2 and SMARTvector Inducible Non-targeting control vector ({"type":"entrez-protein","attrs":{"text":"VSC11653","term_id":"1639798197","term_text":"VSC11653"}}VSC11653) were purchased from GE Dharmacon. Lentiviral particles were prepared for CNR1 and CNR2 shRNA and non-targeting shRNA expression using HEK293T cells as the packaging cells. UM-SCC-47 and UD-SCC-2 cells were infected with viral supernatants containing CNR1 or CNR2 shRNA with Polybrene, followed by selection using 1 μg/ml Puromycin (InvivoGen, San Diego, CA, USA). After infection by virus, the cells were cultured in DMEM with 10% Tet-System-Approved FBS (Takara Bio USA, Inc., Mountain View, CA, USA). The shRNA expression of CNR1 and CNR2 were induced with 1 μg/ml doxycycline (Sigma Aldrich).