On targetplus human plk4 sirnas

For PLK4 knockdown (KD), ON-TARGETplus human PLK4 siRNAs (Dharmacon) were transfected according to the manufacturer's protocol. Briefly, cells at approximately 70% confluency were transfected with 30 pmol PLK4 siRNA under hypoxic (CoCl₂) or normoxic conditions. HIF1α gene knockout (KO) was performed using KN2.0 non–homology-mediated CRISPR KO kit (KN402461, Origene) according to the manufacturer's protocol. The target sequence of the HIF1α guide RNA (gRNA) vector (KN402461G1) was TTCTTTACTTCGCCGAGATC. The linear donor DNA (KN402461D) contained a LoxP-EF1A-tGFP-P2A-Puro-LoxP sequence. This single vector containing Cas9 and single-guide RNA (sgRNA) sequences was cotransfected with linear DNA (donor DNA) as per the manufacturer's protocol. The Cas9-mediated genome cutting was repaired by the integration of predesigned linear donor DNA containing the selection (Puromycin) and a reporter gene (GFP) via nonhomologous end joining repair. Cells at approximately 70% confluency were transfected with 1 μg each of guide RNA and donor DNA diluted in 250 μL OPTI-MEM (Invitrogen) using Lipofectamine LTX. After 48 hours, cells were split (1:5) and grown for 3 days. Cells at passage five were treated with 0.5 to 1 μg/mL puromycin to select for the clones with stable integration of the donor cassette. HIF-1α KO in puromycin-resistant clones was confirmed by qRT-PCR.