On target plus smartpool sirna reagents

The human-specific siRNAs targeting SREBF1 (6720) and SREBF2 (6721) were pre-designed ON-TARGETplus SMARTpool siRNA reagents from Dharmacon. Each ON-TARGETplus SMARTpool siRNA was a mixture of four siRNA duplexes. siRNAs were suspended in RNase-free 1 × siRNA Buffer (Dharmacon) to yield 20 μM stock solutions. HT-144 cells were transfected with siRNAs at a final concentration of 50 nM using Lipofectamine RNAiMAX transfection reagent (Thermo Fisher Scientific) at a density of 2 × 10⁵ cells/well in a 6-well plate. For each transfection, 2.5 μl of siRNA stock solution was mixed with 4 μl of Lipofectamine RNAiMAX in 200 μl of Opti-MEM I medium and then incubated for 10–20 minutes at room temperature. HT-144 cells were diluted in 10% FBS/RPMI-1640 medium without antibiotics so that 800 μl medium contains 2.5 × 10⁵ HT-144 cells. 200 μl of siRNA/Lipofectamine RNAiMAX complexes was mixed with 800 μl of the diluted HT-144 cells in one well of a 6-well plate. Transfected HT-144 cells were incubated at 37°C for 16 hours before medium changes.

Knockdowns were performed using the following On-Target Plus SMARTpool siRNA reagents from Dharmacon, with a nontargeting SMARTpool siRNA (D-001810-10) used as a control. The siRNAs for VPS35 were 010894–05 (GAACAUAUUGCUACCAGUA), 010894–06 (GAAAGAGCAUGAGUUGUUA), 010894–07 (GUUGUAAACUGUAGGGAUG), 010894–08 (GAACAAAUUUGGUGCGCCU); for AP5Z1, they were 025284–17 (GGGACUUCGGUGCAGAUUA), 025284–18 (GUUCCUGGGCAGCGUGAAU), 025284–19 (GGAGGUGGCCUUCGAGUAC), 025284–20 (CCACAGACUUCUUCACGGU); and for SORT1, they were 010620–05 (GAGACUAUGUUGUGACCAA), 010620–06 (GAGCUAGGUCCAUGAAUAU), 010620–07 (GAAGGACUAUACCAUAUGG), 010620–08 (GAAUUUGGCAUGGCUAUUG). The custom oligo to knock down VPS26 was a gift from Matthew Seaman [55 (link)]. All siRNAs were used at a concentration of 25–50 nM in a 2-hit, 5-day protocol according to manufacturer’s instructions (Dharmacon). Knockdown efficiencies were determined by western blotting and showed >80% depletion for VPS26 and >90% for all other knockdowns (quantified by ImageJ).