RNP8-Mediated Silencing of Plk1 in LNCaP Cells

RNP₈ was assembled by incubating the dsRBD octamer and siRNA-DUPA in DPBS for 5 min under sterile condition. 2 × 10⁵ LNCaP cells were treated with fresh RNP₈ for 12 h in RPMI 1640 media plus 10% Q-serum^{42 (link), 54 (link)}, followed by a media exchange. As a positive control, cells were treated with 100 nM siRNA in the presence of Lipofectamine 2000 according to the manufacturer’s recommended protocol (Thermofisher, Waltham, MA). Thirty-two hours post-treatment, cells were harvested for RT-PCR and immunoblotting. Briefly, total RNA was isolated and reverse transcribed into cDNA using random hexamer and Taqman^® Reverse Transcription reagents (Thermofisher, Waltham, MA). mRNA expression was measured using SensiFast SYBR kit (Bioline, Taunton, MA) on Chromo4™ Real-Time system (Bio-Rad, Hercules, CA). Plk1 mRNA expression level was normalized to peptidylprolyl isomerase A (PPIA) and expressed as the percentage of negative control. Immunoblotting was performed as previously reported^{55 (link)} and all antibodies were purchased from ThermoFisher. The immunoblots were imaged on an Odyssey^®CLx imaging system (Li-cor, Lincoln, NE) and analyzed with Image Studio Lite.