Dharmafect 2

For EphA2 silencing studies, PC-3 and DU145 cells were seeded into 6-well plates at a density of 1.5 × 10⁵ cells/1.5 mL and 1.75 × 10⁵ cells/1.75 mL, respectively. Cells were cultured until they reached 70 % confluency. Three hours before transfection, complete medium was replaced with fresh medium supplemented with 5 % FBS. siRNA complexes were prepared at optimal N/P ratios immediately before treatment as described in "Gel retardation (complexation) assay" section. Commercial transfection reagent Dharmafect 2 (#T-2002-01, Dharmacon) was used as a positive carrier control according to the manufacturer’s instructions. The complexes of siEphA2 (50 nM; ON-TARGETplus SMARTpool #L-003116-00-0020, Dharmacon) were formed with 5 µL DDAB-cSLN (N/P = 10), 4.4 µL DOTMA-cSLN (N/P = 8) and 3 µL Dharmafect 2. For other assays, the volumes of the carriers were adjusted by fixing the concentration of siRNA to 50 nM. JIB-04 (260 nM) was dissolved in DMSO (Sigma-Aldrich), gently mixed with antibiotic-free medium (± siRNA complexes), and this mixture was added into the appropriate wells. In all experiments; untreated cells (UT), and cells treated with DMSO, empty carriers, or carriers with control siRNA (siControl; ON-TARGETplus Non-targeting Control Pool #D-001810-10-05, Dharmacon) were used as controls.

To assess the Ago2 knockout efficiency, AF525 and AF5 cells were seeded in a 24-well plate (1.5 × 10⁵ cells/well). After 24 h, cells were transfected using 2 µL of DharmaFECT 2 (Horizon Discovery Ltd., Cambridge, UK) per well. Sixty nanograms of pIZ-Fluc and 10 ng pAcIE1-Rluc were co-transfected either with 1 ng siRNA (targeting FFluc or non-specific hygromycin B resistance gene as control [19 (link)]) or 10 ng dsRNA (targeting Rluc or eGFP (control)). For Ago2 reconstitution assays, AF5 and AF525 cells at a density of 2.4 × 10⁵ cells per well in poly-l-lysine-coated wells were transfected 24 h after seeding using 2 µL of DharmaFECT 2 (Horizon Discovery Ltd., Cambridge, UK) per well. Cells received 100 ng of pIZ-Fluc and 100 ng pAcIE1-Rluc, which were co-transfected with 10 ng dsRNA (targeting FFluc or non-specific lacZ). Additionally, cells were co-transfected with 500 ng per well plasmids expressing either myc-Ago2 or myc-eGFP (control). At 48 h post transfection (hpt), cells were lysed, and relative luciferase activity was measured using Dual-Luciferase Reporter Assay System (Promega Corp., Fitchburg, WI, USA).

SiHa and CaSki cells were transiently transfected with 30nM of miRIDIAN microRNA mimics for hsa-miR-129-2-3p (C-301063-01), hsa-miR-129-5p (C-300539-03), hsa-miR-137 (C-300604-07), hsa-miR-3663-3p (C-301543-00), hsa-miR-3665 (C-301545-00), hsa-miR-4281 (C-3018242-00), hsa-miR-935 (C-301264-01) or negative control #2 (CN-00200-01; GE Healthcare Dharmacon Inc., Lafayette, CO 80026, USA) according to the manufacturer's instructions. Cells were transfected for 18 hours using Dharmafect 2 (GE Healthcare Dharmacon Inc.).