Mission esirna human prox1

The PROX1 gene was silenced in the CGTH cells by transfection with siPROX1 targeting human PROX1 (MISSION esiRNA human Prox1, Sigma-Aldrich, Darmstadt, Germany) using the Ambion Silencer siRNA Transfection II Kit (ThermoFisher Scientific) according to the manufacturer’s protocol. Cells were trypsinized and suspended in complete growth medium (RPMI-1640 medium supplemented with 10% FBS) to a concentration of 1 × 10⁵ cell/mL. The cells were kept at 37 °C while the transfection complexes were prepared. Next, 100 µL of the transfection mix (30 nM siRNA in OptiMEM (ThermoFisher Scientific) plus 5 μL of Lipofectamine 2000 (Life Technologies, Carlsbad, CA, USA)) was added to 2 mL of 1 × 10⁵ CGTH cells (in complete RPMI-1640 medium supplemented with 10% FBS) and the cells were seeded in 6-well plates. Cells were incubated for 48 or 72 h at 37 °C in a humidified 5% CO₂ atmosphere. Control cells were transfected with negative siRNA (siNEG; MISSION siRNA, SIC-001, Sigma-Aldrich). Cells transfected with the transfection mix without any siRNA (Lipofectamine only, Life Technologies) were used as a technical negative control. The experiments were conducted in triplicates and at least three times. Before further processing cells were silenced for 48h, unless otherwise indicated. Each time PROX1 knockdown was verified by qPCR.

Cells were transfected with siRNA (final concentration 30 nM) targeting human PROX1 (MISSION esiRNA human PROX1, Sigma-Aldrich, USA, termed further siPROX1 (SA) or with PROX1 siRNA (h), sc-106451, Santa Cruz Biotechnology, California, USA, termed below siPROX1(SC)) using Lipofectamine 2000 (Life Technologies, Carlsbad, CA, USA) in Opti-MEM medium (ThermoFisher Scientific, Waltham, MA, USA), according to manufacturer′s recommendations. Scramble siRNA was used as the negative control (siNEG; MISSION siRNA, SIC-001, Sigma-Aldrich, USA). The experiments were conducted in triplicates and at least three times. Before further processing cells were silenced for 48 h unless otherwise indicated. Each time PROX1 knockdown was verified by RT-qPCR.