ALDH1A1 knockdown by siRNA

Cells were transfected with ALDH1A1 siRNA (Santa Cruz Biotechnology, TX) or a negative control siRNA (Cell Signaling Technology, MA) in Opti-MEM with lipofectamine RNAi-MAX (Invitrogen, CA) according to the manufacturer’s manual. For transfection, 200 nM of siRNA was diluted in 25 μl Opti-MEM transfection medium (solution A). Solution B was prepared by adding 1.5 μl RNAimax transfection reagent to 25 μl Opti-MEM transfection medium. Solutions A and B were mixed and incubated for 5 min at room temperature. This mixture was added to 96-well plates and incubated in a CO₂ incubator at 37°C. Cells were further maintained for 48 h before drug treatment.