Validated PIM3 Expression Vector Construction and Transfection

The PIM3 expression vector, pcDNA3.1/V5-His-PIM3, was a generous gift from Dr. Jussi Taipale and was generated by PCR amplification and cloning into the pcDNA3.1/V5-HisC vector (19 (link)). The plasmid was sequenced for verification (Genomics Core, UAB). Empty vector (EV, pcDNA3.1/V5-HisC) was used as a control. Transfection was carried out using FuGENE^® HD Transfection Reagent (Promega) per the manufacturer’s protocol. Briefly, HuH6 PIM3 KO cells were plated on the day prior to transfection. The appropriate plasmid was incubated for 15 minutes at room temperature in Opti-MEM^™ media (Thermo Fisher Scientific) with FuGENE^® HD Transfection Reagent (Promega) in a 3:2 ratio of transfection reagent to DNA with 7.5 μg DNA per 1 × 10⁶ cells and added to the cells while swirling the flask. Cells were transfected with either pcDNA3.1/V5-HisC (EV) or pcDNA3.1/V5-HisC-PIM3 (PIM3 expression vector) for 48–72 hours prior to use in experiments. Lysates were made to perform immunoblotting, as described above, to assess for PIM3 expression.