Figure 6. Effects of knockdown of FRα in IGROV1 cells on in vitro efficacy of 6-substituted pyrrolo[2,3 d]pyrimidine antifolates.

FRα was knocked down in IGROV1 with lentivirus shRNA and clones (KD-4 and KD-10) were selected with puromycin. A scrambled shRNA was used as a non-targeted control (NTC). Results of 2∼3 replicates are shown for FRα and PCFT transcript levels in IGROV1 NTC cells and KD-4 and KD-10 cells, measured by real time RT-PCR for FRα (Panel A, left) and for PCFT (Panel A, right). IGROV1 FRα KD clones were functionally characterized for FRα binding and uptake of [³H]folic acid (upper Panel B) and [³H]AGF154 (lower Panel B) at 37°C to measure total cellular (T) and intracellular (I) levels of [³H]substrate, and at 0°C to measure surface (S) FRα-bound [³H]substrate. These experiments were performed in the absence or presence of excess unlabeled folic acid (10 µM) as a competitor of FRα-mediated binding and uptake. IGROV1 FRα KD clones were also assayed for PCFT uptake (Panel C) with [³H]AGF154 at pH 5.5 at 37°C, in the absence and presence of 10 µM unlabeled AGF94. Results in Panels B and C are expressed as mean values +/− range (n=2). PCFT protein levels for the cell lines were measured in crude membranes by SDS-PAGE and Western blotting with PCFT antibody (Panel D). β-Actin was used as a loading control. The molecular mass markers for SDS-PAGE are noted. Densitometry was performed using Odyssey software, and PCFT protein expression was normalized to β-actin. Representative densitometry results for the blot shown are noted. Variations in densitometry values between different blots (n=2) were within 10%.