Skip to main content
. Author manuscript; available in PMC: 2012 Oct 27.
Published in final edited form as: J Med Chem. 2011 Sep 22;54(20):7150–7164. doi: 10.1021/jm200739e

Figure 4. Transport assays for RFC and PCFT cellular uptake.

Figure 4

Panel A: PC43-10 cells ectopically expressing hRFC but no FRs or hPCFT were assayed for [3H]MTX (0.5 uM) uptake at pH 7.2 in the presence of compounds 2–4, or the established RFC substrates, 9, PMX, RTX, LMTX, or LCV (each at 10 µM). Results are compared to those for transporter-null R2 (VC) cells (labeled “vector”). Panel B: Substrate-induced currents (nA) were recorded in individual Xenopus oocytes injected with wild type hPCFT cRNA and voltage clamped to a holding potential of −90 mV. Oocytes were perfused with ND90 solution at pH 5.5 with PMX, compound 3, or compound 4 (all at 5 µM). The design of these experiments was identical to those previously reported15,25. Panel C: R2/hPCFT4 cells, ectopically expressing hPCFT, were assayed for cellular uptake of [3H]MTX (0.5 µM) from pH 5.5 to pH 7.2 in the presence of PMX, 3, 4, or 9 (each at 10 µM). For panels A and C, the results are expressed as percent of control (absence of inhibitors) and as mean values plus/minus SEM from 3 experiments. Details for all the transport and binding assays are in the Experimental Section.