Figure 1.

Synthesis of [¹¹C]TMP, Microscopy, Western Blot, and In Vitro Cellular Uptake in HCT116 and HEK293 Cell Lines

(A) TMP is converted to the phenol with heat and hydrobromic acid (HBr). The ¹¹C methyl iodide under basic conditions with heating allows conversion to [¹¹C]TMP that is then purified by HPLC. Typical yields for each step were 50%–60% and end product specific activity was 500–1,000 Ci/mmol. (B) Fluorescence microscopy of Ec-dhfr-YFP retrovirally transduced HCT116 and HEK293 cells. Scale bar, 20 μm. (C) Immunoblot probing for YFP of Ec-dhfr-YFP fusion proteins with GAPDH loading control. The expected fusion protein molecular weight is 45 kDa. (D) In vitro cell uptake studies. HCT116 cells and HEK293 cells were incubated with of [¹¹C]TMP (2 million CPM) in PBS for 30 min, washed ×2, and assayed for uptake with and without cold TMP (10 μM). The experiment was performed in triplicate with a single experiment shown. Error bars, SD (n = 3).