Long-time scale labeling with ProCho results in PLD-independent formation of alkynyl lipids. (A) Schematic of extended HeLa cell labeling with ProCho (100 μM). (B and C) Cells were labeled with ProCho as in panel A, and lipid extracts were generated, tagged with Az488 via CuAAC, and analyzed by HPLC. Shown are representative HPLC traces (B) and quantification (area under curve) (C). (D) Cells were labeled with ProCho as in panel A, fixed, tagged with Az545 via CuAAC, and imaged by confocal microscopy: green for CuAAC-derived fluorescence and blue for DAPI. n = 3; *p < 0.05. Error bars indicate the standard error of the mean.