Fig. 8.

Platelet-derived growth factor (PDGF) receptor activation leads to intracellular PLD activity. NIH 3T3 cells were labeled for RT-IMPACT by treatment with the indicated PLD inhibitor or DMSO for 30 min (A only). Cells were stimulated with PDGF or PMA for the indicated time (0 to 20 min) followed by addition of (S)-oxoTCO–C1 in the continued presence of PDGF or PMA (5 min) and then rinsed (1 min), and the IEDDA reaction with Tz–BODIPY (0.33 µM) was performed for 1 min followed by flow cytometry analysis (A), with mean fluorescence intensity in arbitrary units (AU) indicated, or (B and C) in real time with time-lapse confocal images taken (B) and quantified (C) 9 s after the addition of Tz–BODIPY, using the PM/Mix/Intra rubric described in Fig. 5. (Scale bars: 20 µm.) For A, statistical significance was assessed by using 1-way ANOVA followed by Games–Howell post hoc analysis. Asterisks directly above data points (*P < 0.05 and **P < 0.01) denote statistical significance compared with the first sample (−inhibitor), asterisks above horizontal lines denote statistical significance comparing the 2 indicated samples, and error bars represent SD. For C, statistical significance was assessed by using a χ² test for independence, with the χ² value (df, degrees of freedom) and associated P value indicated. Each bar contains data of 3 to 4 biological replicates with n = 58 to 78 total cells for PDGF and 3 to 6 biological replicates, with n = 41 to 95 cells for PMA.