Figure 8

Kinetics of LD540 transport for different LD size fractions. 3T3-L1 adipocytes were pretreated with the indicated substances for 1 or 3 days post differentiation or left untreated (control cells), labeled with LD540 for 5 minutes, rinsed, and chased without labeling for an additional 30 min. Single LDs were bleached with an intense laser pulse (405 nm, 1 sec) and fluorescence recovery of the bleached LD was detected for 10 minutes, using confocal microscopy. LDs were subdivided into small (3–6 µm diameter) and large (8–12 µm diameter) fractions. Representative fluorescence images of cells with small (A) or large (B) LDs are shown at the indicated time points before and after photobleaching. Scale bar = 10 µm. Normalized mean fluorescence recovery curves of bleached small LDs pretreated for 1 and 3 days post differentiation with the indicated extracts are shown in (C,D), respectively. (E) Calculated exchange rates from a single exponential fit of fluorescence traces are shown in (C,D). Normalized mean fluorescence recovery curves of bleached large LDs pretreated for 1 and 3 days post differentiation with the indicated extracts are shown in (F,G), respectively. (H) Calculated exchange rates from a single exponential fit of fluorescence traces are shown in (F,G). Error bars are based on the SEM of at least 19 analyzed cells from three individual experiments. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001, significantly reduced exchange rate of cells treated with the indicated substances compared to untreated control cells.