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. 2015 Oct 22;71(2):403–412. doi: 10.1093/jac/dkv340

Figure 4.

Figure 4.

Uptake of MIPS-9541 in HEK293 cells transfected with PEPT2 or the vector alone. (a) Accumulation of MIPS-9541 in HEK293 cells overexpressing PEPT2. The vector- and transporter-expressing cells were incubated with 10 μM MIPS-9541 (in PBS, pH 5.5). Fluorescent signals accumulated in cells were measured using a Tecan Safire II microplate reader over 60 min. Background counts of vector-transfected cells were subtracted from all uptake data and the result was standardized to fluorescence counts/100 μg of protein. (b) Kinetic parameters of MIPS-9541 uptake via PEPT2. The kinetic parameters of MIPS-9541 uptake were derived in HEK293 cells transiently transfected with PEPT2 or the vector. Uptake was assessed with various concentrations of MIPS-9541 (ranging from 0 to 500 µM), subtracting the background of vector-transfected control cells. Km and Vmax values of MIPS-9541 uptake were derived by GraphPad Prism 6.0 software. (c) Inhibition by Gly-Sar, colistin or polymyxin B of PEPT2-mediated MIPS-9541 uptake. MIPS-9541 uptake was measured in the absence or presence of 10 µM Gly-Sar, colistin or polymyxin B in cells overexpressing human PEPT2 or transfected with the vector alone. Background counts of vector-transfected cells were subtracted from all uptake data and the standardized result is presented as a percentage of the control (i.e. no inhibitors). All experiments were conducted independently three times with three replicates in each experiment and values are expressed as the mean ± SEM. Significant difference from the control: **P < 0.01.