Quantitative Amino Acid Uptake Analysis

Waters TargetLynx V4.2 software (Waters, Milford, MA, USA) was used to fit calibration curves based on 1/x²-weighted linear regressions of the peak area ratios of [¹³C₆, ¹⁵N]-L-leucine and the IS. GraphPad Prism 9 software (version 9.3.1) was used for data visualization, statistical analysis (unpaired t-test), and to determine the IC₅₀ values of JPH203 and BCH (nonlinear regression, log(inhibitor) vs. response with variable slope (four parameters)). The obtained IC₅₀ values were converted to the corresponding inhibition constant K_i with the IC-50-to-K_i web-based tool [49 (link)]. Therefore, the mean of reported K_m values for L-leucine uptake by LAT-1_HD (32 µM) was used [1 (link),2 (link),50 (link)], and classical competitive inhibition was assumed. Microsoft Excel 2010 (Mountain View, CA, USA) was used for general calculations and for the normalization of the [¹³C₆, ¹⁵N]-L-leucine cell homogenate amount of substance to protein content [pmol/mg]. Western blot band intensities were quantified with Fiji (Fiji is just ImageJ 2.0.0) [54 (link)].