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. 2019 Sep 19;9:13555. doi: 10.1038/s41598-019-49929-w

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Concentration-dependence of [³H]2-chloroadenosine uptake. PK15-NTD and PK15-hENT4 cells were exposed to the indicated concentrations of [³H]2-chloroadenosine for 5 min at pH 7.5 or pH 6.0, in the presence of 50 nM ABT-702. Each point represents the mean ± S.E.M of at least 6 independent experiments conducted in duplicate. Panel a shows the data for the full concentration range used. Panel b is an expanded view of the data for the concentration range of 1–200 µM from Panel a. Panel c: To assess the kinetics of the acidic pH-dependent transport component, uptake (from Panel a) by PK15-hENT4 cells at pH 7.5 was subtracted from that obtained at pH 6.0 The resulting data set was best described by an allosteric sigmoidal non-linear model with the kinetic parameters shown. Panel d is an expanded view of the data for the concentration range of 1–200 µM in Panel c. Data in this range were best described by a non-cooperative Michaelis-Menten relationship. Due to the large errors that resulted from the data subtraction, only approximate kinetic constants could be obtained in this case. Panel e: To assess the kinetics of the ENT4-mediated transport at pH 7.5, uptake (from Panel a) by PK15-NTD cells at pH 7.5 was subtracted from that using the PK15-hENT4 cells at pH 7.5. This transport component fit best to a regular Michaelis-Menten relationship. Panel f is an expanded view of the data for the concentration range of 1–200 µM in Panel e. These data were best described by a linear relationship. Panels g and h are the Hill Plot derivations of the data shown in Panels c and e, respectively. The slopes of the fitted lines, which represent the Hill coefficients, are indicated.