Nucleoside transporters (NTs) play an important role in the metabolism of nucleoside substances and the efficacy of nucleoside drugs. Its spatial information related to biofunctions at the single-molecule level remains unclear, owing to the limitation of the existing labeling methods and traditional imaging methods. Therefore, we synthesize the inhibitor-based fluorescent probe SAENTA-Cy5 and apply direct stochastic optical reconstruction microscopy (dSTORM) to conduct refined observation of human equilibrative nucleoside transporter 1 (hENT1), the most important and famous member of NTs. We first demonstrate the labeling specificity and superiority of SAENTA-Cy5 to the antibody probe. Then, we found different assembly patterns of hENT1 on the apical and basal membranes, which are further investigated to be caused by varying associations of membrane carbohydrates, membrane classical functional domains (lipid rafts), and associated membrane proteins (EpCAM). Our work provides an efficient method for labeling hENT1, which contributes to realize fine observation of NTs. The findings on the assembly features and potential assembly mechanism of hENT1 promote a better understanding of its biofunction, which facilitates further investigations on how NTs work in the metabolism of nucleoside and nucleoside analogues.

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