Preparation of MDCKII Cells Stably Expressing GFP-Tagged Rmate1

Ohta et al., Functional characterization of multidrug and toxin extrusion protein 1 as a facilitative transporter for fluoroquinolones. JPET

SUPPLEMENTAL DATA

Methods

Preparation of MDCKII Cells Stably Expressing GFP-tagged rMATE1

To generate a GFP-tagged rMATE1 (GFP-rMATE1), the coding sequence of rMATE1 was amplified from the pCI-neo-based plasmid carrying the cDNA, using a forward primer containing an XhoI restriction site (underlined), 5'- GGC TCG AGC CGC CAC CAT GGA GGT CTT GGA GG -3', and the reverse primer for pCI-neo vector, 5'- TTC ACT GCA TTC TAG TTG TGG -3'. Then the amplified product was introduced at the XhoI and XbaI sites into pEGFP-C1 vector (Clontech). MDCKII cells were transfected with the generated plasmid, and geneticin-resistant clones were selected. The expression of GFP-rMATE1 was confirmed by fluorescence from GFP-rMATE1 and transport of [14C]TEA.

Immunofluorescence Analysis

The localizations of GFP-rMATE1 and immunofluorescent-labeled Na+/K+ ATPase were examined by using a confocal laser-scanning microscope, as reported previously (Nakai et al., 2007).

Transcellular Transport Study

The basal-to-apical transcellular transport of NFX was also examined in MDCKII cells expressing GFP-rMATE1 (GFP-rMATE1 cells) as in rMATE1 cells.

Supplemental Figure

Cellular localization of GFP-rMATE1 in MDCKII cells. A, GFP-rMATE1 (green) was localized predominantly at the apical membrane in GFP-rMATE1 cells cultured on the permeable membrane filter (TranswellTM), whereas Na+, K+-ATPase (red) was localized at the basolateral membrane. This apical localization of GFP-rMATE1 is consistent with that reported for mouse rMATE1 in the renal tubular epithelial cells by immunohistochemical assessment (Otsuka et al., 2005). B, the basal-to-apical transcellular transport of NFX in GFP-rMATE1 cells was much greater than that in mock cells and extensively inhibited by corticosterone, consistent with what was observed in rMATE1 cells. All these supplemental results support our suggested role of rMATE1 in apical efflux of NFX.

REFERENCES

Nakai Y, Inoue K, Abe N, Hatakeyama M, Ohta KY, Otagiri M, Hayashi Y and Yuasa H (2007). Functional characterization of human proton-coupled folate transporter/heme carrier protein 1 heterologously expressed in mammalian cells as a folate transporter.

J Pharmacol Exp Ther. 322: 469-76.