Jump-In T-Rex HEK293 cells with doxycycline inducible expression of SLCO2A1 (HEK293-JumpIn-SLCO2A1) were generated by and obtained via the Resolute consortium (http://re-solute.eu). Human umbilical vein endothelial cells (HUVEC, CRL-4053), vascular cell basal medium and endothelial cell growth kit - VEGF were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). Dulbecco’s modified Eagles medium (DMEM), G418 and gelatin were purchased from Merck (Darmstadt, Germany). PGE₂ was purchased from Selleck chemicals GmbH (Cologne, Germany) and olmesartan was obtained from MedChemExpress (South Brunswick, NJ, USA). cDNA encoding the prostaglandin receptor EP3 (NM_198714.1) or EP4 (NM_000958.3) with N-terminal Myc-tag in pcDNA3.1(+) were purchased from Genscript Biotech B.V. (Leiden, the Netherlands). The PGT inhibitor 4-((4-((2-(2-(2-Benzamidoethoxy)ethoxy)ethyl)amino)-6-((4-hydroxyphenyl)amino)-1,3,5-triazin-2-yl)amino)benzoic acid (T26A) was synthesized in house according to procedures adapted from previously reported literature (For more detail see SI) (Min et al., 2007; Schuster et al., 2015). All other resources were of analytical grade and purchased from standard commercial resources.

SLCO2A1 mutants A396E, A396T and L563P were generated using QuickChange II site-directed mutagenesis kit (Qiagen) using pDONR221-SLCO2A1 (Addgene, plasmid #131949) as template DNA and primers were obtained from Integrated DNA Technologies (IDT, Leuven, Belgium). Successful mutant generation was confirmed by Sanger sequencing at LGTC (Leiden, the Netherlands). Next, to enable doxycycline-inducible expression of the generated mutants gateway cloning was used to transfer SLCO2A1 mutants from the pDONR vector to a pJTI-R4-DEST-CMV expression vector containing a tet-operon for doxycycline-induction and C-terminal twin-Strep-tag and hemagglutinin (HA)-tag. In brief, 150 ng entry vector pDNOR221-SLCO2A1-mutant (i.e., A396E, A396T or L563P) and 150 ng destination vector pJTI-R4-DEST-CMV were incubated with Gateway LR Clonase II enzyme in TE buffer for 1h at 25°C. The mixture was incubated with Proteinase K solution for 10 min at 37°C prior to transformation into XL-1 blue competent cells. Plasmids were isolated using NucleoBond xtra midiprep kit (Macherey-Nagel, Germany) and sequences were again confirmed by Sanger sequencing. HEK293-JumpIn-parental cells were stably transfected with cDNA of PGT mutants in pJTI-R4 vector using lipofectamine as described previously (Gorostiola González et al., 2023).

HEK293-JumpIn-SLCO2A1 cells (HEK-JI-SLCO2A1) were cultured in DMEM high glucose supplemented with 10% fetal calf serum (FCS), 2 mM glutamax, penicillin (100 μg/mL) and streptomycin (50 μg/mL) at 37°C with 5% CO₂. Cells were transiently transfected with 2.5 µg prostaglandin receptor EP3 or EP4 cDNA and 2.5 µg empty pcDNA3.1 vector using the 25 kDa linear PEI method as previously described (Mocking et al., 2019). Cells were cultured for an additional 24 h before use in assays. HUVECs were cultured in vascular cell basal medium supplemented with endothelial cell growth kit–VEGF, 5% FCS, 2 mM glutamax, penicillin (100 μg/mL) and streptomycin (50 μg/mL). For experiments culture medium was replaced with medium containing 2% FCS.

The Transport activity through receptor activation (TRACT) assays were performed using xCELLigence real-time cell analyzer SP or MP system. The xCELLigence system measures changes in cellular impedance resulting from cell adhesion, cell proliferation or changes in cell morphology. HEK293-JumpIn-SLCO2A1 cells transiently transfected to express prostaglandin EP3 receptor (EP3) or prostaglandin EP4 receptor (EP4) were seeded (70.000 cells/well) in a 96 well E-plate PET in absence or presence of 1 μg/mL doxycycline (dox) to induce PGT expression. After ∼29 h, cells were pretreated with 100 µM olmesartan, 5 µM T26A or vehicle (PBS +0.1% DMSO) for 1 h prior to stimulation of the cells with increasing concentrations of PGE₂. For inhibition-response curves cells were pretreated with increasing concentrations of inhibitor for 1 h prior to stimulating with 1 nM PGE₂ (i.e., the concentration that provides best window between -dox and +dox presence). Cellular responses were measured every 30 s for 30 min followed by every minute for 30 min, every 5 min for 1 h and every 15 min up to a total span of 3 h.

The impedance-based scratch-induced wound healing assay measures the increase in cellular impedance (CI) on a E-wound plate, that only contains electrodes in the scratch area, therefore the CI directly reflects the number of cells that have infiltrated the scratched area. For impedance-based wound healing experiments HUVECs were seeded (40.000 cells/well) onto a 96 well E-wound plate coated with 1% gelatin. The plate was rested for 30 min at room temperature to allow cells to settle before being transferred into the xCELLigence MP cradle in a 37°C 5% CO₂ incubator. Cell growth and adhesion were monitored every 15 min for a period of 18 h. Next, the E-wound plate was removed from the incubator and homogenous scratches were generated using the AccuWound 96 tool (Agilent, CA, USA). Cell debris was removed by washing the cells with culture medium containing 2% FCS prior to treatment of the cells with 100 nM PGE₂, 5 μM T26A, 100 nM PGE₂ and 5 µM T26A or vehicle in medium containing 2% FCS. We selected 100 nM PGE₂ as this excess concentration was previously shown to successfully induce wound healing for up to 2 days (Syeda et al., 2012; Liu et al., 2015). The plate was returned to the cradle and cellular impedance was monitored every 15 min for a period of 72 h.

An HA-tag enzyme-linked immunosorbent assay (ELISA) was performed to confirm expression of wildtype and mutant PGT variants stably transfected into HEK-JumpIn cells upon induction with 1 μg/mL doxycycline. HEK-JumpIn-SLCO2A1 wildtype, A396E, A396T or L563P were seeded (60.000 cell/well) on a poly-D-lysine coated, transparent, flat-bottom 96 well plate in medium with or without 1 μg/mL dox and grown for an addition 30 h at 37°C 5% CO₂. Cells were washed with PBS, fixated for 10 min with 4% formaldehyde and permeabilized and blocked using Tris-buffered saline (TBS) with 2% BSA and 0.2% saponin for 1 h. Next, cells were incubated with 1:2500 primary Rabbit anti-HA polyclonal antibody (Invitrogen, Carlsbad, CA, United States) in assay buffer (TBS with 0.1% BSA) for 1 h at RT. Cells were washed with assay buffer prior to incubation with 1:6000 Goat-anti-rabbit HRP-conjugate antibody (Brunschwig Chemie, Amsterdam, Netherlands) in assay buffer for 30 min at RT. Immunoreactivity was detected by incubating with 3,3′,5,5′-tetramethylbenzidine (TMB) for 2 min before quenching with equal volume of 1M H₃PO₄. Absorbance was measure at 450 nm using a Wallac EnVision multimode plate reader (PerkinElmer, Groningen, the Netherlands).

All data was analyzed using Graphpad prism v9.0 (Graphpad software Inc, San Diego, CA, USA). xCELLigence data was recorded and normalized to the timepoint prior to stimulation or scratch to obtain normalized cell index (nCI) or delta CI (ΔCI) using RTCA v2.0 (SP) or RTCA software Pro V2.8.0 (MP) for TRACT and wound healing assays, respectively. TRACT-data was corrected for any ligand-independent effects by subtraction of the vehicle-induced response. From the vehicle-corrected nCI data the net area under the curve (AUC) over the first 3 h was calculated to generate concentration-response curves. To obtain potency (pEC₅₀ or pIC₅₀) values, concentration-response data was fitted to a non-linear regression three parameter response model. pIC₅₀ stands for −log(IC₅₀) where IC₅₀ is the concentration (in mol/L) required to inhibit the maximal response by 50%. For impedance-based wound healing assays data was analyzed by calculating either the AUC from scratch up to 12, 24 or 72 h after scratch or by taking the ΔCI at timepoint 24 h after scratch and calculating the % wound healing, where maximal ΔCI at 72h was set at 100%. Statistical analyses were performed using Graphpad Prism v9.0 and specified in the legend of each figure.

To investigate the potential of a label-free impedance-based TRACT assay for PGT using endogenously expressed prostanoid receptors, we selected HEK293-JumpIn-SLCO2A1 cells with dox-inducible expression of the prostaglandin transporter and endogenous expression of prostaglandin E2 receptor EP2 (EP2). Cells were seeded onto a 96 well E-plate and the next day cells were stimulated with increasing concentrations of PGE₂. PGE₂ addition to non-induced cells resulted in a clear dose-dependent cellular response in the impedance-based assay (Supplementary Figure S1A). The endogenously expressed EP2 receptors did not allow to detect a sufficient decrease in PGE₂ levels upon induction of PGT, as no clear rightward shift of the PGE₂ concentration-response was observed (Supplementary Figure S1). Therefore, HEK-JumpIn-SLCO2A1 cells were transiently transfected to either express the high affinity prostaglandin receptor EP3 or EP4 as these receptors could act as more sensitive sensors to detect changes in extracellular PGE₂ levels. Interestingly, the obtained cellular impedance traces upon treatment with PGE₂ were different for activation of G_i-coupled EP3 receptor and G_s-coupled EP4 receptor. For EP3, addition of PGE₂ induced an initial drop in nCI followed by a recovery to baseline within the first 10 min, which was then followed by a gradual decrease in vehicle-corrected nCI up to 180 min (Figure 1A). In contrast, EP4 activation by PGE₂ mediated a steep increase in vehicle-corrected nCI reaching plateau after approximately 30 min followed by a gradual steady decrease in nCI back to baseline up to 180 min (Figure 1B). Thus, overexpression of EP3 and EP4 in HEK-JI-SLCO2A1 cells resulted in a clear dose-dependent decrease or increase in cellular response for EP3 and EP4, respectively, as measured by xCELLigence (Figures 1A, B). Next, the effect of PGT expression on these cells was investigated by dox-induction, which reduced the concentration-dependent cellular PGE₂ response (Figures 1C, D). Both EP3 and EP4 expressing cells displayed an over 10-fold reduced potency for PGE₂, as demonstrated by a rightward shift of the PGE₂ concentration-response curve upon induction of PGT (Table 1; Figures 1E, F). Taken together, PGT-mediated PGE₂ uptake can be detected as attenuation of the GPCR response using either EP3 or EP4 receptors as PGE₂ sensor in the impedance-based assay.

To validate that the observed potency shift could be attributed to PGT-mediated PGE₂ uptake in these cells, dox-induced HEK-JumpIn-SLCO2A1 cells expressing either EP3 or EP4 were pretreated with PGT inhibitor olmesartan prior to stimulation with increasing concentration of PGE₂. Indeed, upon pretreatment with 100 µM olmesartan a leftward shift of the PGE₂ concentration-response curve was observed with a 5.2-fold and 4.2-fold shift in potency for EP3 and EP4, respectively (Figures 2A, C; Table 1). The angiotensin II receptor type 1 (AT1R), the high-affinity target of olmesartan, is not expressed on HEK-JumpIn cells according to transcriptomics data (BioSamples database: SAMN11893676, SAMN11893683 and SAMN11893690). In agreement, pretreatment of either induced or non-induced HEK-JumpIn-SLCO2A1 cells with increasing concentrations of olmesartan did not induce changes in cellular impedance (Supplementary Figure S2). In addition, dox-induced cells pretreated with 5 µM of selective PGT inhibitor T26A displayed a modest 2.5-fold and 3.2-fold increased potency for EP3 and EP4, respectively (Figures 2B, D). The ability of olmesartan and T26A to partially revert the PGE₂ response back towards a non-induced response (i.e., absence of PGT), corroborates the establishment of a TRACT assay for PGT.

Next, we investigated if the presented TRACT assay could be used to determine the potency of PGT inhibitors utilizing the EP4 receptor as signaling partner. Of interest, EP4 was selected over EP3 to determine inhibitory potencies as the observed response was more robust and due to its link to impaired wound healing (vide infra). We selected 1 nM PGE₂ as preferred concentration to stimulate EP4 expressing cells in inhibition experiments as this concentration resulted in the largest shift (window) in PGE₂-mediated AUC cellular response between uninduced (-dox) and PGT expressing cells (+dox) (Figure 1F). HEK-JumpIn-SLCO2A1 cells transiently expressing EP4 receptor were pretreated with increasing concentrations of PGT inhibitor olmesartan or T26A for 1 h prior to stimulating the cells with 1 nM PGE₂ (Figure 3). Both inhibitors displayed dose-dependent inhibition of PGT, observed as an increase in cellular response, with pIC₅₀ = 5.0 ± 0.0 for olmesartan and pIC₅₀ = 6.7 ± 0.2 for T26A, potencies that are comparable to values of traditional [³H]PGE₂ uptake assays (Chi et al., 2011; Kamo et al., 2017). This shows that the impedance-based TRACT assay is a useful (orthogonal) assay to determine the potency of PGT inhibitors.

Next, it was assessed whether natural and disease-related PGT variant activity can be studied using the established label-free impedance-based TRACT assay. To this end, HEK-JumpIn-SLCO2A1 cells were generated that expressed either PHO mutant L563P, thiazide-induced hyponatremia (TIH) mutant A396T or A396E, i.e., a phospho-mimic of A396T that was reported to have decreased uptake activity). Stable transfection of each of the three PGT variants into HEK-JumpIn cells resulted in similar expression levels to wildtype PGT upon dox-induction as confirmed by HA-tag ELISA (Supplementary Figure S3). Both TIH-mutant A396T and its phosphomimic A396E display a 6-fold (pIC₅₀ shift 0.8) reduced potency of PGE₂ upon dox-induced PGT expression indicating that they were still capable of transporting PGE₂ (Figures 4A, B, D). Moreover, the observed potency shift for these mutants was not significantly different from wildtype. In contrast, the loss of function mutant L563P abrogated the shift in PGE₂ potency upon dox-induction, consistent with loss of PGE₂ transport, and this loss was not related to less L563P expression as confirmed by whole cell ELISA (Figure 4C; Supplementary Figure S3). Together, these data confirm that the TRACT assay can be utilized to assess uptake activity of PGT disease-related and natural variants.

Lastly, we explored the opportunity to detect wound healing in real-time with the impedance-based technology using HUVECs endogenously expressing prostanoid receptors and PGT. HUVECs were seeded onto an E-wound plate and grown for 18 h until a plateau was reached (Figure 5A; Supplementary Figure S4). Identical scratches were induced by the AccuWound 96 tool and scratch closure was followed in real-time for 3 days in the presence of 100 nM PGE₂, 5 μM T26A, both 100 nM PGE₂ and 5 µM T26A or vehicle (Figure 5A; Supplementary Figure S4). As HUVECs also express the Angiotensin II type 1 receptor we omitted olmesartan in this wound healing assay to avoid non-PGE₂ mediated effects. Treatment with either PGE₂, T26A or both accelerated invasion of the lesion area by cells and thus scratch closure within the first 24 h after scratch generation as compared to vehicle treated cells (Figures 5A–C). However, calculating the AUC after 24 and 72 h (i.e., total measured time) only PGE₂ addition mediated a significantly increased scratch closure as the effect of T26A seemed to diminish after 48 h (one-way ANOVA with Dunnett’s post-hoc test) (Figures 5A, D). However, when comparing the % wound healing at 24h after scratch generation T26A and co-addition of PGE₂ and T26A presented a clear trend of increased scratch closure compared to vehicle treated cells (Figure 5E). Interestingly, PGE₂ accelerated the scratch repair, as a fully healed scratch apparent from the curve plateau was evident at approximately 48 h, while vehicle-treated cells did not reach plateau until after 72 h (Figure 5A). Taken together, enhanced activation of prostanoid receptors by either addition of extracellular PGE₂ or reduced uptake and breakdown due to PGT inhibition with T26A accelerated wound healing.