HEK293 cells expressing hAQP9 or hDAT reveal higher sensitivity to MPP⁺.

A-B) Immunofluorescence images of HEK293 cells expressing EGFP-hAQP9 (A) or YFP-hDAT (B) grown on coverslips confirm plasma membrane localization of the respective constructs (identified by antibodies to AQP9 or DAT). The cells were counterstained with Hoechst to visualize nuclei. C-G) Native HEK293 cells and HEK293 cells expressing EGFP-hAQP9 or YFP-hDAT were grown in 96-well plates and exposed to different concentrations of MPP⁺ (four wells for each concentration). Cell viability was assessed after 24 hours using the MTT assay. Data were collected from independent plates (n = 3 for each construct) and normalized to respective untreated cells. Native HEK293 cells show sensitivity to MPP⁺ only at very high concentrations (~100 μM). Cells expressing hDAT become sensitive at 1 μM MPP⁺, compared with 0.1 μM for cells expressing hAQP9. Overlay of the dose/response curve for the three groups (D) and the individual curves for native HEK293 cells (E), YFP-hDAT expressing (F) and EGFP-hAQP9 expressing HEK293 cells (G) are shown. Bars are mean ± SEM. Asterisks: significantly different from untreated controls; *p<0.05, **p<0.01, ***p<0.001; crosses: significantly different from previous data point: ++<0.01.