Figure 4

NPT2c variants do not inhibit either WT NPT2c or WT NPT2a phosphate transport activity. (a) Na-dependent Phosphate uptake comparison between non transfected HEK cells (No transfection), HEK cells stably transfected with WT NPT2c (WT) and HEK cells transfected stably with WT NPT2c and transiently transfected with either WT NPT2c (WT + WT NPT2c) or non-transporting NPT2c variants (WT + variant). Transiently transfected cells with 1 μg of DNA and 1 μl of lipofectamine 2000 per well. Expression of non-transporting NPT2c variants (WT + variant) did not decrease sodium phosphate transport compared to the HEK cells expressing WT NPT2c alone (WT). Quantitative data represent 3 independent experiments. Statistical analysis was performed using a non-parametric Kruskall–Wallis test followed by Dunn’s multiple comparison post-hoc test. *p < 0.05, **p < 0.01. Simple line represents Kruskall–Wallis test and zig-zag line represent Dunn’s multiple comparison post-hoc test. (b) Similar experiment with variants that do transport phosphate. With the HEK cells transfected stably with WT NPT2c and transiently with NPT2c variants that do transport phosphate (WT + variant), phosphate uptake is similar to HEK cells transfected stably and transiently with WT NPT2c (WT + WT NPT2c) and higher than WT NPT2c alone (WT). So, expression of the transporting NPT2c variants did not decrease phosphate transport compared to the HEK cells expressing WT NPT2c. Transiently transfected cells with 0.5 μg of DNA and 1 μl of lipofectamine 2000 per well. Quantitative data represent 3 independent experiments. Statistical analysis was performed using a non-parametric Kruskall–Wallis test followed by Dunn’s multiple comparison post-hoc test. *p < 0.05, ^nsp not significant. Simple line represents Kruskall–Wallis test and zig-zag line represent Dunn’s multiple comparison post-hoc test. (c) Similarly, we checked whether non transporting NPT2c variants could decrease phosphate transport in HEK cells expressing WT NPT2a. Again, transient expression of the non-transporting NPT2C variants in HEK Cells stably transfected with WT NPT2a (WT + variant) did not decrease phosphate transport compared to the HEK cells expressing stably WT NPT2a (WT). Transiently transfected cells with 0.5 μg of DNA and 1 μl of lipofectamine 2000 per well. Quantitative data represent 3 independent experiments. Statistical analysis was performed using a non-parametric Kruskall–Wallis test followed by Dunn’s multiple comparison post-hoc test. *p < 0.05. Simple line represents Kruskall–Wallis test and zig-zag line represent Dunn’s multiple comparison post-hoc test.