HEK293T cells were obtained from ATCC and grown in DMEM/high glucose/sodium pyruvate (Invitrogen) containing 10% FBS. Culture conditions were in a 5% CO2 incubator at 37°C. Cell viability was measured using the trypan blue exclusion method.
Plasmids and transfection
NHE9-DsRed was cloned into FUGW-lentiviral vector into the BamHI site. QuikChange Lightning site-directed mutagenesis kit (#21051, Agilent Technologies) was used to engineer P117T, D496N, Q609K, and S438P missense substitutions into NHE9. Resulting plasmids were confirmed by sequencing. HEK293T cells were transfected using Lipofectamine 2000 reagent, as per the manufacturer's instructions. Empty vector transfection was used as control. Transfection efficiency was determined by visualizing DsRed fluorescence.
Endosomal pH measurement
Endosomal pH was measured using flow cytometry, as previously described. Briefly, cells were rinsed and incubated in serum-free medium for 30 min, to remove residual transferrin and then incubated with pH-sensitive FITC-Transferrin (#T2871, Thermo Fisher Scientific) (75 μg/ml) together with pH non-sensitive Alexafluor 633-Transferrin (#T23362, Thermo Fisher Scientific) (25 μg/ml) at 37°C for 55 min. Transferrin uptake was stopped by placing the cells on ice. Excess transferrin was removed by washing with ice-cold serum-free DMEM and PBS, whereas bound transferrin was removed by washing with ice-cold pH 5.0 PBS and pH 7.0 PBS.
Cells were trypsinized and pH was determined by flow cytometry analysis of ∼10,000 cells gated for NHE9 positivity in biological triplicates using the FACSAria instrument (BD Biosciences). A 4–point calibration curve with different pH values (4.5, 5.5, 6.5 and 7.5) was generated using Intracellular pH Calibration Buffer Kit (#P35379, Thermo Fisher Scientific) in the presence of 10 μM K+/H+ ionophore nigericin and 10 μM K+ ionophore valinomycin.
Subcellular localization and functional evaluation of variants was performed using steady state transferrin uptake assay. Briefly, cells were rinsed and incubated in serum-free medium for 30 min, to remove residual transferrin and then incubated with Alexa Fluor 633 or 488-Transferrin (Thermo Fisher Scientific) (100 μg/ml) at 37°C for 60 min. Transferrin uptake stopped by placing the cells on ice. Excess transferrin was removed by washing with ice-cold serum-free DMEM and PBS, whereas bound transferrin was removed by 2× washing with ice-cold pH 5.0 PBS and pH 7.0 PBS.
Cells were trypsinized for flow cytometry transferrin uptake analysis of ∼10,000 cells gated for NHE9 positivity in biological triplicates using the FACSAria instrument (BD Biosciences). Alternatively, cells were fixed with a solution of 4% paraformaldehyde, for confocal imaging using the LSM 700 Confocal microscope (Zeiss). Expression of NHE9 (WT and variants) was detected using the DsRed fluorescence. Nuclei were stained with DAPI. Fractional colocalization was determined from Manders' coefficient using the JACoP ImageJ plugin that measures the direct overlap of pixels in the confocal section, and represented it as mean ± S.E.
Cells were lysed using 1% Nonidet P-40 (Sigma) supplemented with protease inhibitor cocktail (Roche). Cells were sonicated and then centrifuged for 15 min at 18,000× g at 4°C. Protein concentration was determined using the BCA assay. Dithiothreitol (DTT) (NuPAGE) was used to reduce protein samples for protein gel electrophoresis. Equal amounts of protein were separated by polyacrylamide gel (NuPAGE Novex) and then electrophoretically transferred onto nitrocellulose membranes (Bio-Rad). Next, the membranes were treated with the blocking buffer containing 5% milk, followed by overnight incubation with primary Anti-DsRed antibody (#ab62341, Abcam, 1:1,000) and 1 h incubation with HRP-conjugated secondary antibodies (GE Healthcare, 1:1,000). SuperSignal West Pico substrate was used for detection. Fujifilm LAS 3000 imaging system was used to capture images. Tubulin (Sigma T9026, 1:5,000) or GAPDH (Sigma G9295, 1:10,000) was used as a loading control. Densitometry analyses was performed with ImageJ software.
Evolutionary conservation scores were calculated using the Bayesian method using the Consurf web server (http://consurf.tau.ac.il/) that analyses the evolutionary pattern of the amino acids to reveal functionally important regions. The physiochemical and functional consequences of NHE9 substitutions was predicted using tools from the Human Gene Mutation Database (http://www.hgmd.cf.ac.uk/). In silico intrinsic disorder with annotated protein-binding activity was predicted for the NHE9 C-terminal tail using the DISOPRED3 program (http://bioinf.cs.ucl.ac.uk/psipred/).
Data were analyzed statistically by Student's t test using GraphPad Prism and presented as mean ± SD.