Effect of imipramine and of cAMP-dependent PKA phosphorylation on SLC41A1-related [Mg²⁺]_i changes, cell adhesion, and cell proliferation in +tet p.A350V cells and wt cells.

<p>A: Summary of [Mg²⁺]_i changes after resuspension of Mg²⁺-loaded +tet p.A350V cells and wt cells in completely Mg²⁺-free Na⁺-containing solutions with or without (control) the Na⁺/Mg²⁺ exchanger inhibitor imipramine (250 µM). Values have been corrected for [Mg²⁺]_i changes in -tet cells and given as means ± SE; N_p.A350V  = 26 & N_wt  = 21 single experiments per condition; *P = 0.03; **P<0.005. B: Summary of [Mg²⁺]_i changes after resuspension of Mg²⁺-loaded +tet p.A350V cells and wt cells in completely Mg²⁺-free Na⁺-containing media with or without (control) the Na⁺/Mg²⁺ exchanger activator dB-cAMP (100 µM). Values have been corrected for [Mg²⁺]_i changes in -tet cells and are means ± SE; Np.A350V  = 15 & Nwt  = 15 single experiments per condition; **P = 0.01. C: Original growth curves of +tet p.A350V cells and wt cells under control conditions and after application of 250 µM imipramine and of 100 µM dB-cAMP. Cells were seeded at a density of 10×10⁵ per well, induced with tetracycline, and allowed to attach and proliferate for 24 h prior to treatment with the compounds (indicated by the arrow). The Cell Index, a dimensionless parameter reflecting cell adherence and number, was normalized (nCI) to the time just before modulator application. Values are means ± SD; N = 6 single experiments per condition; **P<0.001.</p

[Mg²⁺]_i (mM) of uninduced (-tet) and induced (+tet) HEK293-(HA-strep-SLC41A1), and HEK293-(HA-strep-SLC41A1-p.A350V) cells.

<p>[Mg²⁺]_i values for cells successively incubated for 20 min in solutions containing 10 mM Mg²⁺ (loading conditions) and for 10 min in completely Mg-free media (efflux conditions) are given. Data are presented as means ± SE. N is being indicated. **P<0.001 vs. control (-tet cells); ^aP<0.001 vs. loaded +tet HEK293-(HA-strep-SLC41A1) or HEK293-(HA-strep-SLC41A1-p.A350V) cells.</p

Confocal immunolocalization of HA-strep-SLC41A1

<p>(<b>wt</b>) <b>and HA-strep-SLC41A1-p.A350V in +tet</b> (<b>24 </b><b>h</b>) <b>cells.</b> Strep-tagged wt and p.A350V were immunolabeled with primary mouse anti-strep and secondary GAM Alexa-488 antibodies (green signal). Plasma membranes were fluorescently contrasted with wheat germ agglutinin (WGA) conjugated to Alexa-647 (red signal). Nuclei were stained with DAPI (blue signal). The merged images show that both Alexa-488 and Alexa-647 signals co-localize in +tet cells. Scale bar indicates 10 μm.</p

Immunodetection of phosphorylated recombinant flag-SLC41A1

<p>(<b>wt</b>)<b>, HA-strep-SLC41A1</b> (<b>wt</b>)<b>, and HA-strep-SLC41A1-p.A350V with the PhosphoProtein purification kit</b> (<b>Qiagen</b>)<b>.</b> Total (T), flow-through (U; containing unphosphorylated proteins) and elution (P; containing phosphorylated proteins) fractions were probed with antibodies against strep- or flag-tag. A signal specific for phosphorylated wt or p.A350V was detected in all three cell lines. The specificity of the fractionation was controlled with an antibody against phosphorylated Akt.</p

(A) Quantitative real time PCR analysis of <i>SLC41A1</i> (<i>wt</i>) expression in -tet and +tet cells. The ddCt values of three independent +tet samples and three independent -tet samples are shown. Each biological sample was loaded in triplicate. IRC indicates inter-run control. (B) Quantitative real time PCR analysis of <i>SLC41A1</i>-(<i>c.1049C>T</i>) expression in -tet and +tet cells. The ddCt values of three independent +tet samples and three independent -tet samples are given. Each biological sample was loaded in triplicate. IRC indicates inter-run control. (C) Immunodetection of recombinant HA-strep-SLC41A1(wt) and HA-strep-SLC41A1-p.A350V in total protein isolate from -tet and +tet (24 h) cells. Strep-tagged wt and p.A350V were detected only in tet-induced cells. Positive control: flag-tagged SLC41A1 isolated from HEK293 cells, clone 17, which was extensively characterized in [20], [27]. Loading was controlled by immunodetection of RPL19 protein. (D) Immunodetection of recom

<p>(A) Quantitative real time PCR analysis of <i>SLC41A1</i> (<i>wt</i>) expression in -tet and +tet cells. The ddCt values of three independent +tet samples and three independent -tet samples are shown. Each biological sample was loaded in triplicate. IRC indicates inter-run control. (B) Quantitative real time PCR analysis of <i>SLC41A1</i>-(<i>c.1049C>T</i>) expression in -tet and +tet cells. The ddCt values of three independent +tet samples and three independent -tet samples are given. Each biological sample was loaded in triplicate. IRC indicates inter-run control. (C) Immunodetection of recombinant HA-strep-SLC41A1(wt) and HA-strep-SLC41A1-p.A350V in total protein isolate from -tet and +tet (24 h) cells. Strep-tagged wt and p.A350V were detected only in tet-induced cells. Positive control: flag-tagged SLC41A1 isolated from HEK293 cells, clone 17, which was extensively characterized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071096#pone.0071096-Kolisek1" target="_blank">[20]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071096#pone.0071096-Kolisek2" target="_blank">[27]</a>. Loading was controlled by immunodetection of RPL19 protein. (D) Immunodetection of recombinant HA-strep-SLC41A1(wt) and HA-strep-SLC41A1-p.A350V in soluble and membrane-protein-enriched fractions isolated from -tet and +tet (24 h) cells. Strep-tagged wt and p.A350V were detected almost exclusively in tet-induced cells and predominantly in membrane (M) protein fractions and in much lower quantities in soluble (S) protein fractions. Positive control: flag-tagged SLC41A1 isolated from HEK293 cells (clone 17). Soluble RPL19 was used to control the specificity of the separation between soluble and membrane proteins. (E) Immunodetection of recombinant HA-strep-SLC41A1(wt) and HA-strep-SLC41A1-p.A350V in subcellular protein fractions isolated from +tet (24 h) cells. Wt and p.A350V were predominantly detected in membrane (M) protein fractions with much lower quantities in cytosolic (C) protein fractions and also for p.A350V in traces in the nuclear (N) protein fraction. Transgenic variants were not detected in cytoskeletal (S) fractions. Specificity of the fractionation was controlled on a parallel blot by immunodetection of PMCA4 (M).</p

SLC41A1-related Mg²⁺ efflux in p.A350V cells compared with wt cells.

<p>Before measurements, cells were pre-loaded with Mg²⁺ as described in Material and Methods ([Mg²⁺]_e  = 10 mM). The [Mg²⁺]_i change obtained after 10 min in media containing 145 mM Na⁺ is given for the following conditions: (1) completely Mg²⁺-free media (N_p.A350V  = 113 & N_wt  = 82); (2) media supplemented with 5 mM Mg²⁺ (N_p.A350V  = 13 & N_wt  = 14); and (3) media supplemented with 10 mM Mg²⁺ (N_p.A350V  = 13 & N_wt  = 14). Values have been corrected for [Mg²⁺]_i changes in -tet cells and are given as means ± SE; **P<0.001.</p