Sustained OHT-independent flipping of the floxed cassette in 27Flip/Cre20 cells is a stable, ongoing process.

<p>mCherry (+) and mCherry (−) 27Flip/Cre20 cells were separated by FACS and grown in culture for ∼7 weeks. During this time period, a steady decrease in the purity of the sorted populations was observed by flow cytometry. X-axis, mCherry fluorescence, Y-axis, cell number.</p

DT40 B lymphocytes transfected with a reversible switching construct and expressing mer-Cre-mer show 4-hydroxytamoxifen independent Cre activity.

<p><i>A</i> Schematic of the “Flip” construct. Exon 4 of NUDT9 was replaced with a CMV promoter, an in-frame region (containing a NUDT9 cDNA and an inverted mCherry cDNA, each followed by a poly-A region) flanked by loxp sites, and a neomycin selection cassette. Treatment of cells stably expressing the construct with 4-hydroxytamoxifen (OHT) should lead to reversible flipping of the floxed region, as shown in the cartoon, and subsequent expression of mCherry red fluorescent protein in ∼50% of the cells. <i>B</i> Stable integration of the Flip construct was verified in clone #27 via genomic PCR. The positive control represents a different construct that could be amplified with the same primer set. <i>C</i> Following stable transfection of clone #27 with a mer-Cre-mer (mCrem) expressing vector, cells from various clones were lysed and 50 µg of protein from each were run on an 8% SDS-PAGE gel. mCrem was visualized with polyclonal rabbit anti-Cre antibody (1∶2000, Novagen), and a highly expressing clone, 27Flip/Cre20 was selected for further study (red circle). <i>D</i> 27Flip and 27Flip/Cre20 cells were analyzed by flow cytometry, as indicated. In contrast to the 27Flip parental line, the mCrem expressing 27Flip/Cre20 cells showed robust red fluorescence in a large subpopulation despite the absence of the mer- ligand OHT.</p

OHT-dependent flipping of the floxed cassette saturates by 10 nM OHT.

<p>27Flip/Cre clones were either left untreated or treated with 1, 10, or 100 nM OHT for 48 hours and then allowed to grow in culture for 5 days from the onset of OHT treatment: subsequently all clones were analyzed by flow cytometry. 27Flip parental cells were used as a negative control. X-axis, mCherry fluorescence, Y-axis, cell number.</p

Sustained OHT-independent flipping of the floxed cassette in 27Flip/Cre20 cells correlates with mer-Cre-mer expression level.

<p><i>A</i> Following stable transfection of 27Flip cells with an mCrem expressing vector, 5 clones expressing various amounts of mCrem were selected. Cells from the clones were lysed and 50 µg of protein of each were run on an 8% SDS-PAGE gel. MCrem was visualized with polyclonal rabbit anti-Cre antibody (1∶2000, Novagen), with β-actin visualized with a polyclonal mouse antibody (1∶40000, Sigma) as a loading control. 27Flip/Cre15 and -20 were designated “high expressors”, and 27Flip/Cre26, -31, and -35 were designated “low expressors”. <i>B</i> 27Flip/Cre clones were either left untreated or treated with 1 µM OHT for 48 hours and then allowed to grow in culture for 18 days: all clones were subsequently analyzed by flow cytometry. The 27Flip parental cell line was used as a negative control.</p

Efficiency of OHT-dependent flipping of the floxed cassette correlates with duration of OHT exposure in low mCrem expressors.

<p><i>A</i> 27Flip/Cre clones were either left untreated or treated with 1 nM OHT for 24, 10, 5, 2, or 1 hours, and then allowed to grow in culture for 5 days: subsequently all clones were analyzed by flow cytometry. 27Flip parental cells were used as a negative control. <i>B</i> 27Flip/Cre31 cells were either left untreated or treated with 100 nM OHT for 1, 2, 5, 10, 30, 60, 120, or 240 minutes and then allowed to grow in culture for 5 days: subsequently all clones were analyzed by flow cytometry. X-axis, mCherry fluorescence, Y-axis, cell number.</p

The <em>B. subtilis</em> MgtE Magnesium Transporter Can Functionally Compensate TRPM7-Deficiency in Vertebrate B-Cells

<div><p>Recent studies have shown that the vertebrate magnesium transporters Solute carrier family 41, members 1 and 2 (SLC41A1, SLC41A2) and Magnesium transporter subtype 1 (MagT1) can endow vertebrate B-cells lacking the ion-channel kinase Transient receptor potential cation channel, subfamily M, member 7 (TRPM7) with a capacity to grow and proliferate. SLC41A1 and SLC41A2 display distant homology to the prokaryotic family of Mg²⁺ transporters, MgtE, first characterized in <em>Bacillus subtilis</em>. These sequence similarities prompted us to investigate whether MgtE could potentially compensate for the lack of TRPM7 in the vertebrate TRPM7-deficient DT40 B-cell model system. Here, we report that overexpression of MgtE is able to rescue the growth of TRPM7-KO DT40 B-cells. However, contrary to a previous report that describes regulation of MgtE channel gating by Mg²⁺ in a bacterial spheroplast model system, whole cell patch clamp analysis revealed no detectable current development in TRPM7-deficient cells expressing MgtE. In addition, we observed that MgtE expression is strongly downregulated at high magnesium concentrations, similar to what has been described for its vertebrate homolog, SLC41A1. We also show that the N-terminal cytoplasmic domain of MgtE is required for normal MgtE channel function, functionally confirming the predicted importance of this domain in regulation of MgtE-mediated Mg²⁺ entry. Overall, our findings show that consistent with its proposed function, Mg²⁺ uptake mediated by MgtE is able to restore cell growth and proliferation of TRPM7-deficient cells and supports the concept of functional homology between MgtE and its vertebrate homologs.</p> </div

C-terminal MYC-tagged WT MgtE and ΔN domain mutant display surface expression.

<p>(<b>A</b>) Representative flow plots showing surface expression of the MYC-tagged WT MgtE and the N domain deletion mutant (HA-tagged at the N-terminus and MYC-tagged at the C-terminus) in stably transfected TRPM7-KO cells stained with MYC directly conjugated to PE. While the untransfected TRPM7-KO cells and the uninduced cells did not display any MYC-PE fluorescence, both the WT MgtE and the N domain deletion mutant demonstrated increased mean fluorescence intensities post staining. Sorting and analysis of WT MgtE cells based on the surface expression of MYC showed a significant shift to higher fluorescence intensity. (<b>B</b>) Uninduced and doxycycline induced cells in (A) were fixed & permeabilized and probed with anti-HA/anti-mouse FITC for analysis of the N-terminal HA-tag. A strong shift in fluorescence was observed in both induced WT MgtE and N-terminal mutant expressing cells relative to the uninduced controls. A small change in the fluorescence intensity of the uninduced cells was also observed in both the cases indicating a leaky promoter. Plots are representative of at least two independent experiments.</p

Downregulation of both MgtE expression and growth occurs in presence of high magnesium.

<p>(<b>A</b>) TRPM7-KO cells expressing HA-MgtE were induced either in presence or absence of 15 mM supplemental Mg²⁺ and analyzed for expression after staining with anti-HA and species-specific FITC on BD LSRII at 24 and 48 hours. Cells induced in regular cell culture media had higher MgtE expression compared to cells induced in presence of 15 mM Mg²⁺. (<b>B</b>) Cells from similar conditions as mentioned in (A) were analyzed for expression at 5 days and 4 weeks post-induction and displayed fluorescence shifts similar to what was observed in (A) with significantly lower MgtE expression in cells induced in presence of 15 mM Mg²⁺ at 4 weeks. All data are representative of results obtained from at least three independent experiments. (<b>C</b>) <i>Left panel</i>: TRPM7-KO cells expressing MgtE were induced either in presence or absence of 15 mM supplemental Mg²⁺ and their growth was determined for a period of 98 h. Growth curves represent the average of three independent experiments and the error bars correspond to mean ± SEM. Cells induced in presence of 15 mM Mg²⁺ exhibited diminished growth compared to cells induced in regular cell culture media. <i>Right panel</i>: Cell size analysis of samples in the left panel at 98 h. TRPM7-KO cells expressing MgtE were kept either uninduced or doxycycline-induced in absence/presence of 15 mM Mg²⁺ and acquired on BD LSRII. Cells induced in presence of 15 mM Mg²⁺ were bigger in size compared to the uninduced and induced cells. Results are representative of three independent experiments.</p

Expression of the N domain deletion mutant results in stunted growth and reduced cell size.

<p>(<b>A</b>) <i>Left panel</i>: TRPM7-KO cells expressing the HA-tagged N domain MgtE deletion (Δ1–137) mutant were either kept uninduced, doxycycline - induced or induced in presence of 15 mM supplemental Mg²⁺ following which their growth was monitored at 24, 48, 72 and 96 hours, respectively. TRPM7-KO cells expressing the HA-tagged WT MgtE were used as controls under similar growth conditions. Growth curves represent the average of three independent experiments and the error bars represent standard error of the mean. <i>Right panel</i>: Cell size analysis of TRPM7-KO cells expressing the HA-tagged N domain deletion mutant at 96 hours. Cells were either kept uninduced or induced in absence/presence of 15 mM magnesium and acquired on BD LSRII. Data is representative of at least three independent experiments. (<b>B</b>) TRPM7-KO cells expressing the HA-tagged N domain deletion mutant were analyzed for total protein expression by staining with anti-HA and FITC, post fixation and permeabilization. Single stained cells were used as controls and cells were acquired on BD LSRII followed by data analysis using FlowJo software. Comparable expression of the mutant was observed in cells induced in presence/absence of Mg²⁺. Results are representative of three independent experiments. (<b>C</b>) TRPM7-KO cells expressing the N domain deletion mutant were maintained under similar conditions as mentioned in (B) and analyzed for total protein expression (anti-HA/FITC) at four weeks. While the cells induced in presence of supplemental Mg²⁺ exhibited slightly increased expression (left panel), cells induced in regular cell culture media for up to 4 weeks displayed significantly lower expression levels compared to the ones induced for only 48 hours (right panel). In both (B) and (C), TRPM7-KO cells expressing the WT MgtE had highest expression at all time points. Results are representative of at least two independent sets of experiments.</p

Membrane orientation and cellular localization analysis of HA-MgtE in TRPM7-KO cells.

<p>(<b>A</b>) Two clones of TRPM7-KO cells expressing HA-tagged MgtE (#3 and #1) were doxycycline-induced and either stained live with anti-HA (upper panel) or fixed & permeabilized and labeled with anti-HA (lower panel). Following staining with species-specific FITC, cells were acquired on BD LSRII. Data analysis by FlowJo demonstrated that the N-terminal HA-tag was only accessible intracellularly. (<b>B</b>) Both uninduced (left panel) and doxycycline inducible TRPM7-KO cells expressing HA-tagged MgtE (clone#3; right panel) were fixed & permeabilized followed by anti-HA and FITC staining for immunolocalization analysis of MgtE. The induced cells expressing the protein were clearly discernible from the uninduced cells on the basis of fluorescence. The upper panel shows staining of multiple cells in a single field and the lower panel shows a magnified view of two cells from the upper panel. All data are representative of results obtained from at least two independent experiments.</p