Mutations in the mitochondrial thioredoxin reductase gene TXNRD2 ...

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Figure 4 Expression of wild-type (wt) Txnrd2 and the novel mutations (A59T and G375R) in Txnrd2 +/+ cells. Immunoblotting using a
Txnrd2-specific antibody (A) and confocal microscopy of immunocytochemical staining of transduced cells with an anti-Prx III-specific antibody
(Alexa488) for mitochondria, an anti-FLAG-specific antibody (Alexa568) for reconstituted wild-type (wt) Txnrd2 and the A59T and G375R
mutants and a nuclear counterstain with DAPI (B) revealed comparable expression of wt Txnrd2 and A59T in wild-type cells, whereas the
G375R mutant was barely detectable in Txnrd2 +/+ cells. (B) Wild-type Txnrd2 and the A59T mutant predominantly localized to mitochondria
in Txnrd2 +/+ cells as shown in the false colour merge. The low expression of mutant G375R in Txnrd2 +/+ cells, however, precluded any conclusion
regarding its subcellular localization (for details regarding staining and illustration, see Figure 3 and the Methods section). Mock ¼ empty virustransduced
cells. Scale bars, 10 mm. (C) Cell survival of Txnrd2 +/+ cells transduced with A59T or G375R was impaired in response to increasing
L-buthionine sulfoximine concentrations, indicating that both forms harbour a dominant-negative function. The fact that both mutants had a
similar impact on cell survival despite different levels of expression suggests that mutant G375R is more toxic and that a non-toxic threshold of
expression is selected for upon retroviral transduction Bars represent means and error bars standard deviations derived from cell counting of
three different wells for each condition. P-values ,0.05 were considered as significant and are marked with an asterisk, and P-values ,0.001
were considered as highly significant and are marked with two asterisks (for experimental details and statistical analysis, see the legend of Figure 3).
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