Figure 3 | Cell Death & Differentiation

Figure 3

From: The ALS-linked E102Q mutation in Sigma receptor-1 leads to ER stress-mediated defects in protein homeostasis and dysregulation of RNA-binding proteins

Figure 3

mSigR1 induces mitochondrial toxicity and fails to mobilize IP3R and SOCE-mediated Ca2+ signalling. (a and b) MCF-7 cells were transiently transfected as previously described; 48 h later cells were loaded with Fura-2AM for 30 min, washed twice and then stimulated with 10 μM BDK under Ca2+-free conditions. Average traces of the BDK-induced increase in [Ca2+]i from the ER store through IP3R are represented. Note the increase of [Ca2+]i in wtSigR1-transfected cells (green curve) compared to a significant decrease in mSigR1-transfected cells (red curve). Results are expressed as mean±S.E.M. of ~30 cells. Mean changes in peak [Ca2+]i measured are given. The asterisks denote a statistically significant difference (*P<0.05). (c) Representative SOCE elicited in transfected cells loaded with Fura-2AM as described above. SOCE was triggered by the addition of 2 μM ionomycin under Ca2+-free conditions. SOCE developed (vertical dot line) after the addition of Ca2+ is depicted. (d) STIM1 immunoblot analysis from lysates obtained from pcDNA, wtSigR1 and mSigR1-transfected MCF-7 cells. Note the significant down-regulation of STIM1 in mSigR1-transfected cells. The fold change below represents the quantification of band intensities normalized against α-tubulin. Values derived from three independent experiments. *P<0.05. (e) Significantly decreased STIM1 levels in E102Q-SigR1 fALS lymphoblastoid cell lysates compared to healthy control lymphoblastoid cells. The fold change below represents the quantification of band intensities normalized against α-tubulin. Values derived from three independent experiments. *P<0.05. (f) Significantly reduced mitochondrial membrane integrity and ATP production in mSigR1 expressing MCF-7 cells compared to wtSigR1 expressing cells measured by the tox glow assay. Values derived from three independent experiments. *P<0.05. (g) JC-1 staining of HeLa cells transfected with wtSigR1 or mSigR1. Note the reduced mitochondrial potential in mSigR1 expressing cells. Scale bar, 10 μm. (h) JC-1 staining of lymphoblastoid cells obtained from E102Q-SigR1 fALS patients and healthy controls. Note the decreased membrane potential in mSigR1 expressing cells. Scale bar, 10 μm. (i) Cytochrome C immunolabelling of HeLa and MCF-7 expressing wtSigR1 or mSigR1. Note the Cytochrome C release in cells showing mSigR1 aggregates (arrowheads). Scale bar, 10 μm

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