Figure 2

mSigR1 is abnormally accumulated in the ER and induces cellular toxicity in E102Q-SigR1 fALS patient lymphoblastoid cells. (a) Immunoreactivity of globular SigR1 aggregates (arrows) in E102Q-SigR1 fALS patient lymphoblastoid cells compared to the healthy control. Note the co-localization of SigR1 aggregates with the nuclear envelope marker emerin (arrowhead). Scale bar, 15 μm. (b and c) Co-localization of mSigR1 aggregates with the ER markers KDEL and (c) calreticulin in E102Q-SigR1 fALS patient lymphoblastoid cells compared to the healthy control. Scale bars, 15 μm. (d and e) Upregulation and co-localization of the ER stress markers GRP78 (d) and pPERK (e) with mSigR1 aggregates in E102Q-SigR1 fALS patient lymphoblastoid cells compared to the healthy control. Scale bars, 15 μm. (f) Immunoblot analysis of SigR1 and established ER stress and UPR markers in healthy control and E102Q-SigR1 fALS lymphoblastoid cell lysates. Dot blot analysis (top) shows the presence of triton-X insoluble aggregates in E102Q-SigR1 fALS lymphoblastoid cells. (g) Quantification of the band intensities normalized with α-tubulin depicted in f. Values represent the mean±S.D. of three independent experiments. *P<0.05. (h–i) RT-PCR analysis of the UPR pathways in three healthy control lymphoblastoid cell lines compared to two E102Q-SigR1 fALS patient lymphoblastoid cell lines. E102Q-SigR1 fALS patient’s lymphoblastoid cells showed a significant increase in ATF4 mRNA expression. *P<0.05, # not significant. Note that SigR1 gene expression does not differ between controls and E102Q-SigR1 fALS patients. (j) E102Q-SigR1 fALS patient’s lymphoblastoid cell (n=2) and control lymphoblastoid cell (n=3) lysates were subjected to chymotrypsin-like proteasomal activity and caspase-3 activity assays as described in material and methods. Values are derived from the average of three control lymphoblastoid cell lines compared to average of two E102Q-SigR1 fALS patient’s lymphoblastoid cell lines from three independent experiment,**P<0.005. (k) GM130 and SigR1 immunolabelling in E102Q-SigR1 fALS and control lymphoblastoid cells. Scale bar, 15 μm. (l) Immunoblot analysis using SigR1 antibody to compare SigR1 levels in lymphoblastoid cells from E102Q-SigR1 patient lymphoblasts and healthy controls as well as transiently transfected MCF-7 cells. Quantification of the band intensities normalized with α-tubulin is shown below. (#) denotes absence of significant differences