Journal: Cell Death & Disease

Article Title: Fluvoxamine alleviates ER stress via induction of Sigma-1 receptor

doi: 10.1038/cddis.2014.301

Figure Lengend Snippet: Flv induces Sig-1R expression without activating the unfolded protein response (UPR) during ER stress. ( a ) Flv (10 μ g/ml) or tunicamycin (Tm; 1 μ g/ml) was added to Neuro2a cells, and changes in ATF6 α processing were monitored over the time course shown in the figure. The amount of processed ATF6 α protein increased with Tm treatment but not with Flv treatment. Processed ATF6 α /total ATF6 α was measured with densitometry. The results are shown in the bar graph. The 0 h time point was normalized to 1. Values are the mean±S.D. (* P <0.05, Student's t -test; n =3). ( b ) Flv (10 μ g/ml) or Tm (1 μ g/ml) was added to Neuro2a cells, and changes in XBP-1 splicing were measured by semiquantitative RT-PCR over the time course shown in the figure. XBP-1 splicing was observed with Tm treatment but not with Flv treatment. ( c ) Flv (10 μ g/ml) was added to Neuro2a cells, and changes in the expression of GRP78/BiP and GRP94, which react with anti-KDEL antibodies, were monitored over the time course shown in the figure. No change in the expression of GRP78/BiP or GRP94 as a result of Flv treatment was observed. ( d ) Flv (10 μ g/ml) was added to Neuro2a cells, and the expression of total eIF2 α and phosphorylated eIF2 α was measured over the time course shown in the figure. No change in the amount of total eIF2 α (w-eIF2 α ) or phosphorylated eIF2 α (p-eIF2 α ) as a result of Flv treatment was observed. p-eIF2 α /w-eIF2 α was measured with densitometry. The results are shown in the bar graph. The 0 h time point was normalized to 1. Values are the mean±S.D. (* P <0.05, Student's t -test; n =3). ( e ) Mouse embryonic fibroblasts (MEFs) from PERK wild-type (PERK +/+ ) and PERK knockout (PERK −/− ) mice were treated with 10 μ g/ml of Flv. The expression of Sig-1R and GAPDH was monitored over the time course shown in the figure. Flv treatment induced Sig-1R expression in MEFs from PERK +/+ and PERK −/− animals. ( f ) Densitometry analysis of ( e ) showed that relative intensity of Sig-1R/GAPDH increased significantly in both PERK +/+ and PERK −/− MEFs in response to Flv treatment. Left graph, PERK +/+ MEFs; right graph, PERK −/− MEFs. Values are the mean±S.D. (* P <0.05, Student's t- test; n =3)

Article Snippet: Antibodies against the following proteins were used at the dilutions indicated: Sig-1R (ab53852; 1:250; Abcam, Cambridge, UK), eukaryotic initiation factor-2 α (eIF2 α ) (#9722, 1:1000; Cell Signaling Technology, Danvers, MA, USA), phospho-eIF2 α (#9721; 1:1000; Cell Signaling Technology), activating transcription factor 4 (ATF4) (sc-22800; 1:1000; Santa Cruz Biotechnology, Dallas, TX, USA), ATF6 α (sc-22799; 1:1000; Santa Cruz Biotechnology), X-box binding protein 1 (XBP-1) (sc-7160; 1:1000; Santa Cruz Biotechnology), KDEL ER marker (sc-58774; 1:1000; Santa Cruz Biotechnology), GFP (D153-9; 1:5000; MBL, Woburn, MA, USA), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (MA1-10036; 1:2500; Thermo Scientific, Waltham, MA, USA), caspase-4 (M029-3; 1:1000; MBL), caspase-12 (#2202; 1:1000; Cell Signaling Technology), CHOP (sc-7351; 1:1000; Santa Cruz Biotechnology), calnexin (ADI-SPA-860; 1:500; Stressgen, VIC, Canada), and GM130 (#610822; 1:500; BD Transduction Laboratories, Franclin Lakes, NJ, USA).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Knock-Out