Figure 4 | Cell Death & Disease

Figure 4

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

Figure 4

Flv induces ATF4 via Sig-1R. (a) Neuro2a cells were treated with Flv (10 μg/ml) or Flv (10 μg/ml) and NE-100 (NE-100a Sig-1R antagonist; 1 μg/ml). The expression of ATF4 and GAPDH was monitored over the time course shown in the figure. (b) The amount of ATF4 and GAPDH in (a) was quantified with densitometry. Relative intensity of ATF4/GAPDH in cells treated with Flv+NE-100 was significantly suppressed compared with in cells treated with Flv alone. The 0 h time point was normalized to 1. Values are the mean±S.D. (*P<0.05, Student’s t-test; n=3). (c) Flv (10 μg/ml) was added to Sig-1R+/+ MEFs and Sig-1R−/− MEFs. Changes in ATF4 and GAPDH expression were monitored over the time course shown. (d) The amount of ATF4 and GAPDH in (c) was quantified with densitometry. Relative intensity of ATF4/GAPDH showed that ATF4 induction in Sig-1R+/+ MEFs treated with Flv was not observed in Sig-1R−/− MEFs treated with Flv. The 0 h time point was normalized to 1. Values are the mean±S.D. (*P<0.05, **P<0.01, Student’s t-test; n=3). (e) To investigate the regulation of ATF4 translation by Flv, we used the reporter assay system shown in the figure.11 One construct contained ATF4 uORF1 fused with a GFP coding region (uORF1-GFP). A second construct contained a fusion of ATF4 uORF1, uORF2, the ATF4 N terminus, and a GFP coding region (5′ATF4-GFP). (f) HEK293 cells were transfected with the uORF1-GFP reporter construct or the 5′ATF4-GFP reporter construct shown in (e). Thereafter, Tm (1 μg/ml), Flv (10 μg/ml), or Flv (10 μg/ml) and NE-100 (1 μg/ml) were added. Protein expression over the time course shown in the figure was assessed by immunoblot analysis. Changes in GFP expression were monitored. With Tm treatment, GFP expression in uORF1-GFP cells decreased, whereas GFP expression in 5′ATF4-GFP cells increased. On the other hand, with Flv treatment, GFP expression increased in both uORF1-GFP cells and 5′ATF4-GFP cells. The increase in GFP expression observed in uORF1-GFP and 5′ATF4-GFP cells treated with Flv was suppressed when the cells were treated with Flv+NE-100. (g) Cell transfected and treated as described for (f) were observed by fluorescence microscopy. In DMSO-treated cells, uORF1-GFP expression was observed, but 5′ATF4-GFP expression was not detected. In contrast, in Tm-treated cells, uORF1-GFP expression decreased, and 5′ATF4-GFP expression increased, suggesting that ER stress selectively promoted the translation of ATF4. In contrast, increased GFP expression was observed in both uORF1-GFPand 5′ATF4-GFP-transfected cells after Flv treatment (24 h). In both cell lines, the increase in GFP expression was suppressed by treatment with Flv+NE-100 (24 h), consistent with the results of the immunoblot analysis in (f)

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