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. 2012 Jun 28;3(6):e333. doi: 10.1038/cddis.2012.74

Figure 2.

Figure 2

Perk-dependent increases in the activity of Bim 3′UTR reporter construct during conditions of ER stress. (a) pCDH and miR-106b-25 cells were either untreated (Un) or treated with (0.25 μM) Tg for indicated time points. Total protein was isolated and immunoblotting was performed using antibodies against Bim and β-actin. (b) MCF-7 cells were transfected with Bim 3′UTR reporter plasmid containing binding sites for members of miR-106b-25 (Bim UTR-wt) or Bim 3′UTR reporter plasmid with mutated binding sites for members of miR-106b-25 (Bim UTR-mt). At 24 h after transfection cells were left untreated (Un) or treated with (2.0 μM) Tg and (2.0 μg/ml) Tm, (50 μg/ml) Eto, or (600 μM) H2O2. Luciferase activity was measured 48 h after transfection using Dual-Glo assay system and normalized luciferase activity (Renilla/Firefly) is shown. Error bars represent mean±S.D. from three independent experiments performed in duplicate. (c) PC12 cells were transfected with Bim 3′UTR reporter plasmid containing binding sites for members of miR-106b-25 (Bim UTR-wt). At 24 h after transfection cells were left untreated (Un) or treated with (0.25 μM) Tg and (2.0 μg/ml) Tm. Luciferase activity was measured 48 h after transfection using Dual-Glo assay system and normalized luciferase activity (Renilla/Firefly) is shown. Error bars represent mean±S.D. from three independent experiments performed in duplicate. (d) MCF-7 cells were transfected with Bim 3′UTR reporter plasmid containing binding sites for members of miR-106b-25 (Bim UTR-wt) in combination with pcDNA3 (pcDNA), WT Ire1, dominant-negative Ire1 (ΔC Ire1), WT Perk or dominant-negative Perk (K618A Perk) expression plasmids. At 24 h after transfection cells were left untreated (Un) or treated with (2.0 μM) Tg and (2.0 μg/ml) Tm. Luciferase activity was measured 48 h after transfection using Dual-Glo assay system and normalized luciferase activity (Renilla/Firefly) is shown. Error bars represent mean±S.D. from three independent experiments performed in duplicate. (*P<0.05, two-tailed unpaired t-test compared with untreated cells)