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. 2019 Oct 17;20(6):4893–4904. doi: 10.3892/mmr.2019.10755

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Copyright: © Li et al.

This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

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Figure 3. — Direct inhibition of Nrf2 expression by miRNA-128-3p via targeting of the 3′ UTR in NSCs. (A) Western blot analysis shows the Nrf2 levels 24 h after cerebral I/R in the MCAO model with or without theaflavin treatment (0, 10 and 50 mg/kg). β-actin was used as the internal control for normalization. (B) RT-qPCR analysis shows Nrf2 mRNA levels 24 h after cerebral I/R in the MCAO model with or without theaflavin treatment (0, 10 and 50 mg/kg). GAPDH mRNA was used as the internal control for normalization. (C) Western blot analysis shows Nrf2 levels in NSCs subjected to OGD/R with or without theaflavin treatment (0, 2 and 10 µM). β-actin was used as the internal control for normalization. (D) RT-qPCR analysis shows Nrf2 mRNA levels in NSCs subjected to OGD/R with or without theaflavin treatment (0, 2 and 10 µM). GAPDH mRNA was used as the internal control for normalization. (E) Sequence alignment between miRNA-128-3p and the 3′UTR of Nrf2 mRNA. (F) RT-qPCR analysis shows miRNA-128-3p levels 24 h after cerebral I/R in the MCAO model with or without theaflavin treatment (0, 10 and 50 mg/kg). U6 was used as the internal control for normalization. (G) RT-qPCR analysis shows miRNA-128-3p levels after transfection of miRNA mimics or miRNA inhibitor. (H) RT-qPCR analysis shows miRNA-128-3p levels in NSCs subjected to OGD/R with or without theaflavin treatment (0, 2 and 10 µM) or with 10 µM theaflavin and miRNA mimic. U6 was used as the internal control for normalization. (I) The luciferase reporter gene assay shows the effect of miRNA-128-3p on luciferase activity. (J) RT-qPCR analysis shows Nrf2 mRNA levels in NSCs subjected to OGD/R after transfection of mimics (K) Western blot analysis shows the Nrf2 levels in NSCs subjected to OGD/R after transfection of mimics. All assays were performed in triplicate. Data are expressed as mean ± SD. **P<0.01. Nrf2, nuclear factor (erythroid-derived 2)-related factor 2; UTR, untranslated region; NSCs, neural stem cells; I/R, ischemia-reperfusion; MCAO, middle cerebral artery occlusion; OGD/R, oxygen-glucose deprivation and reoxygenation.

Figure 3. — Direct inhibition of Nrf2 expression by miRNA-128-3p via targeting of the 3′ UTR in NSCs. (A) Western blot analysis shows the Nrf2 levels 24 h after cerebral I/R in the MCAO model with or without theaflavin treatment (0, 10 and 50 mg/kg). β-actin was used as the internal control for normalization. (B) RT-qPCR analysis shows Nrf2 mRNA levels 24 h after cerebral I/R in the MCAO model with or without theaflavin treatment (0, 10 and 50 mg/kg). GAPDH mRNA was used as the internal control for normalization. (C) Western blot analysis shows Nrf2 levels in NSCs subjected to OGD/R with or without theaflavin treatment (0, 2 and 10 µM). β-actin was used as the internal control for normalization. (D) RT-qPCR analysis shows Nrf2 mRNA levels in NSCs subjected to OGD/R with or without theaflavin treatment (0, 2 and 10 µM). GAPDH mRNA was used as the internal control for normalization. (E) Sequence alignment between miRNA-128-3p and the 3′UTR of Nrf2 mRNA. (F) RT-qPCR analysis shows miRNA-128-3p levels 24 h after cerebral I/R in the MCAO model with or without theaflavin treatment (0, 10 and 50 mg/kg). U6 was used as the internal control for normalization. (G) RT-qPCR analysis shows miRNA-128-3p levels after transfection of miRNA mimics or miRNA inhibitor. (H) RT-qPCR analysis shows miRNA-128-3p levels in NSCs subjected to OGD/R with or without theaflavin treatment (0, 2 and 10 µM) or with 10 µM theaflavin and miRNA mimic. U6 was used as the internal control for normalization. (I) The luciferase reporter gene assay shows the effect of miRNA-128-3p on luciferase activity. (J) RT-qPCR analysis shows Nrf2 mRNA levels in NSCs subjected to OGD/R after transfection of mimics (K) Western blot analysis shows the Nrf2 levels in NSCs subjected to OGD/R after transfection of mimics. All assays were performed in triplicate. Data are expressed as mean ± SD. **P<0.01. Nrf2, nuclear factor (erythroid-derived 2)-related factor 2; UTR, untranslated region; NSCs, neural stem cells; I/R, ischemia-reperfusion; MCAO, middle cerebral artery occlusion; OGD/R, oxygen-glucose deprivation and reoxygenation.