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. 2021 Mar;9(6):448. doi: 10.21037/atm-20-5729

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2021 Annals of Translational Medicine. All rights reserved.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0.

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RGS12 can strengthen the transcriptional regulation of COX2 via NF-κB. (A) Western blot analysis of RGS12 expression. Primary BMMs were transfected with pCMV-empty (control) or pCMV-RGS12 (RGS12 OE) plasmids by Lipofectamine 3000 for 24 hours, and the expression levels of RGS12 were analyzed. **, P<0.01 vs. the control group, n=3. (B) Relative mRNA expression of COX2 was analyzed by real-time PCR. Primary BMMs were treated as described in (A), and the expression levels of COX2 were analyzed. **, P<0.01, ***, P<0.001 compared with the control group. The values are the mean ± SEM (n=5). (C) COX2 expression levels in BMMs were analyzed by ELISA. Primary BMMs were treated as described in (A), and COX2 expression levels were analyzed. Note that RGS12 OE enhanced the expression level of COX2. **, P<0.01, ***, P<0.001 compared with the control group. (D) Relative protein levels of NF-κBp65. RAW264.7 cells were transfected with shControl (pSicoR-empty) or shNF-κB plasmids (pSicoR-shNFκBp65-1 and pSicoR-shNFκBp65-2) for 24 hours. β-Actin was used as an internal reference. The data are presented as the means ± SEM. ***, P<0.001 vs. shControl group, n=3. (E) Western blot analysis of NF-κBp65. RAW264.7 cells were transfected with pcDNA3.1-empty (control) or pcDNA3.1-NF-κBp65 (NF-κBp65 OE) plasmids for 24 hours. β-Actin was used as an internal reference. The data are presented as the means ± SEM. ***, P<0.001 vs. the control group, n=3. (F) RAW 264.7 cells were transfected with pUC-shNF-κBp65 and/or pCMV-RGS12 for 24 hours and then induced with TNFα for 24 hours. Relative mRNA expression was measured by real-time PCR. Note that RGS12 could not increase COX2 expression when NF-κB expression was decreased. The values are the mean ± SEM. ***, P<0.001, n=5. (G) Analysis of the interactions between RGS12-PTB and NF-κBp65 by pulldown assay. RAW264.7 cells were transfected with pCMV-Flag or pCMV-PTB-Flag plasmids, and cell lysates were precipitated with anti-Flag affinity gel and immunoblotted (IB) with anti-NF-κB p65 and anti-Flag antibodies. (H) RGS12 promotes the transcriptional activity of NF-κB and COX2 expression (luciferase assay). Stable knockdown of NF-κB in RAW264.7 cells was achieved via the transfection of pSicoR-shNFκBp65-1 and pSicoR-shNFκBp65-2 vectors. The stably transfected RAW264.7 cells were treated with COX2-Luc, pcDNA3.1-NF-κBp65, pCMV-RGS12 and/or pCMV-PTB for 48 hours. *, P<0.05, ***, P<0.001 versus the control. The data are presented as the means ± SEM. Note that RGS12 or PTB could increase the transcriptional activity of NF-κB.