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. 2022 Jul 21;13:905211. doi: 10.3389/fimmu.2022.905211

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Copyright © 2022 Meng, Liu, Tang, Liu, Zhao, Zhou, Li and Hou

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Silencing NR2F1 increased expression of Occludin and inhibited secretion of IL-6. (A) Fold changes of NR2F1 mRNA after silencing (mean ± SD; **** P<0.0001; n=3/group; one-way ANOVA). (B) Silencing efficiency of NR2F1 using siNR2F1-1. Left, representative western blotting images of NR2F1. Right, quantification of relative expression of NR2F1 (mean ± SD; *** P<0.001; n=3/group; unpaired Student’s t-test). (C) Cotransfection of METTL3- and NR2F1-silenced lentivirus (BF, bright field; EGFP, enhanced green fluorescence protein; MCHERRY, red fluorescence protein; scale bar, 10μm). (D) Protein level of Occludin after METTL3 and NR2F1 silencing in ARPE19 cells. Left, representative western blotting images of Occludin. Right, quantification of relative expression of Occludin (mean ± SD; ** P<0.01, **** P<0.0001; n=3/group; one-way ANOVA). (E, F) The secretion levels of IL-6 and IL-8 in ARPE19 cells after METTL3 and NR2F1 silencing (mean ± SD; ns P>0.05, * P<0.05, *** P<0.001; n=3/group; one-way ANOVA). (G) Protein level of Occludin in the METTL3- and NR2F1-silenced ARPE19 cells with LPS stimulation. Left, representative western blotting images of Occludin. Right, quantification of relative expression of Occludin (mean ± SD; *** P<0.001, **** P<0.0001; n=3/group; one-way ANOVA). (H, I) The secretion levels of IL-6 and IL-8 in ARPE19 cells with LPS stimulation after METTL3 and NR2F1 silencing (mean ± SD; ns P>0.05, ** P<0.01, *** P<0.001; n=3/group; one-way ANOVA). (J) Protein level of Occludin after METTL3 and NR2F1 silencing in hRPE cells with LPS stimulation. Left, representative western blotting images of Occludin. Right, quantification of relative expression of Occludin (mean ± SD; * P<0.05, **** P<0.0001; n=3/group; one-way ANOVA). (K) The secretion level of IL-6 in hRPE cells with LPS stimulation after METTL3 and NR2F1 silencing (mean ± SD; * P<0.05, *** P<0.001; n=3/group; one-way ANOVA). (L) The DNA expression of IL-6 after METTL3 silencing with NR2F1 immunoprecipitation (IgG for negative control; mean ± SD; * P<0.05, ** P<0.01; n=3/group; one-way ANOVA).