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. 2021 Mar 24;28(9):2634–2650. doi: 10.1038/s41418-021-00774-3

Fig. 4. Synage lncRNA regulates Cbln1 mRNA through the AGO2-miR-325-3p pathway.

Fig. 4

a Relative expression level of Cbln1 mRNA in adult WT and KO mouse cerebella. b Relative quantification of integrated intensity of CBLN1 protein in the cerebellar cortex of 3-week-old WT and KO mice detected by immunofluorescence staining after stereotaxic injection of AAV-control or AAV-Synage (Synage OE) into the neonatal mouse cerebellum. c Relative nascent transcript levels of both Synage and Cbln1 using multiple pairs of primers, normalized to housekeeping gene HPRT (hypoxanthine guanine phosphoribosyl transferase). NRO nuclear run on, P primer pairs. d Schematic representation of the predicted binding sites of miR-325-3p on Synage and Cbln1 (CDS coding sequence, UTR untranslated region, E exon). e Relative luciferase activity of vector control and WT Cbln1-3′UTR and mutant Cbln1-3′UTR luciferase reporter gene plasmids upon co-transfection of mimic negative control (NC) or miR-325-3p in the HT-22 cell line. f Relative luciferase activity of vector control and WT Synage and mutant Synage luciferase reporter gene plasmids upon co-transfection of mimic NC or miR-325-3p in the HT-22 cell line. Data are presented as the relative ratio of firefly luciferase activity to Renilla luciferase activity. g Western blot analysis of AGO2 protein immunoprecipitation by AGO2 antibody in AGO2-RNA immunoprecipitation. h The amount of Synage lncRNA binding to AGO2 or IgG was quantified as a percentage of input in IP by RT-qPCR (Synage-P1, a primer pair specifically targeting n264625; Synage-P2, a primer pair targeting both n264625 and n285242). i, j The amount of Synage lncRNA (i) and Cbln1 mRNA (j) binding to miR-325-3p was quantified as a percentage of input in miR-325-3p pull-down assays by RT-qPCR. k–m Relative expression levels of miR-325-3p with in vitro transfection of miR-325-3p mimics and inhibitor in the HT-22 cell line, normalized to U6 (k). Quantification (l) and representative images of Western blots (m) for CBLN1 with in vitro transfection of miR-325-3p mimics and inhibitor in the HT-22 cell line.