Fig 2: hsa_circ_0008362/miR-1251-5p/Runx2 axial regulated VSMC calcification.A Schematic representation of putative target sites between miR-1251-5p and circ_0008362 as well as in Runx2 3′-UTR. B FISH showed the presence of circ_0008362 and miR-1251-5p in VSMCs. Scale bar = 50 μm. C the expression of miR-1251-5p in VSMCs transfected the vector of Circ_Control or Circ_0008362-WT. D the expression of hsa_circ_0008362 was detected by qPCR in VSMCs transfected by miR-1251-5p mimics or inhibitor. E The expression of circ_0008362 was detected by qRT‐PCR after biotin‐labeled miR‐1251‐5p pull‐down assay. F RIP assay showed the binding efficiency of miR‐1251‐5p and circ_0008362 to Ago2 protein in VSMCs. G the expression of Runx2 was measured by WB in VSMCs treated with ECNG-EVs or ECHG-EVs. H Luciferase reporter assays showed the luciferase activities of Runx2 in VSMCs transfected with miR‐1251‐5p mimics or control oligos. I VSMCs were transfected with circ_0008362 plasmid and the level of Runx2 protein was measured by WB. J The level of Runx2 protein was measured by WB in VSMCs with different treatment. K ARS staining showed the mineralized nodules in VSMCs with different treatment, and the calcium content was quantified by spectrophotometry. One-way ANOVA with Tukey’s multiple comparisons test (C, D, E, F, H, I, J, K) and the Student's test (G) were used. Three independent experiments were performed, and representative data were shown. Data were shown as mean ± SD. ****p < 0.0001, ***p < 0.001, **p < 0.005, *p < 0.05. ns: no significance. ECNG-EVs, EVs derived from normal-glucose induced ECs; ECHG-EVs, EVs derived from high-glucose induced ECs; Circ_Control, circular RNA negative control; circ_0008362-WT, circ_0008362-wild type plasmid; ARS, alizarin red s; RISC: RNA-induced silencing complex

Fig 3: TRIM32, LRRK2, and Ago2 form a complex. a HEK293T cells were transfected with plasmids for the overexpression of the indicated constructs. On the left panel, immunoblots of cell lysates, probed with the indicated antibodies, are shown. On the right panel, immunoprecipitations with anti-LRRK2 and anti-Ago2 antibodies are shown. The blots are probed with the indicated antibodies. b On the left panel, immunoblots of lysates from brains of mice at different ages displaying the expression levels of endogenous LRRK2, Ago2, and TRIM32. On the right panel, immunoprecipitations with anti-LRRK2 and anti-Ago2 antibodies are shown. The blots are probed with the indicated antibodies

Fig 4: LINC00992 regulated the expression of GOLM1, a target of miR-3935. a The expression of eight mRNAs in four prostate cancer cell lines and RWPE-1 cells was detected by qRT-PCR. b GOLM1 was overexpressed in prostate cancer tissues according to GEPIA database. c The mRNA and protein levels of GOLM1 were evaluated in prostate cancer cell lines and RWPE-1 cell line by qRT-PCR and western blot, respectively. d The binding sites between GOLM1 and miR-3935 were predicted via TargetScan. e Luciferase reporter assay presented the inhibited luciferase activity of GOLM1-WT reporter in the presence of miR-3935 mimics not NC-mimics. f-g GOLM1 expression in transfected cells was tested by qRT-PCR and western blot analyses. h The combination of GOLM1 with miR-3935 in the anti-Ago2 group was validated by RIP assay. i-j The mRNA and protein levels of GOLM1 in different groups were examined via qRT-PCR and western blot. The full-length gels for western blot data in Fig. 3c, g and j were presented in Supplementary Figure 5. *P < 0.05, **p < 0.01, ***p < 0.001

Fig 5: AGO2 gene and protein expression changes following PD. (A) Diagram indicating retinal regions where histological measures were taken. Lesion site refers to the area most damaged by PD. (B) (i) AGO2 in situ hybridization in dim and PD retinas at the three retinal sites indicated in A. AGO2 mRNA increased at all three locations and is distributed across the GCL, INL in the inferior retina and lesion site. In the superior retina, AGO2 mRNA expression is also visible within the ONL (yellow arrow) and the OLM (orange arrow). (ii) Co-labelling of AGO2 mRNA and glutamine synthetase in the superior retina showing colocalization along the OLM and Müller glia processes. Gray micrographs are DIC images of AGO2 in situ hybridization (as shown in i). Red labelling are pseudo-coloured pixels corresponding to the area positive for AGO2 labelling (dark spots in the adjacent DIC image). (C) (i) Image analysis pipeline used for colocalization analysis. (ii) Immunohistochemistry showed AGO2 protein expression is confined within the GCL, INL and photoreceptor segments in dim retinas. Following PD, AGO2 protein expression is elevated within the INL and photoreceptor segments. Co-localization of AGO2 labelling with Müller glia visualized using a reporter mouse strain showed distinct colocalization along the inferior and superior OLM (inset 2), Müller glia processes (inset 4) and Müller glia cell bodies (inset 3). In the absence of PD limited colocalization is seen in the superior retina (inset 1). (D) (i) Axis description for plots shown in ii. (ii) AGO2 protein expression profile quantified across retinal layers and depicted as pixel intensity (y-axis), along the outer to inner retinal direction (green x-axis) and superior to inferior (orange z-axis). (E) (i) AGO2 western blot and corresponding GAPDH loading control. (ii) GAPDH-normalized densitometry values for AGO2 (Error bars show SEM, * P < 0.05, Student’s t-test)