Fig 1: Metformin inhibited tumor growth in vivo by regulating the circ_0003214-miR-489-3p-ADAM10 pathway. The experimental mice were divided into three groups, including control (nontreatment), metformin (metformin treatment), and metformin + circ_0003214 (metformin treatment and circ_0003214 overexpression). (a) Tumor volume was measured every 4 days. (b) Tumor weight was measured at 27 days after injection. (c, d)) The expression of circ_0003214 and miR-489-3p in the excised tumor tissues was detected by qRT-PCR. (e, f)) The expression of ADAM10 in the excised tumor tissues was detected by qRT-PCR and western blot. *P < 0.05.
Fig 2: The effect of ADAM10 inhibitor (GI254023X) or ADAM17 inhibitor (TAPI-1) on the release of TNF-a, TNFR-1 and IL-6R by constitutively and under inflammation BV2 microglia. BV2 microglia were incubated with GI254023X (5 µM) or TAPI-1 (25 µM) for 24 h in the absence or presence of 1000 ng/mL LPS. The medium was collected for ELISA measurements and cell lysates were lysed for WB analysis. The concentration of TNF-a (A), sTNFR-1 (B), IL-6 (C) and sIL-6R (D) released in medium (n = 6 from 6 independent experiments). (E) The representative immunoblots of ADAM10 protein expression in BV2 cell lysate. (F–H) The quantification of ADAM10 expression in BV2 cell lysate (n = 10 from 5 independent experiments). (I) The representative immunoblots of ADAM17 expression in BV2 cell lysate. (J) The quantification results of ADAM17 in BV2 cell lysate (n = 8 from 4 independent experiments). Calnexin was used as a loading control. Black: constitutive cytokine release or protein expression set at 1; red: LPS-induced cytokine release or protein expression; blue: effect of ADAMs inhibitor on constitutive cytokine release or protein expression; green: effect of ADAMs inhibitor on LPS-induced cytokine release or protein expression. Results were expressed as mean ± SEM. * p < 0.05, ** p < 0.01 *** p < 0.001, **** p < 0.0001.
Fig 3: LncRNA CDKN2B-AS1 inhibits the transcription of ADAM10 via DNMT1-mediated ADAM10 DNA methylation, consequently preventing inflammatory response of atherosclerosis and promoting cholesterol efflux.
Fig 4: Neuronal stimulation by NMDA increases ADAM10-mediated NrCAM shedding ATo genetically validate our findings, ADAM10fl/fl neurons were treated with an iCre, or a control lentivirus at DIV2, to knock out ADAM10. The cells were kept in culture until DIV10; then, the neurons were treated with NMDA (50 µM) or vehicle for 30 min (Wan et al, 2012).BWt neurons were cultured like in (Fig 5A). At DIV10, the cells were pretreated with the NMDA receptor antagonist D-APV (100 µM) or vehicle for 30 min; then, the neurons were treated with NMDA (50 µM) or vehicle for 30 min. Densitometric quantifications of the Western blots are shown. One-way ANOVA with post hoc Dunnett's test (****P < 0. 0001, n = 6). Shown are the mean and SEM. Representative Western blots are shown. Source data are available online for this figure.
Fig 5: CDKN2B-AS1 promotes ADAM10 methylation by recruiting DNMT1. (A) the expression of CDKN2B-AS1 and transcription level of ADAM10 in cells infected with lentiviral vector expressing oe-CDKN2B-AS1 or sh-CDKN2B-AS1 determined by RT-qPCR; (B) Western blot analysis was used to detect the protein band and level of ADAM10 in cells infected with lentiviral vector expressing oe-CDKN2B-AS1 or sh-CDKN2B-AS1; (C) FISH was used to detect CDKN2B-AS1 localization in cells (× 200); (D) MS-PCR was used to detect the electrophoresis band of ADAM10 methylation level in atherosclerotic plaque, IMA tissues and ox-LDL-exposed THP-1 macrophages or ox-LDL-exposed THP-1 macrophages treated with 5-aza-dc or M.SssI; (E) CHIP assay to detect the output percentage of ADAM10 in cells infected with lentiviral vector expressing oe-CDKN2B-AS1 or sh-CDKN2B-AS1; (F) RIP assay to detect the output percentage of CDKN2B-AS1 in cells infected with lentiviral vector expressing oe-CDKN2B-AS1 or sh-CDKN2B-AS1; (G) RNA pull down to detect the DNMT1 protein pulled down by lncRNA CDKN2B-AS1 in cells infected with lentiviral vector expressing oe-CDKN2B-AS1 or sh-CDKN2B-AS1; In panel D, U represents the un-methylated lane, and M is representative of the methylation lane; * p < 0.05 vs. the oe-NC group; # p < 0.05 vs. the sh-NC group; the measurement data were expressed in the form of mean ± standard deviation and analyzed by one-way ANOVA, the experiment was repeated 3 times; THP-1, the human monocytic leukemia cell line; RT-qPCR, reverse transcription quantitative polymerase chain reaction; CDKN, cell-dependent kinase inhibitor; ANOVA, analysis of variance; ELISA, enzyme linked immunosorbent assay; RIP, RNA-binding protein immunoprecipitation; FISH, fluorescence in situ hybridization; CHIP, chromatin immunoprecipitation; MS-PCR, methylation-specific PCR.
Supplier Page from Abcam for Anti-ADAM10 antibody [EPR5622]