Fig 1: Spred2 is a direct target of miR-218 in PC12 cells. (A) Predicted sequence between miR-218 and spred2 using starBase online database. (B) The luciferase report activity of spred2-WT/-MUT in PC12 cells. (C and D) mRNA and protein levels of spred2 in PC12 cells after transfection with miR-218 mimics, miR-218 inhibitor and their controls. (E and F) The mRNA and protein levels of spred2 in PC12 cells after H2O2 treatment. All experiments were repeated three times. Data are expressed as mean ± SD. *P<0.05, **P<0.01. Spred2, sprouty-related EVH1 domainprotein2; miR, microRNA; WT, wild type; MUT, mutant, NC, negative control.
Fig 2: miR-218 enhances H2O2-induced PC12 cell injury by targeting spred2. The protein levels of spred2 were determined in PC12 cells after transfection with si-spred2 or si-NC (A). Cell viability (B), cell apoptosis (C and D) and the protein levels of autophagy-associated proteins (E) were detected in PC12 cells co-transfected with miR-218 inhibitor and si-spred2, followed by treatment with 200 µM H2O2 for 24 h. All experiments were repeated three times. Data are expressed as mean ± SD. *P<0.05, **P<0.01. miR, microRNA; spred2, sprouty-related EVH1 domainprotein2; si-, small interfering RNA-; NC, negative control.
Fig 3: SPRED2 knockdown suppresses the effect of thrombin on inflammatory factor secretion and oxidative stress in H/R induced astrocytes. (A) Western blot and RT-qPCR analysis were performed to determine transfection efficiency of si-SPRED2. (B) The xanthine oxidase method was used to detect SOD activity, the thiobarbituric acid assay was performed to quantify MDA content and CM-H2DCFDA was used to detect ROS generation in H/R induced cells. H/R induced cells were treated with thrombin, and thrombin and 3-MA, and transfected with si-SPRED2 or si-NC. (C) The cell supernatant levels of IL-1ß, IL-6 and TNF-a were detected via ELISA. (D) The MTT assay was performed to assess cell viability. ***P<0.001. SPRED2, Sprouty-related EVH1 domain-2; H/R, hypoxia/reoxygenation; si, small interfering; SOD, superoxide dismutase; MDA, malondialdehyde; ROS, reactive oxygen species; IL, interleukin; TNF, tumor necrosis factor; NC, negative control.
Fig 4: Thrombin aggravates autophagy in H/R induced astrocytes. (A) Western blot analysis was performed to detect the protein expression levels of thrombin, SPRED2, Beclin 1, LC3-II and LC3-I. (B) Reverse transcription quantitative PCR analysis was performed to detect the mRNA expression levels of SPRED2 and thrombin. ***P<0.001. H/R, hypoxia/reoxygenation; SPRED2, Sprouty-related EVH1 domain-2; LC3, microtubule-associated protein light chain 3.
Fig 5: Regulation of thrombin on autophagy in H/R induced astrocytes is suppressed by silencing SPRED2. (A) Western blot analysis was performed to detect the protein expression levels of thrombin, SPRED2, Beclin 1, LC3-II and LC-3I. (B) Reverse transcription-quantitative PCR analysis was performed to detect the mRNA expression levels of SPRED2 and thrombin. ***P<0.001. H/R, hypoxia/reoxygenation; SPRED2, Sprouty-related EVH1 domain-2; LC3, microtubule-associated protein light chain 3; si, small interfering; NC, negative control.
Supplier Page from Abcam for Anti-SPRED2 antibody