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. 2022 Mar 17;298(4):101828. doi: 10.1016/j.jbc.2022.101828

Figure 6.

Figure 6

Effects of circAcbd6-miR-320-5p-Osbpl2 on differentiation of NSCs.A and B, Osbpl2 expression in NSCs, neurons, and astrocytes. C, Osbpl2 expression in different tissues. D, localization of Osbpl2 in the hippocampus. The bar represents 200 μm. The image in the upper right corner of the figure is an enlargement of the typical double-labeled neuron area in the small box of the figure. The bar represents 50 μm. E, effect of miR-320-5p on the abundance of Osbpl2 measured with Western blot. (miR-NC) NSCs treated with NC of miRNA mimic; (miR-320-5p) NSCs treated with miR-320-5p mimic; (miR-NCi) NSCs treated with NC of miRNA inhibitor; (miR-320-5pi) NSCs treated with miR-320-5p inhibitor. F, luciferase reporter assay in HEK-293A cotransfected with Luciferase-miR-320-5p fusion and Osbpl2-Wild or Osbpl2-Mut, 72 h post-transfection. G, expression of Map2, Neurod1, and ChAT detected by RT–qPCR. (LV–NC) NSCs treated with NC of overexpression lentivirus; (LV-Osbpl2) NSCs treated with overexpression lentivirus of Osbpl2; (LV–NCi) NSCs treated with lentivirus-mediated silencing of control; (LV-Osbpl2i) NSCs treated with overexpression lentivirus-mediated silencing of Osbpl2. H, expression of Tuj1 and ChAT detected by Western blot. (LV–NC) NSCs treated with NC of overexpression lentivirus; (LV-Osbpl2) NSCs treated with overexpression lentivirus of Osbpl2; (LV-Osbpl2 + miR-320-5p) NSCs treated with overexpression lentivirus of Osbpl2 and miR-320-5p mimic; (LV-Osbpl2 + LV-circ) NSCs treated with overexpression lentivirus of Osbpl2 and circAcbd6. I, the secretion level of ACh measured with ELISA. J, the percentage of Tuj1-positive cells detected by flow cytometry assay. K, immunofluorescence analysis of Tuj1 (green) and ChAT (red) double-positive cells. Nuclei were stained with Hoechst. The bar represents 100 μm. The data are presented as mean ± SD. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. Acbd6, acyl-CoA-binding domain–containing 6; ACh, acetylcholinesterase; HEK-293A, human embryonic kidney 293A cell line; LV, lentiviral vector; NC, negative control; NSC, neural stem cell; Osbpl2, oxysterol-binding protein–like 2; qPCR, quantitative PCR.