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. 2019 Dec 23;7:342. doi: 10.3389/fcell.2019.00342

FIGURE 2.

FIGURE 2

Role of miR-128-3p in neurogenic differentiation from AECs. (A,B) Neurocyte markers were tested using Western blotting after AECs were exposed to miR-128-3p, miR-128-3p inhibitor, and Jagged 1, respectively. Protein abundance was analyzed using ImageJ tools. The data revealed that Jagged1 could rescue the β III-tubulin expression after overexpressed miR-128 in AECs. ∗P < 0.05; ∗∗P < 0.01; ns, non-significant. (C) Immunoprecipitation of Myc-tagged Argonaute 2 (AGO2) from AECs co-transfected with Myc-AGO2 and either miR-128-3p or Let-7a (negative control). The empty vector (EV) served as the Myc-AGO2-related negative control. Jag 1 and β-actin mRNA levels were quantified using qPCR, and the relative immunoprecipitate (IP)/input (cell total RNA) values were plotted. ∗P < 0.05. (D) The effect of miR-128-3p on JAG1 expression was evaluated using luciferase reporter assays. ∗∗P < 0.01. (E) Western blot analysis of JAG1 expression was performed following siRNA targeting. Protein abundance was analyzed using ImageJ tools. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an endogenous control and Scramble was used as a control. Western blot images are representative of at least three independent replicates. ∗P < 0.05. (F,G) Neurocyte markers were tested using flow cytometry after induced AECs were exposed to various small RNAs or Jagged 1. iAECs, induced AECs; miR-128 i, miR-128 inhibitor; miR Con, miR-128 mimics control.