Skip to main content
. 2015 Apr 19;24(16):1912–1922. doi: 10.1089/scd.2014.0397

FIG. 4.

FIG. 4.

Isoflurane represses self-renewal of mES cells to inhibit neurogenesis by increasing miR-9 and subsequent repressing of E-cadherin (E-cad). (A) Above picture: prediction of the E-cadherin 3′UTR-binding site. Bottom panel means mRNA and protein levels of E-cadherin in mES cells treated with isoflurane detected by qRT-PCR and western blot. Ctrl means mES cells treated with no isoflurane. Data shown are mean±SD (n=3). **P<0.01. (B) Luciferase reporter assays were performed with vectors containing DNA fragments corresponding to the putative wild-type or mutant target sites for miR-9 in the 3′UTRs of the E-cadherin mRNAs. Firefly luciferase activity was normalized to Renilla luciferase activity. E-cad means luciferase reporter gene vector with wild-type E-cad 3′UTR. MT means mutant luciferase reporter gene vector with the mutant miRNA-233-binding site. Pre-miR-29a was used as a negative miRNA control in the assays. Data shown are mean±SD (n=3). **P<0.01. (C) Pre-miR-9 decreases the expression of E-cadherin in mRNA and protein levels. Ctrl means miRNA control. Data shown are mean±SD (n=3). **P<0.01. (D) Detection of mRNA and protein levels of E-cadherin by qRT-PCR and western blot in mES cells. Ctrl means overexpression vector control. Data shown are mean±SD (n=3). **P<0.01. (E) AP detection staining of mES cells that overexpressed E-cadherin and control group. Ctrl means overexpression vector control. (F) Detection of mRNA of stemness markers, Oct4, Sox2, Nanog, and SSEA1, in mES cells. Ctrl means shRNA vector control. Data shown are mean±SD (n=3). *P<0.05. (G) Knockdown of E-cadherin in mES cells detected by qRT-PCR and western blot. Data shown are mean±SD (n=3). *P<0.05. (H) AP detection staining of mES cells, knockdown of E-cadherin and control groups. E-cadherin KO means knockdown of E-cadherin in mES cells. (I) Detection of stemness markers, Oct4, Sox2, Nanog, and SSEA1, of mES cells by qRT-PCR. Data shown are mean±SD (n=3). *P<0.05, **P<0.01. (J) Immunostaining of tuj1. Ctrl means control groups with no isoflurane. The scale bar represents 25 μm. (K) Detection of the mRNA level of neural makers, tuj1, mash1, and map2. Bottom panel is the detection of the expression level of markers of the mesoderm and endoderm. Data shown are mean±SD (n=3). *P<0.05. Color images available online at www.liebertpub.com/scd