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. 2014 Dec 23;172(3):815–828. doi: 10.1111/bph.12947

Figure 4.

Figure 4

Feedback loop of miR-200 family–ZEB–E-cadherin. mESCs were transfected with mmu-miR-141 mimic (200 nM), mmu-miR-141 hairpin inhibitor (200 nM), mmu-miR-200b mimic (200 nM), mmu-miR-200b hairpin inhibitor (200 nM), mimic negative control [mimic (-) control; 200 nM] or hairpin inhibitor negative control [hairpin inh (-) control; 200 nM] for 24 h prior to shh treatment for 24 h. (A) The expression of ZEB1, ZEB2 in nuclear fraction or E-cadherin was detected by immunoblot. The lower panel depicts the mean ± SD of three independent experiments for each condition, as determined from densitometry relative to Lamin A/C or β-actin. *P < 0.05 versus control of ZEB1; **P < 0.05 versus shh of ZEB1; #P < 0.05 versus control of ZEB2; ##P < 0.05 versus shh of ZEB2; &P < 0.05 versus control of E-cadherin; &&P < 0.05 versus shh of E-cadherin. (B) Control ChIP assay. Input (non-immunoprecipitated chromatin lysate), anti-RNA polymerase (positive control) and IgG (negative control) or ZEB1 antibodies, which were immunoprecipitated with chromatin loaded in agarose gel. (C) ChIP assay. E-cadherin gene promoter binding with ZEB1 was assayed. The same input DNA was used for each ChIP assay and the values were normalized by input and the relative control was set at one. *P, #P, &P < 0.05 versus control; **P < 0.05 versus shh. (D) The expression of Snail1 in the nuclear fraction was detected by immunoblot. The lower panel shows the mean ± SD of three independent experiments for each condition, as determined from densitometry relative to Lamin A/C.