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. 2021 Feb 18;10:e66288. doi: 10.7554/eLife.66288

Figure 5. Increased Ago2 leads to significant increase of let-7 miRNAs and accelerated differentiation in mouse embryonic stem cells (mESCs).

(A) Western blotting in mESCs with dox-inducible expression of Ago2. Gapdh was used for normalization in calculating the relative expression of Ago2. (B) Relative levels of miRNAs in mESCs with dox-inducible expression of Ago2. U6 RNA was used for normalization. (C) Relative levels of pri-miRNAs in mESCs with dox-inducible expression of Ago2. 18S rRNA was used for normalization. In (B) and (C), the miRNA and pri-miRNA expression levels in mESCs without dox treatment were set as 1 for determining relative levels. The results are from four biological replicates. (D) Colony formation assay for mESCs with dox-inducible expression of either GFP or Ago2. (E) Exit pluripotency assay for mESCs with dox-inducible expression of either GFP or Ago2. The results in (D) and (E) represent the means (± SD) of six independent experiments. *p<0.05, and n.s. not significant (p>0.05) by the Student’s t-test. (F) Western blot analysis on pluripotency factors during EB formation from the GFP-expressing mESCs and Ago2-expressing mESCs (treated with 50 ng/ml dox).

Figure 5.

Figure 5—figure supplement 1. Increased Ago proteins in mouse embryonic stem cells (mESCs) result in a specific increase of let-7 miRNAs.

Figure 5—figure supplement 1.

(A) Relative expression levels of miRNAs in the mESCs with dox-inducible expression of either GFP or Ago2. U6 RNA was used for normalization. (B) mRNA expression levels of Ago1, Ago2, Ago3, and Ago4 in mESCs. TPM, transcripts per kilobase million reads. (C) Western blot of mESCs with inducible exogeneous Ago1 expression. (D) qRT-PCR on the let-7 pri-miRNAs. The expression level in the WT cells was set as 1 for relative comparison. 18S rRNA was used for normalization. (E) qRT-PCR on let-7 miRNAs and non-let-7 miRNAs. For each miRNA, the expression level in WT cells was set as 1 for relative comparison. U6 RNA was used for normalization. The qPCR results in (A) and (D and E) represent the means (± SD) of three independent experiments.
Figure 5—figure supplement 2. The increased let-7 miRNAs are bound and stabilized by the elevated Ago2 in mouse embryonic stem cells (mESCs).

Figure 5—figure supplement 2.

(A) Specific isolation of both endogenous Ago2 and dox-induced FLAG-Ago2 from mESCs with a FLAG-tag knock-in at the N-terminus of Ago2 locus. Super: supernatant; IP: immunoprecipitated sample. (B) Outline of the RNA immunoprecipitation (RIP) experiment. (C) qRT-PCR quantification of the Ago2-bound RNAs. (D) Outline of the actinomycin-D(5 µg/ml)-mediated transcriptional shut-off experiment to measure RNA stability in mESCs. (E) Stabilities of let-7 pri-miRNAs are not sensitive to Ago2 levels. (F) Increased Ago2 specifically stabilizes let-7 miRNAs in mESCs. The results from (C), (E), and (F) represent the means (± SD) of three independent experiments. *p<0.05, and n.s. not significant (p>0.05) by the Student’s t-test.