Figure 4.

Restoration of miRNA pathway activity in mammalian oocytes. Luciferase reporter activities upon co-injection of mouse (A) or porcine (B) fully grown oocytes with 5 × 10⁵, 2.5 × 10⁵ and 1 × 10⁵ molecules of let-7a mimic and 10,000 molecules of let-7a reporters. (C) Repression of 100,000 reporter molecules in mouse oocytes co-injected with 2.5 × 10⁵let-7a mimic molecules. (D) Repression of 10,000 mir-30c reporter molecules in mouse oocytes co-injected with 2.5 × 10⁵mir-30c mimic molecules. All luciferase data are a ratio of the NanoLuc luciferase reporter activity over a co-injected control firefly luciferase reporter activity; the relative 4×-mutant reporter was set to one. Data are from two independent microinjection experiments. Error bars = SD. Asterisks indicate statistical significance (P-value) of one-tailed t-test, (* < 0.05, ** < 0.01, *** < 0.001). (E) Repression of endogenous let-7 target mRNAs upon injection of 2.5 × 10⁵let-7a mimic molecules. Oocytes were collected at 0 h non-injected or cultured for 20 h in IBMX non-injected or injected with let-7a mimic. Experiments were performed twice, each time in duplicate, i.e. with four independent cDNA. Data are normalized to 0 h, which is set to 1. Error bars = SD. Asterisks indicate statistical significance (P-value) of one-tailed t-test, (* < 0.05, ** < 0.01, *** < 0.001) (F) Analysis of protein levels of endogenous let-7 targets upon injection of 2.5 × 10⁵let-7a mimic molecules. Fifty oocytes were used for each lane of Hif1an and 25 oocytes for Nr6a1. The blots were treated with sodium azide and reblotted for tubulin. Cycloheximide treatment was carried out for 8 h and the oocytes were cultured in IBMX. The experiment was repeated twice.