Skip to main content
. 2014 Nov 10;111(47):E5023–E5028. doi: 10.1073/pnas.1415098111

Fig. 3.

Fig. 3.

H19 silencing in undifferentiated C2C12 cells promotes myogenin mRNA stabilization and maturation of myomiRs from precursors. H19 was transiently silenced in C2C12 cells cultured in GM by using a combination of a sequence-specific siRNA and a GAPmeR (collectively indicated as Sil H19). Parallel cultures were mock-silenced by using a combination of a control siRNA and a control GAPmeR (collectively indicated as Sil C). Cells were used for experiments 48 h after transfection. (A) Cells were treated with 100 μM 5,6-Dichlorobenzimidazole 1-β-D-ribofuranoside (DRB), and total RNA was isolated at different times (as indicated) after the addition of DRB and analyzed by RT-qPCR to detect myogenin mRNA expression. (B) RNA was prepared from transfected C2C12 cells, and myogenin levels were quantified by RT-qPCR. (C) Total cell extracts were immunoprecipitated as indicated. RNA was purified from immunocomplexes and analyzed by RT-qPCR to detect myogenin or primary miRNAs. (D) RNA was prepared from transfected C2C12 cells, and the levels of the indicated miRNAs and primary miRNAs were quantified by RT-qPCR. (E) In vitro pri-miR-206 processing assays performed by using total extracts from C2C12 cells mock- (Sil C) or H19- (Sil H19) silenced and cultured in GM or DM (for 36 h). Internally 32P-labeled pri-miR-206 RNA substrate was added, and its processing was monitored as described under Materials and Methods. A representative autoradiogram is shown. The intensity of background bands is ascribable to the long exposure of gels as a result of the low processing efficiency of pri-miR-206. The arrow points to premiR-206 band that is visible only in experiments performed by using extracts from cells treated with DM/Sil C or GM/Sil H19. The bar graph below the autoradiogram is a quantification of the premiR-206 levels measured in two distinct processing assays. The intensity of the bands corresponding to pri–miR-206 and premiR-206, quantified with ImageJ software (http://rsb.info.nih.gov/ij/index.html), was expressed as percentage (±SEM calculated on two experiments) of premiR-206 generated from pri-miR-206 at each time point. To avoid signal saturation, the quantification was performed on underexposed autoradiograms. The values of RT-qPCR experiments shown are averages (±SEM) of three independent experiments performed in triplicate (*P < 0.01 and **P < 0.001, Student t test).