Alteration of miRNA expression in C2C12 myoblasts post-myotube induction.

<p>(A) 15 down-regulated miRNAs that were negatively correlated with the ATP level (correlation coefficient, r = -0.67 to -0.83, P< 0.05). (B) 9 up-regulated miRNAs that were positively correlated with the ATP level (r = 0.67 to 0.79, P< 0.05).</p

Transcription factors associated with the differential transcript abundance among the experimental groups.

1<p>z-score indications significance of prediction of activation status of the respective transcription factor; it is predicted to be activated at z-score ≥2 or inhibited at z-score ≤−2:</p>2<p>overlap p-value: indicates significance of the association between the respective transcription factor and the predicated target genes showing differential expression in the experiment; threshold p≤5.0E-02.</p

Molecular and cellular functions of transcripts with different changes of abundance between day 3 and day 7 of the estrous cycle in the HR groups.

<p>Molecular and cellular functions of transcripts with different changes of abundance between day 3 and day 7 of the estrous cycle in the HR groups.</p

miR-423-3p as a candidate miRNA for functional validation.

<p>(A) Conservation of miRNA-423-3p across species; Bos taurus (bta), Pan troglodytes (ptr), Mus musculus (mmu), Homo sapiens (hsa), Rattus norvegicus (rno), and Sus scrofa (ssc). The seed region (2–8 nt) is highlighted in a gray box and underlined. (B) Predicted targets of mmu-miR-423-3p in the 3′-UTR of Cox6a2, Ndufb7, and Ndufs5 (RNAhybrid). The prediction criteria include free energy ≥ -20 kcal/mol and allowing the G:U wobble base-pair and bulging nucleotides in the seed region. The seed match is underlined.</p

Knockdown of <i>Cox6a2</i> by RNA interference reveals regulation of ATP levels.

<p>siRNAs were designed to target <i>Cox6a2</i> and transfected into murine C2C12 muscle cells in vitro. Relative mRNA expression of Cox6a2 was measured by qPCR 48 hours after transfection. Expression was normalized to Hprt1 and Ppia internal controls. (A) Expression of <i>Cox6a2</i> was significantly reduced relative to its expression in control cells at 48 hours post-transfection of siRNA. (B) ATP levels and (C) ADP/ATP ratio were measured at D0, D4, and D8. All values are presented as mean ± SEM (n = 3).</p

Characteristics of myogenic differentiation.

<p>(A) Morphological change of C2C12 myoblasts differentiating into myotubes at the post-induction D0 (undifferentiated mononucleated cells), D4 (elongated confluent cells), and D8 (long multinucleated myotubes). (B) Myogenic differentiation was accompanied by the up-regulation of Tnnt1, Myh1, and Myh3. (C) An increase in intracellular ATP level (μmoles) during the course of myogenic differentiation. The ATP level is shown as mean ± SEM (n = 5).</p

Predicted binding sites for miRNAs in the 3′-UTRs of their correlated mRNAs (RNAhybrid).

<p>The number of binding sites at the 3´-UTR of significant correlated mRNAs is presented with the free energy hybridization of miRNA and target in parenthesis. n/a denotes no binding site available (predicted) at the 3´-UTR.</p><p>* denotes that binding sites were predicted while the negative correlation between miRNA and mRNA was not significant.</p><p>Predicted binding sites for miRNAs in the 3′-UTRs of their correlated mRNAs (RNAhybrid).</p

Target genes of TP53 as predicted by IPA.

<p>Target genes of TP53 as predicted by IPA.</p

Confirmation of microRNA-microarray results by qPCR.

<p>miR-423-3p, miR-128-3p, and miR-301a-3p were selected for qPCR validation. Mean ± SEM (n = 4) of the log2 transformed microarray result (–•– on primary y-axis) and relative expression (2-ΔΔCt) derived from qPCR (—•—secondary y-axis) are overlaid. A correlation coefficient (r) and p-value are indicated.</p

List of differentially-expressed genes affecting ATP level during myogenic differentiation in C2C12 cells.

<p>Data are shown as fold regulation levels compared to control group (D0) and normalized to mean of housekeeping genes (<i>Actb</i>, <i>B2m</i>, <i>Gapdh</i>, <i>Gusb</i>, and <i>Hsp90ab1</i>), then assessed for correlation with ATP levels.</p><p>List of differentially-expressed genes affecting ATP level during myogenic differentiation in C2C12 cells.</p