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. 2017 Aug 3;7:7219. doi: 10.1038/s41598-017-07575-0

Figure 5.

Figure 5

miR-23 overexpression negatively regulates TrxR1 levels by targeting its 3′UTR. C2C12 myoblasts were transfected with mimic miR-23a, mimic miR-23b or a scramble miR (scr miR). (a) RT-qPCR showing the expression of miR-23a and miR-23b 48 hours after transfection. (b) Representative western blot displaying TrxR1 protein levels at 48 and 72 hours after transfection. The lysates originated by C2C12 transfected with an siRNA against the TrxR1 mRNA sequence (TrxR1 siRNA) were loaded as positive control of TrxR1 downregulation. (c) Quantification of panel b performed on three independent experiments. TrxR1 proteins levels were normalized to β-actin and the value of scr miR was arbitrarily set = 1. (d) A schematic view of the reporter construct generated by cloning the 3′UTR sequence of TrxR1 mRNA in psicheck vector downstream of renilla luciferase cDNA (psi-wt). Site-specific mutagenesis was performed to generate the reporter construct harbouring the 3′UTR mutated in miR-23 binding sites 1 and 2 (psi mut1-2). (e) Histograms showing the relative amount of Renilla/Ffly ratio measured in lysates of C2C12 cells transfected with psi wt, psi mut1 or psi mut1-2 in the presence of scr miR, mimic miR-23a, mimic miR-23b or both (miR-23a-b). The experiment was performed three times. Each biological assay was done in triplicate.