miR-128 Is Implicated in Stress Responses by Targeting MAFG in Skeletal Muscle Cells

Figure 2

miR-128 downregulates the expression of MAFG and MAFG-regulated genes in human and mouse contexts. (a) HEK293 cells were transfected with either CMV-miR-128 construct or negative control vector (CMV-neo). After 24 h, total RNAs were isolated and the relative mRNA levels of the indicated genes were calculated through quantitative real-time PCR by comparing the levels associated to miR-128 expressing- versus CMV-neo control cells, after normalization with c-ABL. Data were reported as relative to the vector control, which was set equal to 100. Each column in the panel represents the mean ± SD of 3 independent experiments. . (b) HEK293 cells were transfected with either pre-CMV-miR-128 or negative control (CMV-neo). After 22 h, cells were treated for 2 h with 200 μM DEM. Untransfected HEK293 cells were used as positive control for DEM treatment. Nrf2 protein levels were determined by Western blotting on cytosolic [C] and nuclear [N] extracts. UCHL3 was used as a control of extract preparations. (c) CMV-neo or CMV-miR-128 were transiently transfected into C2C12 cells for 30 h, and Western blotting analysis of MAFG and BMI-1 was performed on total protein extracts; vinculin was used as a loading control. (d) mRNA levels of AKR1D1, ALDH3, CCDC53, HMOX-1, Nrf2, PCBD2, and UCHL1 genes were analyzed as described in (a) at 30 h after transfection in C2C12 cells. Each column in the panel represents the mean ± SD of at least 3 independent experiments. .

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