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. 2020 Feb 28;36:100968. doi: 10.1016/j.molmet.2020.02.011

Figure 2.

Figure 2

HuR is required for HG-induced Nox4/ROS increase, MCs injury and maintains Nox4 mRNA stability. (A) HuR and Nox4 protein levels in cells with scramble or siRNA against HuR along with HG exposure for 1 hour. Cells were treated with HG for 1 h before being subjected to cell lysis. GADPH was used as a loading control. (B) HuR and Nox4 protein levels in MC cells with overexpression of Myc-HuR, by standard Lipofactmine 2000 transient transfection. (C, D) Nox activity (superoxide production) was measured in MC cells with scramble or siRNA against HuR after HG exposure for 1 h by lucigenin chemiluminescence (C) and HPLC assay (D) in designed cells. The data are presented as the mean ± S.E of three repeats. ∗∗, p < 0.01 in comparison with cells receiving scramble siRNA and no HG treatment. (E) Fibronectin (FN) and α-smooth muscle actin (SMA) were measured in MCs with siRNA against HuR by western blotting. (F) Polysomes assays were performed in MCs. Nox4 mRNA was measured with Taqman probes and presented here as the percentage in each fraction. (G, H) Nox4 RNA stability was measured by qRT-PCR after MCs were exposed to Actinomycin D (AcD) (5 μg/mL), with control or HG, scramble or siRNA against HuR (G), and MCs with HuR overexpression construct or control vector (H). The data are expressed as the percentage of mRNA molecules before the actinomycin D treatment.