Figure 4.

Nar specifically targets degradation of STAT3 via a ROS-dependent proteasome pathway in MCF-7/TR cells

(A) Protein expression of p-STAT3, STAT3, ERα, and ERβ in MCF-7 and MCF-7/TR cells were determined by western blot. (B) MCF-7/TR cells were treated with different concentrations of Nar for 24 h followed by western blot analysis of total and phosphorylated STAT3. (C) MCF-7/TR cells were treated with Nar at 100 nM for various time points. (D) The expression of STAT3 target genes cyclin D1 and c-Myc were determined by western blot. (E) MCF-7/TR cells were treated with Nar at 100 nM for various time points followed by real-time RT-PCR analysis of mRNAs encoding STAT3 and cyclin D1. (F) STAT3 protein levels in MCF-7/TR cells treated with Nar in the absence or presence of proteasome inhibitor bortezomib (BTZ) determined by western blot. (G and H) MCF-7/TR cells treatment with Nar and cycloheximide (CHX) alone or in combination for the indicated time points, STAT3 protein were determined by western blot and quantified by degradation kinetic curves. (I) MCF-7/TR and MCF-7 cells with Nar treatment for 1 and 3 h, and cellular ROS levels were detected. (J) MCF-7/TR cells were treated with the ROS scavenger N-acetylcysteine (NAC) for 1 h, followed treatment with or without Nar, and the images of cellular ROS levels were captured. (K) STAT3 and p-STAT3 levels in MCF-7/TR cells treated with NAC, Nar, and NAC + Nar. (L) STAT3 levels in MCF-7/TR cells treated with Nar or Nar+ different concentrations of H₂O₂. Data are shown as mean ± SD. ∗∗p < 0.01.