Fig. 6: TRX2 inhibition reverted estrogen-mediated ROS buffering.

a LPO-treated WA01 and WA09 HLC were cultured with or without 1 nM E2 for 48 h and subjected to qRT-PCR and western blotting analysis using the assays described in the figure. The Hsp90 and ACTB are used as protein-loading control normalizers. b ROS levels were measured with CellROX and MitoSOX probes in LPO-treated AML12 and HepG2 cells cultured with or without 50 nM of E2 and/or 250 nM of auranofin (AU) for 48 h. The fluorometric values of the NT and E2-treated AML12 and HepG2 cells are those reported in Fig. 4f, g and have been processed in parallel with AU-treated cells. c LPO-treated WA01 and WA09 cells were cultured with or without 1 nM of E2 and/or 500 nM of auranofin (AU) for 48 h and subjected to confocal analysis. Representative images of MitoSOX-stained cells are shown (Red: MitoSOX; blue: DAPI, nuclei. Scale bar, 25 µm). d Total protein lysates from AML12 and HepG2 cells transfected with 50 nmol/L of siTRX2 or siCTR for 72 h were subjected to western blot analysis, as indicated. The Hsp90 and ACTB are used as protein-loading control normalizers. e Mitochondrial ROS levels were measured using MitoSOX probe in TRX2-silenced AML12 and HepG2 cells treated with or without LPO and/or 50 nM of E2 for 48 h. Data represent means ± SEM. a Mann–Whitney, (b; e-HepG2) Kruskal–Wallis, Dunnett-corrected, (e—AML12) One-way ANOVA, Tukey-corrected, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Each dot represents a biological replicate.