Journal: Cell Death & Disease
Article Title: Estrogen-dependent activation of TRX2 reverses oxidative stress and metabolic dysfunction associated with steatotic disease
doi: 10.1038/s41419-025-07331-7
Figure Lengend Snippet: a Untreated and LPO-exposed WA01 and WA09 HLC were subjected to western blot analysis with the antibodies indicated. ER+ MCF7 breast cancer cell line is used as a positive control (CTR+) for estrogen receptor (ER) expression. The Histone H3 (H3) is used as a protein loading control normalizer. b LPO-treated WA01 and WA09 HLC were cultured with or without 1 nM of 17β-estradiol (E2) for 48 h and subjected to qRT-PCR using the assays described in the figure. The relative quantity is shown using ∆ Ct. c LPO-treated AML12 and HepG2 cells were cultured with or without 50 nM of E2 for 48 h and subjected to ERE-luciferase reporter assay. d LPO-treated WA01 and WA09 HLC were cultured with or without 1 nM of E2 for 48 h and subjected to confocal analysis. Representative confocal images of MitoSOX-stained cells are shown (red: MitoSOX; blue: DAPI, nuclei. Scale bar, 25 µm). e LPO-treated AML12 cells were cultured with or without 50 nM of E2 for 48 h and subjected to confocal analysis. Representative images of CellROX-stained cells are shown (Green: CellROX. Scale bar, 25 µm). Quantification of CellROX intensity is reported. f , g Intracellular ROS levels were measured by CellROX and MitoSOX staining in LPO-treated AML12 and HepG2 cells cultured with or without 50 nM of E2 for 48 h. The fluorometric values of the NT and E2-treated AML12 and HepG2 cells have been used as reference in Fig. . h LPO-treated AML12 cells were cultured with or without 50 nM of E2 for 48 h and subjected to TMRE staining and subsequent quantification of mitochondrial morphology parameters: aspect ratio and form factor (Red: TMRE. Scale bar, 25 µm). The confocal analysis of LPO-treated AML12 cells has been used as reference in Fig. and has been processed in parallel with E2-treated cells. i Representative images of MitoTracker Green-stained LPO-treated AML12 cells, with or without 50 nM of E2 for 48 h, are shown (Green: MitoTracker Green; blue: DAPI, nuclei. Scale bar, 25 µm). Quantification of MitoTracker Green is reported. The confocal analysis of LPO-treated AML12 cells has been used as reference in Fig. and has been processed in parallel with E2-treated cells. Data represent means ± SEM. ( b —GDF15; c ; e ; h ; i ) Student’s t -test, ( b —SLC34A2; f ; g ) Mann–Whitney, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. a – g Each dot represents a biological replicate, h , i three biological replicates in technical triplicate.
Article Snippet: The probes used in the work are: Albumin (Hs00609411_m1), HNF4α (Hs00230853_m1), TXN2 (Hs00429399_g1), PLIN2 (Hs00605340_m1 and Mm00475794_m1), GDF15 (Hs00171132_m1), SLC34A2 (Hs00197519_m1), NRF2 (Hs00975961_g1), NQO1 (Hs01045993_g1).
Techniques: Western Blot, Positive Control, Expressing, Control, Cell Culture, Quantitative RT-PCR, Luciferase, Reporter Assay, Staining, MANN-WHITNEY
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