Fig. 4: Estrogens reduced oxidative stress and altered mitochondrial morphology in LPO-treated hepatocytes. | Cell Death & Disease

Fig. 4: Estrogens reduced oxidative stress and altered mitochondrial morphology in LPO-treated hepatocytes.

From: Estrogen-dependent activation of TRX2 reverses oxidative stress and metabolic dysfunction associated with steatotic disease

Fig. 4

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. 6b. 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. 3d 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. 3e 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.

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