Fig. 6: LN induces NRF2 activation and metabolic reprogramming also in THLE-2 human hepatocytes.

A The RNA derived from siCTR- or siNRF2-transfected THLE-2 cells either NT or treated for 24 hours with LN (500 µM) was analyzed by qRT-PCR analysis using the assay described in the Figure. Relative mRNA expression was calculated using β-actin as endogenous control. Data are presented as fold change relative to the NT condition of siCTR. Data are represented as mean ± SEM. Each dot represents a biological replicate, n = 4. One-way ANOVA followed by Tukey’s correction. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; B Western blot analysis of p62 protein level was performed in siCTR- and siNRF2-transfected THLE-2 cells treated with LN (100 and 500 µM) for 24 hours. β-ACTIN was used as loading control. Western blot quantification of 3 biological replicates was calculated using ImageJ software; C Left: Seahorse XFe96 Mito Stress Test on LN-treated THLE-2 cells (100 µM for 24 hours). OCR was calculated in real-time after administration of ATP synthase inhibitor Oligo (1.5 µM), FCCP (1 µM), and respiratory complex I inhibitor Rot together with respiratory complex III inhibitor AA (Rot/AA, 0.5 µM). Right: Basal and maximal respiration, and ATP production were calculated as described in the Materials and Methods section and normalized on protein content. NT condition was used as comparator in the statistical analysis. Data are represented as mean ± SEM. One representative biological replicate is shown. Three biological replicates were performed in 7 technical replicates. Two-way ANOVA followed by Tukey’s correction (left) and Student t-test (right), *P < 0.05, ***P < 0.001; ****P < 0.0001; D The RNA derived from THLE-2 cells either NT or treated for 24 hours with LN (500 µM) was analyzed by qRT-PCR analysis using the assay described in the figure. Relative mRNA expression was calculated using β-actin as endogenous control. Data are presented as fold change relative to the NT condition. Data are represented as mean ± SEM. Each dot represents a biological replicate, n = 4. Student t-test. *P < 0.05, **P < 0.01, ***P < 0.001; E The RNA derived from siCTR- or siNRF2-transfected THLE-2 cells either NT or treated for 24 hours with LN (500 µM) was analyzed by qRT-PCR analysis using the assay described in the figure. Relative mRNA expression was calculated using β-actin as an endogenous control. Data are presented as fold change relative to the NT condition of siCTR. Data are represented as mean ± SEM. Each dot represents a biological replicate, n = 4. One-way ANOVA followed by Tukey’s correction. **P < 0.01, ****P < 0.0001. Abbreviations: β-actin: beta-actin; FCCP, proton uncoupler carbonyl cyanide p-triflouromethoxyphenylhydrazone; G6PD; glucose-6-phosphate dehydrogenase; GCLC, glutamate-cysteine ligase catalytic subunit; HK2, hexokinase 2; LN, lead nitrate; NQO1, NAD(P)H quinone dehydrogenase 1; NRF2, nuclear factor (erythroid-derived 2)-like 2; NT, untreated; Oligo, oligomycin; OCR, oxygen consumption rate; Rot/AA, rotenone/antimycin A; SLC16A1, Solute carrier family 16 member 1; SLC16A3, Solute carrier family 16 member 3.