Fig. 2: LN induces the expression of NRF2 target genes and reduces ROS generation in RNT cells.

A The RNA derived from RNT cells either NT or treated for 24 h with LN (100 µM) was analyzed by qRT-PCR analysis using the assay described in the figure. Relative gene 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 = 3. Student t-test. **P < 0.01, and ****P < 0.0001; B Western blot analysis of GSTP1, G6PD, and NQO1 protein levels was performed in NT, Veh conditions, and LN-treated RNT cells. Vinculin was used as loading control. Western blot quantification of 3 biological replicates was calculated using ImageJ software; C Immunofluorescence staining of NRF2 was performed in RNT cells NT or treated with LN (100 µM) with or without IVE (5 µM) for 24 hours. Representative confocal images of stained cells (left) are shown (green: NRF2; blue: DAPI, nuclei. Scale bar, 25 µm – inset of a higher magnification cell). Quantification of NRF2 nuclear/cytoplasmic signal fraction (right) is reported. Data are presented as fold change relative to the NT condition. Data are represented as mean ± SEM. Each dot represents a replicate: three biological replicates were performed in technical triplicate, n = 9. One-way ANOVA followed by Tukey’s correction, ****P < 0.0001. D RNT cells were treated with LN (100 µM) for 24 hours and subjected to FACS analysis to measure ROS levels with DCFDA fluorescent probe. 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 < 0.001; E RNT cells were treated with LN (100 µM) for 24 hours and subjected to confocal analysis. Representative pictures of CellRox-stained cells are shown (Green: CellRox. Scale bar, 25 µm). Quantification of CellRox is reported. Data are represented as mean ± SEM. Each dot represents a replicate: three biological replicates were performed in technical triplicate, n = 9. Student t-test, ****P < 0.0001; F ROS levels were measured using DCFDA probe in siCTR- and siNRF2-transfected RNT cells NT or treated with LN (100 µM) for 24 hours. 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 = 3. Two-way ANOVA followed by Tukey’s correction, **P < 0.01; G The GSH/GSSG levels ratio was measured in siCTR- and siNRF2-transfected RNT cells NT or treated with LN (100 µM) for 24 hours. 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 = 3. One-way ANOVA followed by Tukey’s correction, *P < 0.05, ****P < 0.0001; H, I RNT cells were treated with LN (100 µM) for 24 hours and subjected to FACS using BODIPY^581/591-C₁₁, a proxy of lipid peroxidation H, and western blot analyses to assess the levels of 4-HNE I. Actin was used as loading control. Western blot quantification of 3 biological replicates was calculated using ImageJ software. Data are presented as fold change relative to the NT condition. Data are represented as mean ± SEM. Each dot represents a biological replicate, n = 3. Student t-test, **P < 0.01. Abbreviations: β-actin: beta-actin; 4HNE, 4-hydroxynonenal; DCFDA, 5,6-Carboxy-2’,7’-Dichlorofluorescein Diacetate; FACS, cytofluorimetric analysis; GCLC/Gclc, glutamate-cysteine ligase catalytic subunit; GSH/GSSG, reduced glutathione/oxidized glutathione; GSTP1/Gstp1, placental glutathione S-transferase; IVE, ivermectin; LN, lead nitrate; MFI, mean fluorescent intensity; NQO1/Nqo1, NAD(P)H quinone dehydrogenase 1; NRF2, nuclear factor (erythroid-derived 2)-like 2; NT, untreated; Veh, vehicle.