Fig 2: Nfat5 expression in mouse pulmonary artery SMCs. Murine pulmonary artery SMCs (mpaSMCs) from Nfat5fl/fl mice were exposed to normoxia or hypoxia for 24 h. (A) Immunofluorescence-based detection of NFAT5 (red fluorescence) and assessment of cytosolic and nuclear fluorescence intensity (* p < 0.05, *** p < 0.001 vs. normoxia, the results of 19–20 randomly selected regions of interest (ROI) from one experiment are shown, scale bar: 50 µm). (B) Immunodetection of NFAT5 in lysates of mpaSMCs treated with AdCRE adenovirus to knockout Nfat5 (Nfat5−/−) or AdPl adenovirus as a control (Nfat5fl/fl) after capillary electrophoresis. β-actin served as the loading reference (*p < 0.05 as indicated, n = 4). (C,D) mRNA expression of Nfat5 and Ptgs2 was analyzed by qPCR in normoxia- and hypoxia-exposed Nfat5−/− and Nfat5fl/fl with Actb as reference (* p < 0.05, *** p < 0.001 as indicated, n.s.—not significant, n = 3–6).

Fig 3: Nfat5 expression in mouse vascular SMCs. (A) Murine aortic SMCs (maoSMCs) from Nfat5fl/fl mice were exposed to hypoxia for the indicated periods. The mRNA expression of Nfat5 was analyzed by qPCR with Actb as reference (* p < 0.05 vs. normoxia (set to 1: dashed line), one-sample t-test, n = 4–5). (B) Capillary electrophoresis of cytoplasmic and nuclear protein fractions of maoSMCs exposed to normoxia or hypoxia (24 h) and immunodetection of NFAT5 (not significant (n.s.) vs. cytoplasm/normoxia, * p < 0.05 vs. nucleus/normoxia, n = 4). (C) Immunofluorescence-based detection of NFAT5 ((C), red fluorescence) and automated assessment (threshold level was set to 20, dashed red line) of the percentage of NFAT5-positive nuclei in maoSMCs after exposure to hypoxia for 24 h (*** p < 0.001 vs. normoxia, n = 3). (D,E) qPCR-based analysis of Nfat5, Vegfa, and Ptgs2 mRNA expression in maoSMCs exposed to normoxia or hypoxia for 24 h ((D), * p < 0.05, ** p < 0.01 vs. normoxia (set to 1: dashed line), n = 5–6) as well as after genetic ablation of Nfat5 (AdCRE, AdPl treatment served as control) and exposure to hypoxia for 24 h ((E, * p < 0.05, *** p < 0.001, n.s. vs. AdPl/hypoxia (set to 1: dashed line), n = 4).

Fig 4: mRNA and protein expression of Nrf2 and HO-1 in the renal tissue following treatment with PbAc (20 mg/kg) and/or CoQ10 (10 mg/kg). Real-time PCR findings are presented as mean ± SD of triplicate assays and referenced to Actb. a indicates significant difference vs. the control group at P < 0.05; b indicates significant difference vs. the PbAc treated group at P < 0.05.

Fig 5: Levels and mRNA expression levels of the apoptotic proteins (Bcl-2, Bax and caspases-3) in the renal tissue following treatment with PbAc (20 mg/kg) and/or CoQ10 (10 mg/kg). ELISA readings are presented as mean ± SD (n = 7). Real-time PCR results are presented as mean ± SEM of triplicate assays and adjusted to Actb. a indicates significant difference vs. the control group at P < 0.05; b indicates significant difference vs. the PbAc treated group at P < 0.05.