Fig 2: Sevoflurane upregulated p-tau in the fetal brains via the CB1R/CDK5/p25 pathway. Assays of western blotting (n = 3) demonstrating the expression of CB1R, CDK5, p35, p25, tau, and p-tau (A,B) and the value of p25/p35 (C) and p-tau/tau (D). Immunofluorescence double staining of CB1R and Nestin (E, n = 3) and CDK5 and Nestin (F, n = 3) in the fetal rat brains. Quantification of CB1R/Nestin-positive cells (G) and CDK5/Nestin-positive cells (H). Data are manifested as mean ± S.E.M. *p < .05, **p < .01, vs. the Con + Veh group; #p < .05, ##p < .01, vs. the Sevo + Veh group.

Fig 3: HG-promoted oxidative stress-induced apoptosis in podocytes. (A) The effect of HG treatment on protein level of CDK5, p35/25 and apoptosis-related cleaved caspase-3 in MPC-5 cell lines. The cells were treated for 24, 48, and 72 h with LG and HG conditions, respectively. LaminB was regarded as the loading control. (B) The change of mRNA of CDK5 after 24, 48, and 72 h treatment with LG and HG in MPC-5 cells. (C) The detection of CDK5 expression in MPC-5 cells after HG treatment by flow cytometry. The cells were treated for 24 h under HG and LG conditions, and the goat anti-rabbit 488 secondary antibody was used. (D) The effect of HG treatment on the activity of CDK5 at different timepoints. Cdk5 was harvested by C-8 antibody by immunoprecipitation. The immunoprecipitate was then subjected to in vitro histone H1 kinase assays. (E,F) The effect of HG treatment on the level of (E) H2O2 and (F) ROS after 24 h culture. H2O2 was measured by hydrogen peroxide test kit, and ROS level was assessed using the probe 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) following TFP5 treatment. (G,H) The change of the mRNA level of IL-6 (G) and NFKB (H) expression in MPC-5 under HG culture condition. The cells were treated for 24 h under LG and HG conditions. GAPDH was regarded as the reference gene. (I–K). The mRNA level of Bax (I), Bcl-2 (J) and caspase 3 (K) under HG treatment in MPC-5 cells. The cells were treated for 24 h under LG and HG conditions. GAPDH was regarded as the reference gene. (L) The effect of TFP5 treatment on the HG-induced apoptosis in MPC-5 cell lines. Propidium Iodide (PI) was used to separate the viable cells, and Annexin-FITC was used to identify the apoptotic cells. *p < 0.05, **p < 0.01. A p value less than 0.05 was considered as statistically significant.

Fig 4: Regulatory effects of QYD or CDK5 inhibitors on the NFAT5-GEF-H1 signaling pathway. (A) Scatter plot of CDK5 and NFAT5 genes in SAP patients. Quantitative RT-PCR analysis of the relative levels of CDK5, NFAT5 and GEF-H1 (B) mRNA in rat lung tissues. (C and D) Western blot was performed to assess the protein expression levels of CDK5, NFAT5, GEF-H1, and Acetyl-alpha Tubulin in rat lung tissues. GAPDH was used as a loading control. Semi-quantification of protein expression of CDK5, NFAT5, GEF-H1 and Acetyl-alpha Tubulin using histograms. Data are representative images or expressed as mean ± SD of each group of rats from at least three independent experiments; *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 5: Lung tissue immunofluorescence in rats. (A) Immunofluorescence analysis of CDK5 (red), NFAT5 (green), and nucleus (blue) expression in lung tissue (scale bar=50μm). (B and C) Semi-quantitative analysis of CDK5 and NFAT5 expression; *P < 0.05, **P < 0.01, ***P < 0.001.