Fig 2: Enhancement of Bcl2 and MRP4 partially reversed the suppressive effects of up-regulation of miR-125a-5p on paclitaxel resistance in EC cells. (A) The expression of Bcl2 protein in Ishikawa/PA and HEC-1A/PA cells. *P < 0.05 vs. agomiR-NC + pUC group; **P < 0.01 vs. agomiR-NC + pUC group; #P < 0.05 vs. agomiR-125a-5p + pUC group. (B) The expression of MRP4 protein in Ishikawa/PA and HEC-1A/PA cells. *P < 0.05 vs. agomiR-NC + pUC group; **P < 0.01 vs. agomiR-NC + pUC group; #P < 0.05 vs. agomiR-125a-5p + pUC group. (C,D): The IC50 of paclitaxel in Ishikawa/PA and HEC-1A/PA cells. *P < 0.05 vs. agomiR-NC + pUC group; **P < 0.01 vs. agomiR-NC + pUC group; ##P < 0.01 vs. agomiR-125a-5p + pUC group.

Fig 3: Doxorubicin chemoresistance is associated to H19-mediated upregulation of multidrug resistant proteins (MDR)In panels A and B, the levels of ABCB1 and ABCC4 mRNAs, and MDR1 and MRP4 proteins were quantified respectively by real-time RT-PCR and Western blot analysis in various cell lines as indicated. The mRNA levels were expressed as fold of parental MCF-7 control, and the data are the mean ± SEM of three to five independent experiments. The inserts on top of panels A and B, and panel C are representative Western blots analyses of MDR1, MRP4, XPD and TOPIIA proteins, respectively. β-actin was used as an internal loading control in Western blot analysis. Lanes in all Western blots are denoted as: lane 1 – MCF-7, lane 2 – MCF-7/Con1600, lane 3 – MCF-7/Dox1600, lane 4 – MCF-7/Dox/NC and lanes 5 to 7 – Three independent MCF-7/Dox/shH19 clones. *p < 0.05 compared to the corresponding controls.

Fig 4: Proposed mechanism of dysregulated glutathione metabolism and synthesis in cervical spinal cord of wobbler mice. Glutathione metabolism between astrocytes and neurons: astrocytes import glutathione (GSH) via MRP4 (Multidrug Resistance Protein 4), while neurons do not contain an uptake mechanism for GSH and therefore rely on their own GSH synthesis. Both astrocytes and neurons can synthesize GSH from its constituent amino acids. Astrocytes can import cystine via the cystine/glutamate exchanger system Xc-. Astrocytes supply neurons with substrates for GSH synthesis. They export GSH via MRP1 (Multidrug Resistance Protein 1). In the next step, the ectoenzyme GGT (Gamma-Glutamyl Transferase) hydrolyzes GSH. The resulting Cysteinyl–Glycine (Cys-Gly) is cleaved by the ectoenzyme Aminopeptidase N (ANPEP). Next, the resulting amino acids are imported into neurons by different transporters, where they can be used for GSH synthesis. The main transporter for cysteine uptake in neurons is EAAT3 (Excitatory Amino Acid Transporter 3). Proposed mechanism of dysregulated glutathione metabolism and synthesis in the cervical spinal cord: a decreased protein expression of GCL (γ-Glutamyl-cysteinyl-ligase) leads to a decreased synthesis of γ-Glu-Cys (γ-Glutamyl-Cystein), resulting in a decreased GSH synthesis, even though protein expression of GSS (glutathione synthetase) is increased. Thus, less GSH is available for export from astrocytes via MRP1 and cleavage by GGT and ANPEP. As a compensatory mechanism, GGT1 is upregulated. However, EAAT3 is downregulated, which aggravates the deficient cysteine supply to neurons. Overall, this leads to an insufficient GSH synthesis in neurons. Due to the inadequate GSH levels, neurons’ and astrocytes’ defense against ROS (reactive oxygen species) is severely impaired. In astrocytes, increased levels of ROS lead to an increased expression of Slc7a11 encoding for the light chain of system Xc-, enhancing cystine import in astrocytes. Legend: Green arrows indicate an upregulation; red arrows indicate a downregulation of corresponding enzyme on protein or mRNA levels. Cys-Cys: Cystine; Glu: Glutamate; Gln: Glutamine.

Fig 5: Intermittent fasting (IF) suggests reversal of DNA methylation status in the promoter region under chronic cerebral hypoperfusion (CCH). The methylation status of the differentially methylated genes were compared to the methylation levels of the respective time specific IF Sham group. Venn diagram of differentially methylated genes across all gene regions in the AL BCAS mice compared to AL Sham mice (A) and in the IF BCAS mice compared to AL BCAS mice (B) with respect to their corresponding Sham groups. The breakdown of specific methylation trends of differentially methylated genes were represented in Venn diagrams. A total of 9 hypermethylated genes and 7 hypomethylated genes were identified to be overlapping across all three timepoints in the AL BCAS mice and a total of 3 hypermethylated and 18 hypomethylated genes were identified to be overlapping across all three timepoints in the IF BCAS mice. (C) Representative immunoblots and quantification of overlapping differentially methylated genes to validate findings in DNA methylation sequencing. This includes protein abundance of ACTN1 in AL CCH compared to AL Sham mice, GNAS in AL CCH compared to AL Sham mice, MRP4 in AL CCH compared to AL Sham mice and IF CCH compared to IF Sham mice. Vinculin and β-actin were used as loading controls. Data are represented as mean ± SD. n=5-7 mice in each experimental group. Unpaired t-test, Bonferroni correction, *p<0.05 compared to ALB30D. Analysing specific differentially methylated genes in the promoter region by presenting the spread in the form of a Venn diagram in the (D) AL BCAS mice compared to the AL Sham mice and (E) IF BCAS mice compared to the AL BCAS mice. (F) 5 differentially methylated genes were identified to be overlapping across three different timepoints in the AL BCAS mice. The methylation levels of the 5 genes were illustrated in the form of a heatmap to show its temporal regulation which was present in Ddx3y, Gnas and Pax6. (G) 4 differentially methylated genes were identified to be overlapping across the three different timepoints in the promoter region of the IF BCAS mice. The methylation levels of the 4 genes were illustrated in the form of a heatmap to show the temporal regulation which was present in Zrsr1, Gng10, Actg1 and Speg.