Fig 2: RANTES, IFNγ, IL-6, and TNFα median levels (error bars indicating IQR) in the plasma of AIT patient and blood donor groups. Chemokine (RANTES) and pro-inflammatory cytokine (IFNγ, IL-6 and TNFα) levels from SMIA investigations are expressed as pg/mL. IFNγ, IL-6 and TNFα were chosen as they participate in RANTES induction.

Fig 3: MHY1485 upregulates IL6 and RANTES. A An antibody array of 40 inflammatory cytokines (RayBiotech) was performed in the supernatants of C20 human microglial cells treated with 5 µM of MHY1485 for 48 h. Control sup was collected from microglia treated with DMSO for 48 h. The red arrow shows the IL6 expression, whereas the blue arrow indicates the RANTES expression. B Realtime mRNA expressions of IL6 were monitored in microglia treated with 1, 2, and 5 µM of MHY for 5 h under serum-free conditions. **p < 0.01 versus control as measured with the Mann–Whitney non-parametric test. C Realtime mRNA expressions of RANTES were monitored in human microglial cells treated with 1, 2, and 5 µM of MHY1485 for 5 h under serum-free conditions. **p < 0.01 versus control as measured with the Mann–Whitney test. D Immunoblot analysis of IL6 (~ 20 kDa) was performed in human microglial cells after treating increasing doses of MHY1485 for 24 h. The resultant expression was normalized with respective β-actin expressions followed by analyzing E relative density. **p < 0.01 versus control (Mann–Whitney test). F At 14 days, after the last dose of MHY1485 feeding blood collection was carried out via cardiac puncture method, serum was isolated in heparin tube, and then performed a similar antibody array method of 40 inflammatory cytokines. Red and blue arrows indicate IL6 and RANTES expressions, respectively. ELISA analyses of G IL6 and H RANTES in the serum samples were performed (n = 8/group). Non-parametric Mann–Whitney test represents ***p < 0.005 (= 0.0002) versus vehicle. Realtime mRNA expressions of I IL6 and J RANTES were performed in muscle tissue of vehicle and MHY-fed mice (n = 5 per group). The non-parametric Mann–Whitney tests represent **p < 0.01 and *p < 0.05 versus vehicle. Results are mean ± SEM of three different experiments. K IL6 and L RANTES concentrations were also measured by quantitative ELISA methods in sera of n = 8 healthy and age-matched n = 8 ME/CFS patients. Mann–Whitney tests represent *p < 0.05 (= 0.0407) and ***p < 0.005 (= 0.0002) versus the control group. Results are mean ± SEM of three different experiments

Fig 4: The essential role of STAT3 in MHY 1485-mediated expression of IL6 and RANTES. A Immunoblot analyses of mTOR with phosphorylated tyrosine 705 (pY705) and phosphorylated Serine727 (pS727) in the nuclear extracts of human C20 microglial cells treated with 1, 2, and 5 µM of MHY1485. Histone 3 (H3) immunoblot analysis was performed as a control. (Inset) Densitometric analyses were done in ImageJ followed by normalization with respective H3 bands. B EMSA analysis of STAT3 (probed with STAT3 oligonucleotide; Li-Cor Bioscience) in the nuclear extracts of human microglial cells treated with 1 and 2 µM MHY1485 (NC = negative control). The raw blot is shown in Supplementary Fig. 10. C The promoter analysis of the human IL6 promoter at chromosome 7 displays the detailed location and sequence of consensus STAT3 responsive element. D Chromatin immunoprecipitation (ChIP) analysis of STAT3- and IgG-pulled DNA (product length = 123 bp) surrounding STAT3 responsive element of IL6 promoter. Chromosomal DNA was isolated from blue = input; green = control; and red = 5 µM MHY1485-treated microglial cells, performed with ChIP, and the resultant real-time PCR amplification data suggests increased binding of STAT3 at IL6 promoter. Fold increases in E anti-STAT3- and F IgG-pulled down DNA were displayed after normalizing the Ct value of MHY-pulled down DNA with the Ct value of the input. *p < 0.05 versus control and ns = no significance. Results were mean ± SD of three different experiments. G Human RANTES promoter map at chromosome 17 with location and sequence. H Realtime PCR amplification of human RANTES promoter pulled down by anti-STAT3 antibody in human microglial cells treated with 5 µM MHY1485 (red). Control cells were treated with DMSO (green), and input (blue) was no antibody-treated group. Fold-increase of chromosomal DNA surrounding the STAT3-responsive element of the RANTES promoter was quantified after pulling down with I STAT3 antibody and J IgG. The result was further quantified with relative Ct analyses in (K) anti-STAT3 and (L) IgG-pulled DNA after normalizing with input Ct. *p < 0.05 versus control and ns = no significance. Results were mean ± SD after three different experiments. K GFP-reporter assay of wild-type IL6 promoter cloned at STAT3 response element tagged GFP at the N-terminus region (pwtIL6). The promoter clone was transfected in HEK293T cells followed by stimulation with 1, 2, and 5 µM of MHY1485, and the resultant GFP signal was quantified with Ex: Em = 485 nm:535 nm. Results are mean ± SD of three different experiments. *p < 0.05 versus control and ns = no significance. L Site-directed mutagenesis of STAT3-response element of human IL6 promoter followed by the construction of a GFP reporter clone was performed. A dose-dependent effect of MHY1485 on the expression of GFP reporter was evaluated in HEK293T cells. ns = no significance. M DAB immunostaining of Y705PSTAT3 in skeletal muscle tissue (biceps muscle biopsies) of vehicle- and 5 mg/kg MHY1485-fed mice (n = 5/group). The representative image was derived from muscle epithelium. N Dual IF analysis of Y705PSTAT3 and macrophage-marker IBA1 in epithelial tissue of vehicle- and MHY-fed mice (n = 5/per group). Insets are magnified images of respective enclosed areas. Nuclei were stained with DAPI