Fig 2: Relationship between p53 and FN expression. a Unsupervised hierarchical clustering of mRNA expression detected by next generation sequencing in OV-shp53 and control cells (Con). The expression level of each mRNA is colored; red, black, and green indicated high (> 4), neutral (1–4), and low (< 1), respectively. Major clusters are shown as group I to XI. b Analysis of endogenous FN1 mRNA expression by conventional (left) and real time RT-PCR assay (right) in OV-shp53 and control cells (Con). The values of endogenous FN1 mRNA expression detected by conventional RT-PCR assay were normalization to GAPDH. The fold changes in mRNA expression for both assays are presented as means±SDs. The experiment was performed in triplicate. c Western blot analysis for the indicated proteins in total lysates from OV-shp53 and control cells (Con). d OVISE cells were transfected with FN1 reporter constructs, together with either p53wt or p53mt. Relative activity was determined based on arbitrary luciferase light units normalized to pRL-TK activity. The activities of the reporter plus the effector relative to that of the reporter plus empty vector are shown as means±SDs. The experiment was performed in duplicate

Fig 3: Circulating fibronectin in ME/CFS.a. Immunoblot analysis shows overall increase in both plasma fibronectin (plFN1) and cellular fibronectin (clFN1) in ME/CFS patients. Equal amounts of serum proteins from 5 ME/CFS patients with higher serum fibronectin levels were run in parallel with the same from 5 healthy controls. Purified plFN1, recombinant fibronectin isoform FN1.2 and HUVEC cell lysate expressing only cellular fibronectin were used as positive controls. Two different antibodies raised against Extra domain A (EDA) domain and CBD domain of fibronectin was used to differentiate plFN1 from clFN1 as plFN1 lacks EDA domain.b. Distribution of circulating FN1 concentrations in different groups of patients separated by gender. Two-tailed parametric t-test. Healthy control (HC) male vs female, **P = 0.0014; No LC male vs female, *P = 0.0204; severe LC male vs female, *P = 0.0347.c. Comparison of gender-specific circulating FN1 concentrations (log2 values) among different patient groups. Two-tailed parametric t-test. Healthy control (HC) male vs ME/CFS male, ***P = 0.0001; HC male vs mild LC male, *P = 0.012; HC female vs no LC female, *P = 0.0193; HC female vs mild LC female, ***P = 0.0006; HC female vs severe LC female, **P = 0.0016.d. IgM antibody levels against fibronectin (FN1) in ME/CFS patients and healthy controls in the form of a violin plot. IgM levels were determined from microarray studies where each antigen was tested twice separately. Normalized values against signal to noise ratio and net fluorescence intensity is used.e. Immunoblot analysis comparing the various species of circulating fibronectin (FN1) proteins in healthy controls and ME/CFS patient sera. Recombinant FN1.2 (recFN1.2) protein lacking extra domain-A (EDA domain) and HEK293 cell lysate serves as control. Three different antibodies detecting specific protein domains (EDA domain, CBD (cell binding domain) and cellular FN (clFN)) of FN1 was used against the same samples to compare the different species of proteins in the sera.

Fig 4: Autoimmunity, mitochondrial alterations and circulating Fibronectin levels in ME/CFS.a. Confocal images show mitochondrial architecture in primary HUVEC cells exposed to 1μg of purified IgG from patient sera. Two different representative images for each condition is shown.b. Average mitochondrial surface area in primary HUVEC cells exposed to 1μg of purified IgG from patient sera is quantified and compared between healthy controls, mild/moderate, severe ME/CFS. Data from three independent experiments from each serum sample is shown as a violin plot. n=3. Two-tailed non-parametric test. Healthy control vs mild/moderate ME/CFS, *P = 0.0329. Healthy control vs severe ME/CFS, **P = 0.0046. Mild/moderate vs severe ME/CFS, ****P < 0.0001.c. Immunoblot analysis shows decrease in mitofusin1 (Mfn1) and PLD6 protein levels in HUVEC cells exposed to 1μg of purified IgG from patient sera for 12 h. Actin staining was used as a loading control. Fold change values were derived from densitometric analysis of bands after normalization with the same for actin. n=2. HD, healthy donors; CFS, severe CFS patients.d. Heat map of log2 fold LFQ intensities of proteins detected within purified immune complexes from patient sera. Three proteins that showed differential protein levels between healthy controls and ME/CFS patients are shown.e. Multivariate analysis of clusters based on distance metrics derived from IgM antibody levels for a panel of autoantigens. Log-transformed scaled data showing relative differences between different variable in both healthy controls and patients.f. The Variables Factor map for the Principal Components (for Patients and Healthy Controls combined data) shows the projection of the top 10 Autoantigen variables projected onto the plane spanned by the first two Principal Components.g. Serum fibronectin (FN1) levels in patient sera. Log2 values of FN1 are presented as a violin plot. Two-tailed parametric t-test. Healthy control (HC) vs ME/CFS, **P = 0.005.h. Kernel density plot showing the bivariate serum FN1 distributions among healthy controls and ME/CFS patients. FN1 concentrations on both X- and Y-axis are presented as μg/ml.i. Circulating fibronectin (FN1) levels correlates with ME/CFS severity associated Bell score. Log2 fold FN1 vales are presented as a violin plot. Two-tailed parametric t-test. HC vs Bell 020, ****P < 0.0001. Bell 0–20 vs Bell 30–50, **P = 0.0032.j. AUROC analysis for circulating FN1 levels in healthy controls (HC) vs severe ME/CFS patients.k. Serum fibronectin (FN1) levels in different patient groups post SARS-CoV-2 infection. Log2 values of FN1 are presented as a violin plot. Two-tailed parametric t-test. Healthy control (HC) vs mild LC, **P = 0.0032. HC vs severe LC, *P = 0.0488. ns, not significant.

Fig 5: IgM-FN1 and circulating fibronectin as a biomarker in ME/CFS and long COVID.a. Multi-variate AUROC analysis for circulating FN1 and IgM against fibronectin (IgM-FN1) in healthy controls (HC) vs severe ME/CFS patients. Multiple logistic regression. ****P < 0.0001.b. Multi-variate AUROC analysis for circulating FN1 and IgM against fibronectin (IgM-FN1) in healthy controls (HC) vs severe LC patients. Multiple logistic regression. ****P < 0.0001.c. Multiple variable bubble plot with circulating FN1 levels plotted against IgM-FN1 for healthy controls (HC) and severe ME/CFS patients.d. Multiple variable bubble plot with circulating FN1 levels plotted against IgM-FN1 for healthy controls (HC) and total long COVID patients (mild LC + severe LC).e. Multiple variable bubble plot with circulating FN1 levels plotted against IgM-FN1 for ME/CFS and total long COVID patients (mild LC + severe LC).f. Multiple variable bubble plot with circulating FN1 levels plotted against IgM-FN1 for severe ME/CFS (with Bell 0–20) and severe long COVID patients.