Fig 2: K63-linkages activate NFκB and JAK/STAT pathways in AD TauO-induced pathology in vitro.A–E, MAPK phosphorylation profiles in AD TauO-treated K63Ub-iHEK-Tau cells. Cell signaling profile in lysate of iHEK-Tau cells with K63Ub overexpression and control cells was measured with RayBio Human phosphorylation profile array. Bar graphs present protein fold-change of signaling activity involved in (A) MAPK, (B) Akt, (C) JAK/STAT, (D) NFκB, and (E) TGFβ pathways. Statistical analysis was calculated by One-way ANOVA with Tukey test. (∗p < 0.05 compared to UT). F–J, representative chemiluminescent arrays showed in fire lookup tables of (F) MAPK, (G) Akt, (H) JAK/STAT, (I) NFκB, and (J) TGFβ for UT and AD TauO-treated K63-linkage overexpressing-iHEK-Tau cells. The intensities of array dots were quantified in quadruplet per array and were normalized against the positive controls on the blots. AD TauO, AD brain-derived tau oligomers; iHEK-Tau, HEK293 cell line with inducible expression of human 4R tau.

Fig 3: EVs from LPS‐stimulated MCs induce TNF‐α secretion in recipient MCs. BMMCs or PCMCs were cultured in EV‐free medium in the presence or absence of LPS (100 ng/ml ) for an hour. A23187 (0.5 μM) and PBS were used as positive and negative controls, respectively. EVs were separated from the conditioned medium after 24 h. Naïve BMMCs (a) or PCMCs (c) were cultured in EV‐free medium (control) conditioned medium (SN), EV‐depleted conditioned medium (EV‐dep SN), EV‐reconstructed medium (EV‐rec SN) or separated EV containing non‐conditioned medium. For concentration measurements, different ratios of EV donor and acceptor cells were tested (b). Naïve BMMCs were also cultured in the presence of EVs from unstimulated BMMCs incubated in 100 ng/ml LPS for 2 h prior to experiments (d) or large‐, medium size‐ or small‐EVs of stimulated and unstimulated MCs (e). We used GW4869 (10 μM), a neutral sphingomyelinase inhibitor to block small EV generation, 30 min before LPS stimulation of donor cells. As GW4869 was diluted in DMSO, we used DMSO as control. Separated small particles were measured by NTA (f) and were added to naïve cells (same amount as producing cells) after washing for 24 h (g). In inhibition experiments, BMMCs were treated with TAK242 (0.2 μM), dynasore (80 μM) cytochalasin D (10μg/ml ) prior to culture in the presence of separated EVs from conditioned medium of unstimulated (PBS‐EV) or LPS‐stimulated (LPS‐EV) MCs (h). TNF‐α concentration was measured after 24 h with ELISA. Column bars are means of at least three independent experiments (biological replicates, n>3) as the mean and SD of three replicates (n = three, *P ≤ 0.05, **P ≤ 0.01, t test and ANOVA), where one dot indicates the average of three technical replicates. BMMCs were cultured for 24 h in EV‐free complete medium in the presence of isolated EVs derived from LPS‐stimulated (100 ng/ml ) and unstimulated BMMCs. Cells were washed two times lysed and investigated for phosphorylation of ERK1/2, JNK and P38 by RayBio Cell‐based phosphorylation ELISA kits (i). Biological replicates, n≥3, *P ≤ 0.05, **P ≤ 0.01, t test. Schematic picture shows the possible mechanism of MAPK signalling activation (j)

Fig 4: Farnesylthiosalicylate (FTS) inhibits phosphorylation of mitogen‐activated protein kinases (MAPKs) and T‐cell proliferation following T‐cell antigen receptor (TCR) stimulation. Isolated mouse CD4+ T cells were stimulated with anti‐‐CD3/28 antibodies and treated with FTS 10 µM (green) or vehicle (red) for 30 minutes. A, Bar graphs depict the relative phosphorylation level (arbitrary units of intensity per densitometry) of the indicated kinases included in the RayBio Mouse MAPK Pathway Phosphorylation Array, as analyzed per the manufacturer’s protocol. B, Flow cytometry data expressed as overlay histograms depicting phosphorylated (p)‐extracellular signal‐regulated kinase (Erk)1/2, p‐AKT, and p‐p38 levels, determined by phospho‐flow‐cytometry with phospho‐specific antibodies, as detailed in the Methods section. T cells treated with FTS or control media are shown in green and red, respectively, whereas unstained counterpart samples are in grey. C, CD4+ T cells were labeled with carboxyfluorescein succinimidyl ester (CFSE) and activated by plate‐bound anti‐CD3 and soluble anti‐CD28 monoclonol antibodies (mAbs), treated with the indicated concentration of FTS, and cultured for 72 hours. Shown is a representative experiment (CFSE‐dilution flow cytometry histograms) out of two independent experiments, and the bar graphs depict mean ± SD of triplicates. Samples were analyzed for statistical significance by Student's t test (***P < .001, **P < .01, and *P < .05).