U0126

To separate axons from neuronal soma, a microfluidic chamber (Standard Neuron Device, 450 um microgroove barrier, Cat# SND450, Xona Microfluidics, Temecula, CA) was employed. The small dimension of the microgrooves in the chamber allows axons to sprout from the cell seeded compartment (soma side) into the other compartment of the chamber (axon side), but prevents the passage of cell bodies [37 (link)]. Briefly, cleaned, sterilized, and dried chambers were affixed to myelin (10 μg/ml, Sigma St. Louis, MO) and Poly-D-lysine (1 mg/ml, Sigma) coated 6-well plates. The cortical neurons were counted to obtain a density of 3×10⁷ cells/ml, seeded into soma side at a number of 6×10⁵ cells/chamber in DMEM with 5% FBS and incubated in a moist incubator at 37°C/5% CO₂ for an initial 6 hrs. Then the cells were washed and cultured in Neurobasal/B27/AA/Glu medium. Three days later, astrocytes (4x10⁴/chamber, 2 million/ml), MSCs (4x10⁴/chamber, 2 million/ml) or both astrocytes (4x10⁴/chamber, 2 million/ml) and MSCs (800/chamber, 4x10⁴/ml, ratio of MSC to astrocyte = 1/50) were co-seeded in axon side of microfluidic in Neurobasal/B27/Glu/AA containing 2% FBS (Neurobasal/B27/Glu/AA/2% FBS). U0126 (50 μM, InvivoGen), an ERK inhibitor or tPA (10–100 nM, Activase, Genentech, CA) were included in the media in some chambers. The growth media was changed every other day thereafter.

TLR2 ligand Pam3CSK4, TLR3 ligand Poly(I:C), TLR4 ligand LPS, TLR7/8 ligand CL087, TLR9 ligand CpG ODN, p38 inhibitor SB2021980, and ERK1/2 inhibitor U0126 were purchased from InvivoGen (San Diego, CA, USA). High-mobility group box 1 protein (HMGB1) was from Sigma-Aldrich (St. Louis, MO, USA). Kinases inhibitors including SB203580, PD98059, SP600125, and LY294002 were purchased from Calbiochem (San Diego, CA, USA); and antibodies against p38, phospho-p38, ERK1/2, phospho-ERK1/2, Akt, phospho-Akt (Ser473), JNK1/2, phospho-JNK1/2, CREB, phospho-CREB, NF-κB, and phospho-NF-κB were obtained from Cell Signaling (Danvers, MA, USA).

Cells on glass coverslips were exposed to 100 μM H₂O₂ in 10 mM deoxyglucose in RPMI minus glucose (Thermo Fisher Scientific, Waltham, MA) for 1 hour. Afterwards, cells were recovered in Maintenance media for 4 hours. This protocol mimics a transient metabolic/oxidative stress with recovery. Five µL of resuspended exosomes were added to iCMs for 2 hours prior to stress induction. In select experiments, iCMs were treated 30 minutes prior to exosome treatment with inhibitors (Invivogen, San Diego, CA): 5 µM SB203580 and 10 µM U0126.

DPSCs were seeded into 24-well plates at a density of 1 × 10⁵ cells per well. After cells reached 80% confluence, the culture medium was changed for odonto/osteogenic differentiation induction medium with or without IFN-γ and then cultures were continued for another 2 weeks.
To investigate the related signaling pathways involved in the IFN-γ-regulated odonto/osteogenic differentiation of DPSCs, DPSCs were incubated with odonto/osteogenic differentiation induction medium containing IFN-γ 0.5 ng/mL with or without the NF-κB inhibitor (PDTC, Sigma, USA) or MAPK inhibitors (SB203580, U0126, SP600125, all from In vivo Gen, USA) for 2 weeks. The induction medium was changed every 3 days. Finally, cells were stained with alizarin red (pH = 4.1) and staining quantified according to previously published methods55 (link).