Cycloheximide (chx)

Polysome fractionations were performed as described previously [26 ]. Briefly, HepG2 cells (one 10 cm culture dish) were treated with 100 mg/ml cycloheximide (Cayman) for 10 min at 37 °C. Then, cells were harvested and 200 μl of cytoplasmic extract was layered onto 10–50% sucrose gradient and centrifuged at 39,000 rpm in a Beckman SW-41Ti rotor for 3 h at 4 °C. Samples were collected from the top of the gradient into 15 fractions. Collected fractions were then analyzed by qPCR.

Purification of ribosome-associated mRNA was performed as described previously with slight modification [57 (link)]. Mice were decapitated, and the brains were removed immediately. The PrL were dissected in ice-cold PBS. The brain tissue was homogenized in 1 ml Supplemented Hybridization Buffer (25 mM Tris pH 7.0, 25 mM Tris pH 8.0, 12 mM MgCl₂, 100 mM KCl, 1% Triton X-100) containing 1 mM DTT, 1 × protease inhibitors (Roche, Upper Bavaria, Germany), 200 U/ml RNase inhibitor (Promega, Madison, WI, USA), 100 μg/ml cycloheximide (Cayman, Ann Arbor, MI, USA), and 1 mg/ml heparin (Sigma-Aldrich). The supernatant was incubated with 10 μg anti-HA antibody (Sigma-Aldrich, #H6908) and 100 μl Dynabeads Protein G (Invitrogen) for 12 h. Purified mRNA was eluted from the Dynabeads using TRIzol LS (Invitrogen) according to the manufacturer’s instructions with the inclusion of a DNase digestion step. The Agilent RNA 6000 Pico Kit (Agilent, Santa Clara, CA, USA) and Agilent 2100 bioanalyzer were used to evaluate the quality of purified mRNA. Samples with RIN number > 7 were used.

Protein extraction and western blotting methods were performed as described previously [53 (link)]. The antibodies used in this study including anti-β-actin (Santa cruz; sc-47778), anti-p38 (abcam; ab32142), anti-phopho-p38 MAPK(Cell signaling Technology; #9215), anti-SOX2 (Santa cruz; sc-20088), anti-Oct4 (abcam; ab19857), anti-HA (Cell signaling Technology, #3724), anti-c-Myc (Cell signaling Technology; #13987), anti-Nanog (abcam; ab80892), anti-Klf4 (Cell signaling Technology, #4038), anti-ERK (Beijing Zhongshan Golden Bridge Biotechnology Co Ltd, ZS94), anti-Ub (Santa cruz, sc-8017), anti-phospho-ATF2 (Cell signaling Technology, #9221). The anti-p38α, anti-p38β, anti-p38γ, anti-p38δ, anti-MKK3, anti-MKK6 antibodies were previously described by Kwong [26 (link)]. The p38 inhibitor SB203580 was from Sigma (s8307). For the protein stability assay of the stemness protein, cells were treated with cycloheximide (Cayman chemical, 14126) at 20 μg/ml concentration for 0h, 1h, 2h, 6h, 9h, 15h before lysates were collected. For the ubiquitylation assay, cells were treated with MG132 (Sigma, c2211) at 10 μM concentration for 6h before lysates were collected.

MSCs and HUVECs were cultured for 12–16 h in DMEM containing 0.5% FBS and 3–4 h in F-12 containing 1% BSA, respectively. In our previous study, MSC migration can be significantly enhanced by adding 100 ng/ml human recombinant interleukin-1β (IL-1β) [31 ]. In this study, both MSCs and HUVECs were stimulated with 100 ng/ml IL-1β (Peprotech, NJ, USA) for 30 min and 12 h individually. The protein synthesis inhibitor cycloheximide (Cayman, USA) was added to MSCs 60 min prior to IL-1β stimulation at a final concentration of 20 μg/ml. The CXCR3 antagonist (±)-AMG 487 (Tocris, UK) was added to culture MSC medium 2 h prior to stimulation at 500 nM. The p38 MAPK inhibitor SB203580 (Tocris, UK) was added to MSCs 2 h prior to stimulation at concentrations of 25 μM, 10 μM, and 20 μM, respectively.