Figure 5.

TERF1 regulation in neuroblastoma (NBL)–human monocyte cocultures. A) Detection of TERF1 mRNA (by quantitative real-time polymerase chain reaction [qRT-PCR]) and protein (by immunoblotting) levels in SK-N-BE(2) cells alone (control group) or cocultured with human monocytes (coculture group) for the indicated time periods. B) Same experiment described in (A) but in CHLA-255 NBL cells. C) Immunoblotting for TERF1 in SK-N-BE(2) cells cocultured with human monocytes and pretransfected with LNA-anti-Scrambled (anti-scr group) or with LNA-anti-miR-155 (anti-155 group) for the indicated time periods. D) qRT-PCR for miR-155 (upper panel), TERF1 mRNA (middle panel), and immunoblotting for TERF1 protein (bottom panel) in SK-N-BE(2) cells cultured with the supernatant (SN) from SK-N-BE(2)-human monocytes cocultures (normal SN group) or with exosome-depleted SN of the coculture by ultracentrifugation (depleted SN group) for the indicated time periods. E) qRT-PCR for miR-155 (upper panel), TERF1 mRNA (middle panel), and immunoblotting for TERF1 protein (bottom panel) in SK-N-BE(2) cells treated for the indicated time periods with whole exosomes fraction (WEF) isolated from SK-N-BE(2)- human monocytes cocultures (WEF group) or PBS. F) qRT-PCR for TERF1 mRNA in SK-N-BE(2) cells cocultured with human monocytes and treated with Ruxolitinib (Rux), GW4869, or DMSO (as a control) for the indicated time periods. G) Immunoblotting for TERF1 in SK-N-BE(2) cells treated as described in (F). Relative levels of miR-155 were normalized to U6 snRNA, whereas TERF1 mRNA was normalized to GAPDH mRNA. β-actin was used as a loading control for TERF1 protein. Data are presented as mean ± SD of experiments conducted in triplicate. *P < .001, Student’s t test, two-sided.