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. 2015 Nov 2;112(46):E6293–E6300. doi: 10.1073/pnas.1505962112

Fig. 5.

Fig. 5.

Exosome-associated TERRA stimulates inflammatory cytokines. (A) RNA dot blot analysis of sucrose gradient fractionation of LCL-derived exosomes probed for TERRA (Upper), TERRA antisense, 18S rRNA, or alpha-satellite RNA (Lower). (B) Total exosomes (input) or sucrose gradient fractions were incubated with PBMCs for 3 h and then assayed by qRT-PCR for expression of IL6, TNFα, GM-CSF, CXCL10, or control GUSB mRNA. Bar graphs represent qRT-PCR values relative to gapdh mRNA (mean ± SD) from three independent experiments. (C) Schema of VP16-TRF1(ΔN) activation of TERRA. (D) RNA dot blot for TERRA, 18S, or U1 RNA from HCT116 cells (Left) or exosomes (Right) transduced with vector, TRF1(ΔN), or VP16-TRF1(ΔN). (E) HCT116 cells transduced as in D were assayed by Western blot for CD63, TRF1, FLAG, and Actin. (F) qRT-PCR for expression of IL6, TNFα, CXCL10, or control GUSB mRNA for PBMCs treated with exosomes derived from HCT116 cells transduced with vector control (green), TRF1(ΔN) (red), VP16-TRF1(ΔN) (purple), or PBS control (black). (G) Northern blot of in vitro transcribed TelG, TelC, or U6 RNA treated with control or RNaseA and probed for TERRA (Left), TERRA antisense (Center), or U6 (Right). (H) IMR90 cells were treated with liposomes containing TelG or TelC RNA for 24 h and then assayed by qRT-PCR for IL6, TNFα, CXCL10, or control GUSB mRNA. Bar graphs represent qRT-PCR values relative to gapdh mRNA (mean ± SD) from three independent experiments.