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. 2020 Dec 7;117(51):32606–32616. doi: 10.1073/pnas.2013542117

Fig. 6.

Fig. 6.

tRF changes upon LPS stimulation of murine RAW 264.7 macrophages and human CD14+ monocytes and tRF-mimic transfection. (A) LPS stimulation of RAW 264.7 murine macrophage cells induced clear morphologic changes within 18 h. Extracted RNA was subjected to size selection and cDNA synthesized from the ≤50-nt fraction alone. (Scale bar, 100 μm, magnification in the upper panel 2.5×) (B) LPS-stimulated RAW 264.7 cells show dexamethasone-suppressible elevated levels of poststroke induced tRFs. Normalized qRT-PCR values (using mmu-miR-30d-5p, mmu-let7d-5p as housekeeping transcripts) (SI Appendix, Expanded Methods), compared to unstimulated controls. Each dot represents two to four technical replicates, ANOVA with Tukey post hoc, *P < 0.05, **P < 0.01, bar graphs ±SD(lg). (C) Murine Zbp1 sequence carries an 8-nt-long fragment in the 3′UTR complementary to tRF-22-WE8SPOX52. (D) To test the miR-like mechanism of action, RAW 264.7 cells were transfected with ssRNA tRF-22-WE8SPOX mimics or negative control ssRNA, and RNA was extracted 24 h after transfection and subjected to polyA-selected RNA-seq (E) and qRT-PCR (F). (E) Long RNA-seq of cells transfected with ssRNA tRF-22-WE8SPOX52 mimics revealed significantly down-regulated expression of Z-DNA binding protein (Zbp1) as compared to negative control (NC). *P < 0.05, shown is adjusted P value of Wald test via DESeq2, bar graph ±SD. (F) qRT-PCR from an independent cell culture experiment confirmed the down-regulation of Zbp1 expression after ssRNA tRF-22-WE8SPOX52 mimic transfection (relative normalized expression using Gapdh as a housekeeping gene), *P < 0.05 one-way ANOVA, each dot represents a technical cell culture replicate, bar graph ±SD(lg). (G) MACS-sorted CD14+ cells from healthy human donors were stimulated with LPS with or without addition of nicotine and collected 6, 12, and 18 h thereafter. Extracted RNA was subjected to size selection and cDNA synthesized from the ≤50-nt fraction. Timepoints 6 h and 18 h are shown in SI Appendix, Fig. S8. (H) At 12 h after LPS stimulation, human monocytes exhibited up-regulation of poststroke induced tRFs as compared to unstimulated controls or cells treated with nicotine alone. This reaction was boosted by the addition of nicotine. Shown is relative expression (hsa-miR-30d and hsa-let7d-5p were used as housekeeping transcripts) (SI Appendix, Expanded Methods) normalized to the nonstimulated group. Each dot represents one donor. ANOVA with Tukey post hoc, *P < 0.05, **P < 0.01, ***P < 0.001 vs. nonstimulated cells; #P < 0.05, ##P < 0.01, ###P < 0.001 vs. cells upon addition of nicotine, bar graphs ±SD(lg).