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. 2020 Sep 24;219(10):e201912074. doi: 10.1083/jcb.201912074

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© 2020 Wozniak et al.

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Figure 3. — Identification of a cRILP-associated RBP. (A) Knockdown of candidate RBPs showing that shRNA to FMR1 blocks cRILP-associated miR-155 loading into exosomes after inflammasome activation by LPS and ATP, while not affecting miR-19b. TRC, empty vector control. (B) Western blot analysis of exosomes isolated from LPS/ATP-treated THP-1 cells shows that FMR1 is present in exosomes. These vesicles also contain cRILP and the RILP-associated GTPase, Rab7. Ut, untreated. (C) cRILP redistributes FMR1 throughout the cellular periphery. HeLa cells were transfected with HA-FMR with or without cRILP-Flag or ncRILP-Flag. HA-FMR1 (green) localizes loosely near the nucleus. Coexpression of cRILP (red) redistributes this localization toward the cell membrane, while ncRILP sequesters FMR1 in the mitotic center. Pearson’s coefficients for n = 10–15 cells. (D) cRILP is more tightly associated with FMR1 and the ESCRT-related protein Hrs than is ncRILP. cRILP-Flag or ncRILP-Flag was expressed in HeLa cells and immunoprecipitated (IP) using Flag-tagged magnetic beads. (E) FMR1 associates with Hrs, and this association is increased when cRILP is expressed. HA-FMR1 was expressed alone or with cRILP-Flag and immunoprecipitated using HA-tagged magnetic beads. (F) cRILP relocalizes Hrs. HeLa cells were transfected with Hrs-RFP with or without cRILP-Flag or ncRILP-Flag. Hrs localizes in larger structures throughout the cytoplasm. Coexpression of cRILP (green) redistributes this localization into smaller puncta, extending toward the cell membrane. ncRILP has no effect on the distribution of Hrs. Pearson’s coefficients for n = 12–15 cells. (G) cRILP induces the association between Hrs and FMR1. HeLa cells were transfected with Hrs-RFP and HA-FMR1 with or without cRILP-Flag or ncRILP-Flag. Coexpression of cRILP (green) enhances the interaction between Hrs and FMR1, while no association between the two is seen when ncRILP is expressed. Pearson’s coefficients for n = 10–15 cells. (H) miR-155 is slightly enriched in exosomes when FMR1 is overexpressed. This enrichment can be enhanced by treatment with LPS/ATP or blocked by expressing ncRILP. Data are shown as mean ± SD; ns, not significant; *, P ≤ 0.05 for n = 3–5.