Extended Data Fig. 9: SND1 interacts with TSG101 to transport m6A-RNAs into extracellular vesicles.
a, EV RNAs from Alkbh5-KO KPC cells significantly enhanced axonogenesis of sympathetic neurons compared with Alkbh5-WT (n = 5 independent experiments). b, RNA-seq analysis showing expression-upregulated axonogenesis-related genes in neurons after transfection with EV RNAs from KPC cells with or without Alkbh5 KO (3 biological repeats for each setting). c, qPCR assays verify the expression upregulation of the genes in b (n = 5 independent experiments). d, Knockdown Bdnf in DRG neurons partially suppressed the axonogenesis induced by EV RNAs from Alkbh5-KO KPC cells (n = 5 independent experiments). e, Western blot analysis shows that Alkbh5 KO had no significant effect on Snd1 level in KPC cells compared with Alkbh5 WT. f, Immunoprecipitation and immunoblot analysis show that Alix could not interact with Snd1, Hnrnpa1 and Hnrnpa2b1. g and h, Reciprocal immunoprecipitation assays showing the interaction between SND1 and TSG101 in PANC-1 cells. i, Immunofluorescence assays showing that endogenous Snd1 and Tsg101 colocalized in cytoplasm (white arrows) of KPC cells. j, Proteinase protection assays showing that Snd1 and Tsg101 were degraded by proteinase K only after destruction of the membrane structure using Triton X-100 treatment. k and l, Tsg101 Knockdown (KD) decreased Snd1 levels in EVs (k) but not in cells (l). m, Immunofluorescence assays showing that TSG101 and SND1 were co-localized (white arrows) in multivesicular bodies (MVBs) in RAB5AQ79L-transfected ALKBH5-KO MiaPaCa-2 cells. Scale bars, 20 μm (a), 50 μm (d), 5 μm in left picture and 2 μm in right zoom-in picture (i), 2 μm in left picture and 1 μm in right zoom-in picture (m). All bar chart data are the means ± s.e.m., with P by two-tailed Student’s t-test in a–d.
