Fig. 5

SUMOylated hnRNPA2B1 activates ALIX to trigger circTLCD4-RWDD3 loading into EVs in ESCRT-III-dependent manner. a Silver staining after co-IP assay to detect SUMOylated hnRNPA2B1-interacting proteins. b Western blotting analysis to assess the interaction between hnRNPA2B1 and ALIX in circTLCD4-RWDD3-overexpressing A549 cells with or without mutating K108 residue of hnRNPA2B1. c Representative confocal images of PLA between hnRNPA2B1 and ALIX (red signal) in circTLCD4-RWDD3-overexpressing A549 cells with or without mutating K108 residue of hnRNPA2B1. Scale bars, 5 μm. d Schematic illustrating conserved SIM in ALIX sequence. e Western blotting analysis after co-IP assays to investigate the interaction between hnRNPA2B1 and ALIX after mutating K108 residue of hnRNPA2B1 or SIM in ALIX. f Confocal images of PLA between hnRNPA2B1 and ALIX (red signal) in A549 cells with hnRNPA2B1^K108R or mutation of the SIM in ALIX. Scale bars, 5 μm. g Western blotting analysis after co-IP assays to assess the activation of ALIX in A549 cells with hnRNPA2B1^K108R or mutation of the SIM in ALIX. h Representative confocal images of the co-localization between hnRNPA2B1 and the indicated subcellular markers in circTLCD4-RWDD3-overexpressing A549 cells with or without SENP3 treatment. Scale bars, 5 μm. i Pearson correlation coefficients were calculated from the A549 cells expressing hnRNPA2B1 and the indicated subcellular markers. j qRT-PCR analysis for circTLCD4-RWDD3 expression in EVs derived from circTLCD4-RWDD3-overexpressing A549 cells with or without knocking down ALIX. k, l Western blotting analysis for hnRNPA2B1 expression in circTLCD4-RWDD3-overexpressing A549 cells with or without knocking down ALIX (k) or in EVs derived from the indicated A549 cells (l). m qRT-PCR analysis for circTLCD4-RWDD3 expression in EVs derived from A549 cells with hnRNPA2B1^K108R or mutation of the SIM in ALIX. n Western blotting analysis of hnRNPA2B1 expression in EVs derived from A549 cells with hnRNPA2B1^K108R or mutation of the SIM in ALIX. o Detection of intracellular co-localization of ALIX and CHMP4B in A549 cells with hnRNPA2B1^K108R or mutation of the SIM in ALIX. Scale bars, 5 μm. p Pearson correlation coefficients were calculated from the indicated A549 cells expressing ALIX and CHMP4B. q qRT-PCR analysis for circTLCD4-RWDD3 expression in EVs derived from A549 cells with mutation of the SIM in ALIX or knocking down CHMP4B. r Western blotting analysis of hnRNPA2B1 expression in EVs derived from A549 cells with mutation of the SIM in ALIX or knocking down CHMP4B. The statistical difference was assessed by the unpaired Student’s t-test in (j); and one-way ANOVA followed by Dunnett tests in (i, m, p, and q). Error bars show the SD from three independent experiments. *P < 0.05; **P < 0.01