Fig. 4

FMR1-AS1 could be packaged into exosomes and activates TLR7- NFκB-c-Myc signaling. a Outline of the ChIRP-MS workflow. Briefly, cells are crosslinked by 3% formaldehyde for 30 min and solubilized by sonication. FMR1-AS1 are pulled out by biotinylated antisense oligos, and associated proteins are eluted with free biotin. Each size fraction is subjected to LC/MS-MS identification. b Over 60% of FMR1-AS1 was retrieved from the cell by ChIRP, while no Gapdh was detected. RNase treatment eliminates FMR1-AS1 transcripts prior to pull-down. c FMR1-AS1 associated proteins that were identified as exosomal proteins by ExoCarta database. d Functional classification of FMR1-AS1 ChIRP-retrived proteins. e, f Identification of PRDX2 and TLR7 in both even/odd probe group retrieved proteins using Mass spectrometry. g Validation of ChIRP-enriched proteins by immunoblotting using TLR7, hnRNPK, PRDX1, PRDX2, ECM1 and β-actin antibodies. h Western-blotting validation of TLR7 in FMR1-AS1 pulldown protein extractions. i, j RNA-immunoprecipitation (RIP) experiments were performed using TLR2/3/4/7/8 and IgG antibodies to immunoprecipitate and a primer to detect FMR1-AS1 in ECA-109 and KYSE-150 cells. k NFκB activities in FMR1-AS1-upregulated, FMR1-AS1-downregulated and respective control ESCC cells, examined by EMSA. l NFκB activity FMR1-AS1-upregulated, FMR1-AS1-downregulated and respective control ESCC cells, examined by luciferase reporter plasmid with FMR1-AS1 promoter (mean ± SD, n = 6, *p < 0.05). m Western blotting showing nuclear p65, p50 and c-Myc levels in FMR1-AS1-upregulated, FMR1-AS1-downregulated and respective control ESCC cells. n NFκB activity in FMR1-AS1-upregulated, FMR1-AS1-downregulated and respective control ESCC cells, examined by luciferase reporter plasmid with MYC gene promoter (mean ± SD, n = 6, *p < 0.05)