Fig. 2

Biological characterization of FMR1-AS1. a Ribosome occupancy at the FMR1 and FMR1-AS1 locus. The green peaks indicate reads density that mapped at the region. b RNA fluorescence in situ hybridization to localize FMR1-AS1. c Relative abundance of FMR1-AS1 transcript in cytoplasm, nucleus and chromatin in female ESCC cells. GAPDH, XIST and LincRNA-p21 were used as controls respectively. d Expression of FMR1-AS1 in ESCC cells induced by TNF-α with or without NF-kB inhibition by sc-3060 or JSH-23 (mean ± SD, n = 5, *p < 0.01). e Chromatin immunoprecipitation showing p65, p50 and RNAP II occupancy at the FMR1-AS1 locus in ESCC cells. Locations of amplicons (C1–C4) are indicated in the scheme above. Values represent the enrichment of bound protein fractions relative to input (mean ± SD, n = 3). f Luciferase reporter assay in ESCC cells induced by TNF-α with or without NF-kB inhibition by sc-3060 or JSH-23, and the reporter constructs expressing the luciferase gene under FMR1-AS1 promoter segment (mean ± SD, n = 5, *p < 0.01). g, h sXCI detection assays, based on HpaII digested genomic DNA PCR on the highly polymorphic CAG repeat-region of androgen receptor (AR), which amplifies the undigested inactive X chromosome only. i sXCI frequency in the FMR1-AS1 high/low groups of female ESCC tissues (P_χ² < 0.001). j FMR1-AS1 expression in female ESCC patients with or without sXCI (CR ≥ 3). k, l Correlation analysis on the expression level of FMR1-AS1, XIST (R² = 0.012, P < 0.4585) and TSIX (R² = 0.3353, P < 0.001)