Fig. 7

Identification of downstream target for the SLCO4A1-AS1/TOX4 axis. A Screening of candidate genes using intersection of two pairs of RNA-seq data (H1299-mock vs. H1299-TOX4 and H1299-mock vs. H1299-SLCO4A1-AS1). The eight protein coding candidate genes were upregulated in TOX4-overexpressing cells and downregulated in SLCO4A1-AS1-overexpressing cells. B The fold change of candidate genes was validated in the TOX4-overexpressing cells and SLCO4A1-AS1-overexpressing H1299 cells by RT-qPCR assay. CNTSR1 mRNA and protein expression was detected in TOX4-expressing H1299 cells by RT-qPCR and western blotting. RT-qPCR data are presented as mean ± SD (***p < 0.001). DNTSR1 mRNA and protein expression was detected in SLCO4A1-AS1-expressing H1299 cells by RT-qPCR and western blotting. RT-qPCR data are presented as mean ± SD (***p < 0.001). E Immunohistochemistry staining was performed to evaluated NTSR1 expression in mouse tail vein lung metastasis model and scale = 50 μm. F Chromatin immunoprecipitation (ChIP) was performed with an antibody against TOX4 or IgG in H1299-TOX4 and A549-TOX4 cells. The enriched NTSR1 promoter was purified and quantified by RT-qPCR using primers targeting the NTSR1 promoter region (inset). The data of RT-qPCR are presented as mean ± SD (***p < 0.001). G ChIP was performed to assess the effect of SLCO4A1-AS1 overexpression on the interaction of TOX4 and NTSR1 promoter. The NTSR1 promoter was quantified by RT-qPCR using primers targeting the NTSR1 promoter region. The data of RT-qPCR are presented as mean ± SD (***p < 0.001)