Fig. 5

LncHOXA10 interacted with SNF2L. a RNA pulldown was performed and the specific band in lncHOXA10 sample was identified as SNF2L by mass spectrum. b The combination between lncHOXA10 and SNF2L was examined by RNA-pulldown and Western blot. c Diagram showing the procedure of RAP (RNA antisense purification). DNA probes targeting endogenous lncHOXA10 were grouped into Probeset #2. d RAP assay was performed and a specific band in Probeset #2 sample was identified as SNF2L. e Eluate samples of RAP assay were utilized for SDS-PAGE, followed by Western blot for SNF2L detection. f LncHOXA10 truncates were generated (upper panels) and incubated with sphere lysates. The interaction between lncHOXA10 truncates and SNF2L was examined by Western blot (lower panels). g RNA electrophoretic mobility shift assay (RNA EMSA) was performed to validate the combination of lncHOXA10 and SNF2L. h RNA immunoprecipitation (RIP) assay was performed using oncospheres and enrichment of lncHOXA10 and GAPDH mRNA were examined through realtime PCR. IgG was an antibody control. i Double FISH assay showed the co-localization of lncHOXA10 and SNF2L in oncospheres. Scale bars, 10 μm. j LncHOXA10 depleted cells were used for SNF2L, H3K4me3 and RNA polymerase II (RNA PolII) ChIP assays, and HOXA10 promoter enrichment was detected with realtime PCR. (K, L) HOXA10 promoter was cloned into PGL3 luciferase reporter plasmid, and luciferase activity was measured in lncHOXA10 silenced (k) and overexpressing cells (l). m SNF2L knockout cells were generated through CRISPR/Cas9 approach, and the knockout efficiency was confirmed by Western blot. The expression of HOXA10 was also detected by Western blot. n, o Full length (FL) lncHOXA10 or truncated (△#1) lncHOXA10 was overexpressed in WT and SNF2L KO cells, followed by sphere formation assay (N) and transwell invasion assay (o). Data are representative of three independent experiments