Figure 1: LncBRM is highly expressed in HCC tumours and liver CSCs.

(a) The indicated lncRNAs were silenced using pSiCoR lentivirus, followed by sphere formation assays. *, **, for Hep3B cells, Hep3B shlncBRM versus Hep3B shCtrl; #, ##, for Huh7 cells, Huh7 shlncBRM versus Huh7 shCtrl. (b) Total RNAs were extracted from peri-tumour (P) and tumour (T) tissues, followed by northern blotting. ACTB served as a loading control. (c) Primary HCC samples were prepared for examination of lncBRM expression using RT–qPCR. aHCC, advanced HCC; eHCC, early HCC. (d) LncBRM was detected by in situ hybridization. LncBRM highly expressed cells (middle panel) and lncBRM photon intensity (right panel) were calculated by Image-Pro Plus 6 and shown as scatter plot (means±s.e.m.). Scale bars, 100 μm. (e) Liver CSCs (CD13⁺CD133⁺) and non-CSCs (CD13⁻CD133⁻) were sorted from HCC samples, followed by detection of lncBRM using RT–qPCR (left panel). Oncospheres and non-spheres derived from HCC primary tumour cells were analysed similarly. Expression levels of lncBRM were normalized to that of non-tumour sample 17 as a baseline level. (f) lncBRM was examined in oncospheres and non-spheres with northern blotting. N, non-sphere; S, sphere. (g) Non-spheres and spheres were stained with lncBRM probes and CD13 antibody for confocal microscopy. Scale bars, 20 μm. (h) Nucleocytoplasmic fractionation of oncosphere cells was performed and followed by immunoblotting (upper panel) and RT–qPCR (lower panel). U1 RNA served as a nuclear location control and NKILA was used as a cytoplasmic location control. Data are shown as means±s.d. Two tailed Student’s t-test was used for statistical analysis; *P<0.05, **P<0.01, ***P<0.001; ^#P<0.05, ^##P<0.01. Data are representative of at least three independent experiments.