Fig 1: BCL9 and BCL9L are overexpressed in human HCC. aBCL9 and BCL9L expression levels were analyzed using the three public HCC data sets TCGA-LIHC, GSE22058, and GSE25097. BCL9 and BCL9L expression was significantly higher in HCC tissue than in adjacent non-tumorous liver tissue. Tukey box-and-whisker plot; two-tailed Student’s t test. b Western blot analysis of HCC cell lines (HLE, HLF, Huh7, HepG2, Hep3B, and Huh6) and immortalized liver cell lines (THLE-2, THLE-3) for BCL9, BCL9L, and ß-catenin protein expression. HepG2 and Huh6 harbor an activating ß-catenin deletion and mutation, respectively, causing an accumulation of ß-catenin
Fig 2: BCL9 and BCL9L overexpression correlates with poor survival of HCC patients. aBCL9 and BCL9L expression levels of the TCGA-LIHC cohort were divided according to histologic tumor grade and TNM classification parameters [27]. Data are represented as Tukey’s box-and-whisker plot. *p < 0.05, ***p < 0.001; 1-way ANOVA with Tukey’s multiple comparison test. bBCL9 and BCL9L expression levels of the TCGA-LIHC cohort were divided according to tumor etiology. Data are represented as Tukey’s box-and-whisker plot. *p < 0.05, **p < 0.01; one-way ANOVA with Tukey’s multiple comparison test. cBCL9 and BCL9L expression values and survival data of the TCGA-LIHC cohort were retrieved from http://www.oncolnc.org/ [16]. Patients were grouped into low (lower median) or high (upper median) expressions of BCL9 or BCL9L. Kaplan–Meier with log-rank test
Fig 3: Greater matrix stiffness increased glioma stemness by inducing BCL9L. (A) Western blotting analysis of BCL9L expression in tissues derived from the HS and LS groups. (B) Western blotting analysis of BCL9L expression in LN229 cells cultured on gels of different stiffness levels. (C) Flow cytometry analysis of CD133 expression in LN229 cells treated with negative control shRNA (LN229-NC), BCL9L shRNA #1 (LN229-KO1) or BCL9L shRNA #2 (LN229-KO2) and cultured on 16-kPa stiffness gels, and in LN229 cells treated with the control vector (LN229-Vec) or BCL9L overexpression vector (LN229-BCL9LOE) and cultured on flask dishes. (D) The proliferation of LN229-NC, LN229-KO1, LN229-KO2, LN229-Vec or LN229-BCL9LOE cells pre-cultured on 16-kPa stiffness gels, and of LN229-Vec or LN229-BCL9LOE cells cultured on flask dishes. (E) Tumor volumes were measured at various time points in mice injected with LN229-NC, LN229-KO1 or LN229-KO2 cells pre-cultured on 16-kPa stiffness gels, and in mice injected with LN229-Vec or LN229-BCL9LOE cells cultured on flask dishes. (F) The cell colonies formed by LN229-NC, LN229-KO1 or LN229-KO2 cells pre-cultured on 16-kPa stiffness gels and by LN229-Vec or LN229-BCL9LOE cells cultured on flask dishes were detected at various time points. (G) Tumorigenesis of mice subcutaneously injected with 104 LN229-NC, LN229-KO1 or LN229-KO2 cells pre-cultured on 16-kPa stiffness gels, or injected with LN229-Vec or LN229-BCL9LOE cells cultured on flask dishes. *P < 0.05, **P < 0.01, n.s. no significant difference.
Fig 4: Knockdown of BCL9L or BCL9/BCL9L interrupts Wnt/ß-catenin signaling, while knockdown of BCL9 alone is insufficient to decrease Wnt/ß-catenin activity. a Expression analysis of AXIN2 and BAMBI by qRT-PCR using the ??CT method in HLE, HepG2, and Huh6 cells after transfection with siBCL9 and/or siBCL9L. Data are represented as mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, n.s. not significant; one-way ANOVA with Dunnett’s multiple comparison test. b Effects of BCL9 and/or BCL9L knockdown on Wnt/ß-catenin signaling activity of HepG2 and Huh6 cells was determined by Wnt reporter assays. Cells were co-transfected with combinations of siBCL9 and/or siBCL9L and pGL3-OT or pGL3-OF. For normalization, the renilla luciferase vector pGL4.70 was used in all conditions. Data are represented as mean ± SD of at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ns not significant; one-way ANOVA with Tukey’s multiple comparison test
Fig 5: Knockdown of BCL9 and/or BCL9L prevents proliferation and induces apoptosis in HLE cells, but not in HepG2 or Huh6 cells. HLE, HepG2, and Huh6 cells were transfected with 5 nM siRNA against BCL9 and/or BCL9L. Cell viability and apoptosis were determined every 24 h. Data are represented as mean ± SD of at least three independent experiments. a Cell viability was analyzed by WST-1 assay and normalized to siControl (dotted line). b Apoptosis was analyzed by caspase 3/7 activity and normalized to cell viability and siControl (dotted line)
Supplier Page from Abcam for Anti-BCL9L antibody