Fig 1: Enforced miR-424-5p expression or silencing BCL9L suppresses gemcitabine resistance, EMT, stemness, and Wnt/ß-Catenin signaling activation in resistant CCA cells.A HuCCT1-Gem and SNU-245-Gem cells were transduced with sgBCL9L-1, sgBCL9L-2, or sgNC, then BCL9L expression was evaluated by western blot. B HuCCT1-Gem and SNU-245-Gem cells were transduced sgBCL9L-1, sgBCL9L-2, sgNC, miR-424-5p or miR-ctrl, then cells were seeded in 96-well plates (5000 cells/well) and exposed to 0, 1.25, 2.50, 5.0, 6.25, 10, 20, 40, 80, 160, and 320 nM gemcitabine for 6 d, and assayed for cell viability. C–J HuCCT1-Gem and SNU-245-Gem cells were transduced sgBCL9L-1, sgBCL9L-2, sgNC, miR-424-5p or miR-ctrl, the cells were used for sphere formation assay (C, D), transwell cell migration (E, F) and invasion (G, H) assay, and western blot analysis of indicated genes in Wnt/ß-Catenin pathway (I, J). Scale bars = 100 µm for sphere formation assay, and scale bars = 50 µm for transwell cell migration and invasion assay. ***P = 0.05.
Fig 2: Silencing LINC00665 represses Wnt/ß-Catenin signaling and BCL9L expression in gemcitabine-resistant CCA cells.A, B expression of indicated genes in Wnt/ß-Catenin pathway (A) downstream targets (B) was evaluated by western blot or qRT-PCR. C HuCCT1-Gem and SNU-245-Gem cells were transduced with sh-LINC00665-1, sh-LINC00665-2, or sh-ctrl, then the expression of indicated genes in Wnt/ß-Catenin pathway was evaluated by western blot. D immunofluorescent staining showed the subcellular location of ß-Catenin in HuCCT1-Gem and SNU-245-Gem cells after transducing with sh-LINC00665-1, sh-LINC00665-2, or sh-ctrl. DAPI was used to stain the nucleus. Scale bars = 50 µm. E, F expression of BCL9L in HuCCT1-Gem and SNU-245-Gem cells transduced with sh-LINC00665-1, sh-LINC00665-2, or sh-ctrl was evaluated by qRT-PCR (E) and western blot (F). ***P = 0.05.
Fig 3: LINC00665 regulates BCL9L expression by acting as a molecular sponge for miR-424-5p.A the putative binding sites for LINC00665 and miR-424-5p were shown. B HuCCT1 and SNU-245 cells transduced with LINC00665 expression lentivirus or EV, then relative miR-424-5p expression was evaluated by qRT-PCR. C HuCCT1-Gem and SNU-245-Gem cells were transduced with sh-LINC00665-1, sh-LINC00665-2, or sh-ctrl, then relative miR-424-5p expression was evaluated by qRT-PCR. D HEK293T cells transduced with wt LINC00665 or mt LINC00665 expression pMIR-REPORT vector were used for luciferase reporter assay. Cells were co-transfected with miR-424-5p or miR-ctrl expression vector as indicated. E Relative expression of LINC00665 in the bound RNAs pulled down by biotinylated wild-type (wt) miR-424-5p, mutant (mt) miR-424-5p, and non-targeting control (NC) were evaluated by qRT-PCR. F the putative binding sites for BCL9L and miR-424-5p were shown. G HuCCT1-Gem and SNU-245-Gem cells transduced with miR-424-5p expression lentivirus or miR-ctrl, then relative BCL9L expression was evaluated by qRT-PCR. H HEK293T cells transduced with wt BCL9L or mt BCL9L expression pMIR-REPORT vector were used for luciferase reporter assay. Cells were co-transfected with miR-424-5p or miR-ctrl expression vector as indicated. I Relative expression of BCL9L in the bound RNAs pulled down by biotinylated wild-type (wt) miR-424-5p, mutant (mt) miR-424-5p, and non-targeting control (NC) were evaluated by qRT-PCR. J HuCCT1 and SNU-245 cells were transduced with LINC00665 expression lentivirus, EV, miR-424-5p, or miR-ctrl as indicated, then expression of BCL9L was evaluated by western blot. ***P = 0.05.
Supplier Page from Abcam for Anti-BCL9L antibody [3B9C1]