Fig 1: shRNA-mediated decrease in LGR5 expression results in inhibition of cell migrationTPC-1 clonal cells harboring stable expression for either non-targeting (Ctrl) shRNA A. or targeting human LGR5 shRNA B. were subjected to a wound healing assay for 24 and 48 h. Representative images are shown. Leading edges of repairing cell monolayers are indicated by red bars. Briefly, cells were grown to 80% confluence at which time cells were removed with a P200 tip. Images were then acquired at 0 h, 24 h, and 48 h and analyzed by Olympus software to measure ability of the cells to repair the wound. C. Cellular migration was quantified and represented graphically with results expressed as mean ± SEM of three independent measurements. Significance p < 0.05*.
Fig 2: TPC-1 cells demonstrate native Wnt/β-catenin signaling with pharmacologic manipulationA. TPC-1 cells were treated either with increasing concentrations of recombinant RSPO1 for 24, 48 and 72 hours and LGR5 expression was examined by qRT-PCR. Results represent the mean of three independent experiments expressed as fold over vehicle (VEH) control for each time point. *P < 0.05; **P ≤ 0.01 B. TPC-1 cells were transfected with TOPFLASH or FOPFLASH as indicated and treated with either vehicle (DMSO), 0.5 uM, or 2.0 uM niclosamide for either 24 or 48 hours. Renilla luciferase plasmid was as normalization control and results are expressed as mean fold-change from baseline (vehicle; n = 2). C. TPC-1 cells were treated with compounds to either inhibit (niclosamide) or activate (RSPO) the Wnt pathway. TPC-1 cells were treated either with recombinant RSPO1 or niclosamide at the indicated doses for 48 hours and mRNA expression levels for LGR5, Axin2 and β-catenin were quantified. Values were normalized to β-actin housekeeping gene expression and expressed as the fold over DMSO control (mean ± SEM; n = 3). Significance p < 0.05**.
Fig 3: LGR5 and RSPO are induced in human thyroid cancerTotal RNA was isolated from Nthy-ori 3–1, a normal human thyroid cell line, and PTC cell lines TPC-1 and KTC-1. Expression of A. LGR5 and B. RSPO1–3 were quantified by qRT-PCR. Results are normalized to β-actin gene expression and expressed as mean fold-over Nthy-ori-3-1 for each gene (n = 3 independent isolations). C. LGR5 mRNA expression in normal frozen human tissue samples (n = 4 samples) and papillary thyroid cancer samples (n = 6 samples). D. Representative immunohistochemistry confirming elevated protein expression of both LGR5 and RSPO2 in patients with pathologically characterized PTC relative to normal thyroid tissue (magnification x20). Significance p < 0.05**.
Fig 4: LGR5 tumoral positivity is associated with the BRAFV600E mutationA. Expression of LGR5 in frozen normal human thyroid tissue (n = 4 normal samples) and in BRAFV600E tumor negative and positive samples (n = 3 samples/group). B. Representative IHC tumor samples for LGR5 (brown DAB staining) in BRAFV600E negative patients (1–3) and BRAFV600E positive patients (4–6). C. In a cohort of 33 human patients with known BRAFV600E mutational status, LGR5 positivity was strongly associated with the BRAFV600E mutation (p = 0.005**). In those who were BRAFV600E positive, 92% were also LGR5 positive (black) while 8% were LGR5 negative (grey). Significance p < 0.05**.
Supplier Page from OriGene Technologies for LGR5 Human shRNA Plasmid Kit (Locus ID 8549)