Article Title: TAZ induces lung cancer stem cell properties and tumorigenesis by up-regulating ALDH1A1
Figure Lengend Snippet: Establishment of an in vivo xenograft TAZ-overexpressing mouse model ( A ) Overexpression of TAZ-S89A in mouse immortalized lung epithelial cells (E10) caused highly malignant NSCLC tumor formation. Tumorigenesis assay was performed by subcutaneously injecting about 3 × 10 6 E10-TAZ-S89A cells into two-sides of nude mice. E10-TAZ-S89A cells caused large tumors (i) in two weeks. Two week later, the tumors were fixed, sectioned, and subjected to H E staining and IHC. H E staining with antibody incubation of E10-TAZ-S89A tumor section showed high-grade, poorly-differentiated carcinoma (ii). IHC staining for TAZ expression using TAZ antibody (1:300 dilution, BD Biosciences) showed that TAZ was overexpressed in the nuclei (iii). Pictures were taken using TE200 Nikon Inverted Fluorescent Microscope (Nikon, Montreal, Canada) as 20× magnification. ( B ) Western blot analysis of TAZ-S89A expression. Ten μg of cell lysate extracted from E10-TAZS89A-P or E10-TAZ-S89A-T cells in the absence (−) or presence (+) of Dox were subjected to WB analysis using anti-TAZ (1:1000, BD Biosciences) and anti β-actin (1:10,000 Sigma, Oakville, Canada) antibodies. β-actin was used as an internal loading control. ( C ) Cell proliferation assay. Triplicates of 1.5 × 10 4 E10-TAZ-S89A-P or tumorigenic E10-TAZ-S89A-T cells were seeded into each well of 12-well plates, then untreated (−) or treated (+) with Dox. Cell numbers were counted on days 1, 2, 3, 4, 5, and 6 after plating. The experiments were repeated at least three times. Data are shown as means ± S.D. “*” represent significant difference. ( D – E ) Soft agar assay. Triplicates of 2×10 3 TAZ-S89A-P or E10-TAZ-S89A-T cells were mixed with 0.4% agarose in growth media and overlaid on 0.8% agarose in 6-well plates, followed by incubation in the absence or presence of Dox (2 μg/ml). Colony formation was examined after culturing for 18 days (D). Colony numbers were counted by Bio-Rad Gel Doc System (Bio-Rad, Mississauga, Canada). Data are shown as means ± S.D. “*” represent significant difference ( P
Article Snippet: Pictures were taken using TE200 Nikon Inverted Fluorescent Microscope at 40× magnification.
Techniques: In Vivo, Over Expression, Mouse Assay, Staining, Immunohistochemistry, Incubation, Expressing, Microscopy, Western Blot, Proliferation Assay, Soft Agar Assay