human snai1 cdna  (Addgene inc)

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: MiR-137 and miR-34a directly target Snail and inhibit EMT, invasion and sphere-forming ability of ovarian cancer cells

doi: 10.1186/s13046-016-0415-y

Figure Lengend Snippet: MiR-137 and miR-34a modulate EMT, invasion and sphere-forming ability of OC cells through targeting Snail. MiR-137 or miR-34a inhibitor or Neg inhibitor was co-transfected into SKOV-3 cells, together with (or without) Snail siRNA. MiR-137 or miR-34a mimic or Neg mimic was co-transfected into ES-2 cells, together with (or without) Snail cDNA vector lacking the 3′-UTR region. Cell invasion assay ( a ), sphere formation assay ( b ) and Western blotting analysis of indicated proteins ( c ) in OC cells treated as described above were performed. ** P < 0.01

Article Snippet: MiRNA mimic and miRNA inhibitor for miR-137 or miR-34a (30 nM, Ambion), Snail siRNA (5 nM, Ambion) and Snail cDNA plasmids (OriGene) were transfected using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol.

Techniques: Transfection, Plasmid Preparation, Invasion Assay, Tube Formation Assay, Western Blot

Journal: The Journal of Clinical Investigation

Article Title: ZEB1 drives prometastatic actin cytoskeletal remodeling by downregulating miR-34a expression

doi: 10.1172/JCI63608

Figure Lengend Snippet: (A–D) 344SQ_miR-34a cells and 344SQ_vector cells were cultured in the presence or absence of doxycycline (Dox). (A) Q-PCR analysis of miR-34a levels. (B) Cell numbers in monolayer. Migrating (C) and invading (D) cells in Boyden chambers were photographed and counted. Scale bars: 100 μm. (E) Primary tumor weight and total lung metastases from flank tumors in syngeneic mice (mean ± SD, n = 5). P values were determined by 2-tailed Student’s t test. (F) MDA-MB-231 cells were transiently transfected with a random sequence miR precursor molecule control or with pre–miR-34a precursor. Shown are Q-PCR analysis of miR-34a levels, expressed relative to control transfectants (set at 1.0), and migration and invasion assays in Boyden chambers. (G and H) Q-PCR analysis of epithelial (Cdh1 and Scrib) and mesenchymal (Cdh2 and Vim) markers and their transcriptional regulators (Zeb1, Zeb2, Snai1, Snai2, and Twist1) in 344SQ_vector and 344SQ_miR-34a cells (G) and in MDA-MB-231 cells transiently transfected with pre-miR control or pre–miR-34a precursor (H). Results are expressed relative to control transfectants (set at 1.0). Data are mean ± SD (n = 3). *P < 0.01. (I) Kaplan-Meier analysis of 3 independent cohorts of lung cancer patients (33–35), comparing the differences in risk between tumors with high (>0) or low (<0) scores (36), reflecting the presence or absence, respectively, of overlap with the murine miR-34a signature. P values from log-rank (differences between arms) and univariate Cox (gene signature score as a continuous variable) tests are shown.

Article Snippet: Human SNAI1 cDNA (catalog no. 16218), murine Twist1 cDNA (catalog no. 1783; Addgene), murine Arhgap1 cDNA, and murine Arhgap1 shRNA (Origene) were purchased.

Techniques: Plasmid Preparation, Cell Culture, Transfection, Sequencing, Control, Migration

Journal: The Journal of Clinical Investigation

Article Title: ZEB1 drives prometastatic actin cytoskeletal remodeling by downregulating miR-34a expression

doi: 10.1172/JCI63608

Figure Lengend Snippet: (A–D) miR-34a repressed TGF-β–induced invasion in 3D Matrigel cultures. 344SQ_vector cells formed polarized epithelial spheres (A) that became hyperproliferative and invasive in the presence of TGF-β (B). 344SQ_miR-34a cells formed polarized epithelial spheres (C) that did not become invasive in the presence of TGF-β (D). Shown are light (left) and fluorescent (right) microscopic images of structures formed after 10 days in Matrigel containing doxycycline in the presence or absence of TGF-β (10 ng/ml). Blue, Topro-3; red, anti–α6 integrin; green, anti–ZO-1. Scale bars: 200 μm (light); 50 μm (fluorescent). (E) miR-34a did not abrogate TGF-β–induced EMT. Q-PCR analysis of epithelial markers (Cdh1, Scrib, and Crb3) and mesenchymal markers (Cdh2 and Vim) and their transcriptional regulators (Zeb1, Zeb2, Snai1, and Snai2) in 344SQ_vector and 344SQ_miR-34a cells after 10 days in Matrigel cultures containing doxycycline in the presence or absence of TGF-β. Results are expressed relative to empty vector transfectants treated without TGF-β (set at 1.0). Data are mean ± SD (n = 3).

Article Snippet: Human SNAI1 cDNA (catalog no. 16218), murine Twist1 cDNA (catalog no. 1783; Addgene), murine Arhgap1 cDNA, and murine Arhgap1 shRNA (Origene) were purchased.

Techniques: Plasmid Preparation