human snai1  (Addgene inc)

Journal: Cancers

Article Title: The Epithelial–Mesenchymal Transcription Factor SNAI1 Represses Transcription of the Tumor Suppressor miRNA let-7 in Cancer

doi: 10.3390/cancers13061469

Figure Lengend Snippet: Growth factor treatment results in increased SNAI1, increased stemness and decreased let-7 expression. MCF-7, PANC-1 were treated with TGFB1; OVCAR8, OVSAHO were treated with EGF. Levels of control group (cells treated with vehicle control) were normalized to 1. Values for RT-qPCR are shown on a log scale. ( A , B ) RT-qPCR analysis for mRNA expression level of SNAI1 ( A ) and of stemness markers (( B ), LIN28A, NANOG, POU5F1 and HMGA2 ). ( C , D ) The quantification of Western blot analysis for protein expression of SNAI1 ( C ) and HMGA2 ( D ). ( E ) Left panel: The quantification of number of spheroids per 3000 cells (both first passage and second passage) is shown. Right panel: Phase contrast images of spheroids formed from cells (first passage) as indicated are presented. In each panel, the spheroids formed from control group are presented on the left, those from the treatment group are on the right. Scale bar = 100 μm ( F ) RT-qPCR analysis for let-7 miRNA ( let-7a , let-7e , let-7g and let-7i) expression. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: The cDNA of human SNAI1 was subcloned from Flag-Snail WT (Addgene 16218, Watertown, MA, USA) into pWZL-Blast-GFP (Addgene 12269) after removing GFP using BamH1/Xho1.

Techniques: Expressing, Control, Quantitative RT-PCR, Western Blot

Journal: Cancers

Article Title: The Epithelial–Mesenchymal Transcription Factor SNAI1 Represses Transcription of the Tumor Suppressor miRNA let-7 in Cancer

doi: 10.3390/cancers13061469

Figure Lengend Snippet: SNAI1 overexpression results in increased stemness and decreased let-7 expression. Cell lines were transduced with the retroviral expression vector pWZL-Snail or empty vector, pWZL-Empty, in cell lines MCF-7, PANC-1, OVCAR8 and OVSAHO. Levels of control group (cells transduced with pWZL-Empty) were normalized to 1. Values for RT-qPCR are shown on a log scale. ( A , B ) RT-qPCR analysis for mRNA expression of SNAI1 ( A ) and of stemness markers LIN28A , NANOG , POU5F1 and HMGA2 ( B ). ( C , D ) The quantification of Western blot analysis for protein expression of SNAI1 ( C ) and HMGA2 ( D ). ( E ) The quantification of number of spheroids formed per 3000 cells (both first passage and second passage) as indicated. ( F ) RT-qPCR analysis for let-7 miRNA ( let-7a , let-7e , let-7g and let-7i) expression. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001

Article Snippet: The cDNA of human SNAI1 was subcloned from Flag-Snail WT (Addgene 16218, Watertown, MA, USA) into pWZL-Blast-GFP (Addgene 12269) after removing GFP using BamH1/Xho1.

Techniques: Over Expression, Expressing, Transduction, Retroviral, Plasmid Preparation, Control, Quantitative RT-PCR, Western Blot

Journal: Cancers

Article Title: The Epithelial–Mesenchymal Transcription Factor SNAI1 Represses Transcription of the Tumor Suppressor miRNA let-7 in Cancer

doi: 10.3390/cancers13061469

Figure Lengend Snippet: SNAI1 knockdown reverses stemness and restores let-7 expression. Mesoporous silica nanoparticles coated with hyaluronic acid (HA-MSN) were used to deliver siRNA (siSnail and siControl) in MCF-7, PANC-1, OVCAR8, and OVSAHO. Levels of control group (cells treated with siControl) were normalized to 1. Values for RT-qPCR are shown on a log scale. Samples were harvested after 24 h (MCF-7, OVCAR8 and OVSAHO) or 72 h (PANC-1). ( A , B ) RT-qPCR analysis for mRNA expression of SNAI1 ( A ) and of stemness markers LIN28A , NANOG , POU5F1 and HMGA2 ( B ). ( C , D ) The quantification of Western blot analysis for protein expression of SNAI1 ( C ) and HMGA2 ( D ). ( E ) The quantification of number of spheroids formed per 3000 cells (both first passage and second passage) as indicated. ( F ) RT-qPCR analysis for let-7 miRNA ( let-7a , let-7e , let-7g , and let-7i ) expression. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001

Article Snippet: The cDNA of human SNAI1 was subcloned from Flag-Snail WT (Addgene 16218, Watertown, MA, USA) into pWZL-Blast-GFP (Addgene 12269) after removing GFP using BamH1/Xho1.

Techniques: Knockdown, Expressing, Control, Quantitative RT-PCR, Western Blot

Journal: Cancers

Article Title: The Epithelial–Mesenchymal Transcription Factor SNAI1 Represses Transcription of the Tumor Suppressor miRNA let-7 in Cancer

doi: 10.3390/cancers13061469

Figure Lengend Snippet: SNAI1 knockdown reduces stemness in patient-derived cells in vitro. HA-MSN were used to deliver siRNA (siSnail and siControl) in PDX cells in vitro. Levels of control group (cells treated with siControl) were normalized to 1. Values for RT-qPCR are shown on a log scale. ( A , B ) RT-qPCR analysis for mRNA expression of SNAI1 ( A ) and of stemness markers LIN28A , NANOG , POU5F1 and HMGA2 ( B ). ( C , D ) The quantification of Western blot analysis for protein expression of SNAI1 ( C ) and HMGA2 ( D ). ( E ) The quantification of number of spheroids per 3000 cells formed from PDX6 in vitro. ( F ) RT-qPCR analysis for let-7 miRNA ( let-7a , let-7e , let-7g, and let-7i ) expression. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: The cDNA of human SNAI1 was subcloned from Flag-Snail WT (Addgene 16218, Watertown, MA, USA) into pWZL-Blast-GFP (Addgene 12269) after removing GFP using BamH1/Xho1.

Techniques: Knockdown, Derivative Assay, In Vitro, Control, Quantitative RT-PCR, Expressing, Western Blot

Journal: Cancers

Article Title: The Epithelial–Mesenchymal Transcription Factor SNAI1 Represses Transcription of the Tumor Suppressor miRNA let-7 in Cancer

doi: 10.3390/cancers13061469

Figure Lengend Snippet: SNAI1 knockdown in vivo reduces stemness gene expression and tumor burden. HA-MSN were used to deliver siRNA (siSnail and siControl) via IV injection to orthotopic PDX in vivo. Tumor samples were harvested and analyzed at necropsy. Levels of control group (cells treated with siControl) were normalized to 1. Values for RT-qPCR are shown on a log scale. ( A , B ) RT-qPCR analysis for mRNA expression of SNAI1 ( A ) and of stemness markers LIN28A , NANOG , POU5F1 and HMGA2 ( B ), in tumors. ( C , D ) The quantification of Western blot analysis for protein expression of SNAI1 ( C ) and stemness markers LIN28A and HMGA2 ( D ), in tumors. ( E ) RT-qPCR analysis for let-7 miRNA ( let-7a , let-7e , let-7g and let-7i ) expression in tumors. ( F ) Left panel: Representative images of xenograft mice. siControl (upper) and siSnail knockdown (lower). Right panel: Quantitation of bioluminescence at primary sites over six weeks. X axis, days; Y axis, total flux in photons/second relative to day 1. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: The cDNA of human SNAI1 was subcloned from Flag-Snail WT (Addgene 16218, Watertown, MA, USA) into pWZL-Blast-GFP (Addgene 12269) after removing GFP using BamH1/Xho1.

Techniques: Knockdown, In Vivo, Gene Expression, IV Injection, Control, Quantitative RT-PCR, Expressing, Western Blot, Quantitation Assay

Journal: Cancers

Article Title: The Epithelial–Mesenchymal Transcription Factor SNAI1 Represses Transcription of the Tumor Suppressor miRNA let-7 in Cancer

doi: 10.3390/cancers13061469

Figure Lengend Snippet: SNAI1 represses let-7 promoters. ( A ) Schematic representation of the promoter region of let- 7 i (upper) and reporter constructs used in luciferase assays (lower diagrams). E1, E2, E3: E-boxes (sequence: CANNTG); MU: Mutated E-boxes; TSS: Transcription start site ( B ) For luciferase assays, HEK293T cells were co-transfected with two plasmids: (1) let-7 promoter luciferase (let-7i, let-7a1/d/f1, let-7a3/b, let-7c) , and (2) either SNAI1 (constitutively expressed, gray bars) or empty vector (black bars). Luminescence activity was measured 48 h thereafter. ( C ) HEK293T cells were co-transfected with either let-7i lucB or let-7i mlucB with or without SNAI1. Luminescence was measured 24 h later. * p < 0.05; ** p < 0.01.

Article Snippet: The cDNA of human SNAI1 was subcloned from Flag-Snail WT (Addgene 16218, Watertown, MA, USA) into pWZL-Blast-GFP (Addgene 12269) after removing GFP using BamH1/Xho1.

Techniques: Construct, Luciferase, Sequencing, Transfection, Plasmid Preparation, Activity Assay

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