flag snai1  (Addgene inc)

Journal: Blood Advances

Article Title: Frequent mutations of FBXO11 highlight BCL6 as a therapeutic target in Burkitt lymphoma

doi: 10.1182/bloodadvances.2021005682

Figure Lengend Snippet: FBXO11 tumor-derived mutants show impaired ability to induce BCL6 and SNAIL degradation. (A) Representative western blot for FBXO11 and BCL6 protein expression in HEK-293T cells transfected with BCL6 in combination with GFP, WT FBXO11, or FBXO11 mutants at the indicated time points after treatment with CHX. FBXO11 mutants were detected by anti-FBXO11 antibody, and BCL6 was detected by anti-BCL6 antibody. β-actin was used as a loading control. GFP was used for transfection efficiency. One representative experiment of 2 independently performed experiments is shown. (B) Impaired degradation of SNAIL by FBXO11 mutants. Representative western blot for FBXO11 and SNAIL protein expression in HEK-293T cells transfected with SNAIL and WT FBXO11 or the indicated FBXO11 mutants at the indicated time points after treatment with CHX. FBXO11 mutants were detected by anti-Flag antibody. β-actin was used as a loading control. One representative experiment of 2 independently performed experiments is shown. (C) Degradation kinetics of BCL6 by FBXO11 mutants. BCL6 abundance was measured from western blot gels as in (A) by ImageJ software and normalized for the GFP intensity of the corresponding lane. The ratio between the relative levels of BCL6/GFP at each time 0 was set as 100%. Data are from 1 of 2 independent experiments, each with comparable results. (D) SNAIL abundance was measured from western blot gels, as in (B), using ImageJ software and normalized for β-actin intensity of the corresponding lane. The ratio between the relative levels of SNAIL/β-actin at each time 0 was set as 100%. Data are from 1 of 2 independent experiments, each with comparable results.

Article Snippet: FLAG-tagged SNAIL (clone ID: 16218) and FLAG-tagged BCL6 (clone ID: 31391) plasmids were purchased from Addgene (Cambridge, MA).

Techniques: Derivative Assay, Western Blot, Expressing, Transfection, Software

Journal: OncoTargets and therapy

Article Title: Urolithin A Inhibits Epithelial–Mesenchymal Transition in Lung Cancer Cells via P53-Mdm2-Snail Pathway

doi: 10.2147/OTT.S305595

Figure Lengend Snippet: Snail is required for inhibition of EMT by urolithin A in lung cancer cells. ( A ) Western blot demonstrates decreased Snail expression following 5 h of urolithin A (0, 5, 10, 15, 20 and 25 μM) stimulation in H1299 and A549 cell lines compared with Slug, Twist and Zeb1. ( B ) The cells transfected with a control or Snail-specific siRNA. At 48 h post-transfection, cells were stimulated with urolithin A for additional 10 h. Western blotting shows that the expression of E-cadherin was increased in cells transfected with a Snail siRNA. ( C ) A549 and H460 cells were transfected with a Snail cDNA. After 48 h, cells were untreated or treated with the indicated amounts of urolithin A for 10 h. Western blotting shows that the urolithin A-induced levels of E-Cadherin decreased further in the cells transfected with a Snail cDNA. ( D ) The cell migration of A549 and H460 after transfection of Snail cDNAs and urolithin A treatment (urolithin A 0, 10 μM) was assessed by the Wound healing assay. The quantification was present in right panels. (* P <0.01, ** P <0.01, *** P <0.001 for the difference from the control cells). ( E ) The cell invasion and motility of A549 and H460 after transfection of Snail cDNAs and urolithin A treatment were assessed by the Cell Invasion Assay. (** P <0.01, *** P <0.001 for the difference from the control cells).

Article Snippet: The plasmids of Snail promoter (no.31694), Snail cDNA (no.16218), mdm2 cDNA (no.16233),p53 cDNA (no.69003),p53 shRNA (no.28222) were purchased from Addgene.

Techniques: Inhibition, Western Blot, Expressing, Transfection, Control, Migration, Wound Healing Assay, Invasion Assay

Journal: OncoTargets and therapy

Article Title: Urolithin A Inhibits Epithelial–Mesenchymal Transition in Lung Cancer Cells via P53-Mdm2-Snail Pathway

doi: 10.2147/OTT.S305595

Figure Lengend Snippet: Urolithin A induces Snail degradation via mdm2-mediated ubiquitination. ( A ) A549 and H460 cells were treated with urolithin A (0, 10 and 20 μM) for 5 h. The expression of Snail gene was detected by RT-PCR. (ns means no statistical difference). ( B ) A549 and H460 cells were co-transfected with a plasmid of the Snail promoter luciferase reporter gene with a plasmid of control Renilla luciferase reporter gene. At 36 h after transfection, cells were treated with urolithin A (0, 5, 10 and 20 μM) for 5 h, and luciferase activity was detected using the dual luciferase reporter system. (ns means no statistical difference). ( C ) Cells were treated with CHX (Cycloheximide, 50 μg/mL) for the indicated time in the presence or absence of urolithin A. Western blot was used to determine Snail protein levels. ( D ) Western blotting analysis of Snail, p62 and LC3A/B after cells were pre-treated with 20 μM HCQ for 1 h and then treated with urolithin A (0, 10 and 20 μM) for 5 h in A549 and H460 cells. ( E ) Western blotting analysis of Snail, after cells were pre-treated with 20 μM PII for 1 h and then treated with urolithin A (0, 10 and 20 μM) for 5 h in A549 and H460 cells. ( F ) Cells were treated with urolithin A after which cell lysates were immunoprecipitated with anti-Snail antibody and then Western blotted with anti-Ubiquitin. ( G ) Western blot examined mdm2 expression flowing 5 h of urolithin A (0, 5, 10, 15, 20 and 25 μM) stimulation in A549 and H460 cells. ( H and I) After transfection with mdm2 cDNA ( H ) or mdm2 siRNA ( I ) for 48 h, A549 and H460 cells were treated with urolithin A (0, 10 and 20 μM) for 5 h. Western blot was carried out for analysis of Snail levels. ( J ) Cells were treated with urolithin A for 5 h after which cell lysates were immunoprecipitated with anti-mdm2 antibody and then Western blotted with anti-Snail.

Article Snippet: The plasmids of Snail promoter (no.31694), Snail cDNA (no.16218), mdm2 cDNA (no.16233),p53 cDNA (no.69003),p53 shRNA (no.28222) were purchased from Addgene.

Techniques: Ubiquitin Proteomics, Expressing, Reverse Transcription Polymerase Chain Reaction, Transfection, Plasmid Preparation, Luciferase, Control, Activity Assay, Western Blot, Immunoprecipitation

Journal: OncoTargets and therapy

Article Title: Urolithin A Inhibits Epithelial–Mesenchymal Transition in Lung Cancer Cells via P53-Mdm2-Snail Pathway

doi: 10.2147/OTT.S305595

Figure Lengend Snippet: Urolithin A upregulates mdm2 by inhibiting the interaction of p53 and mdm2. ( A ) H1299 cells were treated with different concentrations of urolithin A (0, 5, 10, 15, 20 and 25 μM) for 5 h. Western blot examined the expression of mdm2. ( B ) Western blot demonstrates expression of p53 following 5 h of urolithin A (0, 5, 10, 15, 20 and 25 μM) stimulation in indicated lung cancer cell lines. ( C ) Cells were treated with urolithin A for 5 h after which cell lysates were immunoprecipitated with anti-p53 antibody and then Western blotted with anti-Ubiquitin and anti-mdm2 antibodies. ( D ) Transfection of A549 and H460 cells with p53 shRNA for 48h, and then treated with different concentrations of urolithin A for 5 h, the expression levels of mdm2 and Snail were analyzed by immunoblotting. ( E and F) Indicated cells were transfected with p53 cDNA. After 48 h, cells were treated with urolithin A (0, 10 and 20 μM) for 5 h. The levels of mdm2 and Snail were detected by Western blotting.

Article Snippet: The plasmids of Snail promoter (no.31694), Snail cDNA (no.16218), mdm2 cDNA (no.16233),p53 cDNA (no.69003),p53 shRNA (no.28222) were purchased from Addgene.

Techniques: Western Blot, Expressing, Immunoprecipitation, Ubiquitin Proteomics, Transfection, shRNA

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