snail cdna plasmids  (OriGene)

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: 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: Nucleic Acids Research

Article Title: IGF2BP1 promotes mesenchymal cell properties and migration of tumor-derived cells by enhancing the expression of LEF1 and SNAI2 (SLUG)

doi: 10.1093/nar/gkt410

Figure Lengend Snippet: IGF2BP1 modulates FN1 and SNAI2 (SLUG) transcription via LEF1. ( A ) Schematic of luciferase reporters comprising the full-length in silico predicted (FN-839) or 5′-truncated fragments of the human FN1 promoter. The proposed transcription start is indicated by +1 with a reported 5′-UTR of 266 nt. Putative LEF1-binding sites predicted by ‘PROMO’ are depicted as white boxes with labels ‘1-5’ in 5′-to-3′ direction. ( B ) The Firefly luciferase activity of indicated promoter fragments or empty pGL4 vector was monitored in HEK293 cells on transient co-transfection with RFP or LEF1 for 30 h. Firefly activities were normalized by Renilla activities [relative luciferase units (RLU)], serving as internal controls. All reporters comprising the putative LEF1-binding site four showed promoter activity and were activated by LEF1. ( C and D ) Binding of endogenous LEF1 protein to the human FN1 promoter in HEK293 cells was assessed by ChIP. The association of endogenous LEF1 or histone H3 to the FN1 promoter was monitored by semi-quantitative (C) as well as quantitative PCR (D) using to FN1 promoter specific amplicons (P1 and P2, indicated in lower panel). An intergenic probe served as positive control. IgG-agarose was used to monitor unspecific binding (C, negative control). In (D), the enrichment of indicated genomic DNA fragments (P1 and P2) or the intergenic control (intergenic) was determined relative to the diluted input fraction (I) normalized by IgG-controls using the ΔC t -method. ( E ) HEK293 cells were co-transfected with FN-839 luciferase reporter and IGF2BP1-directed (shI1-1), LEF1-directed (shL1-1) or control shRNA encoding vectors for 48 h. RLUs were determined as described in (B). ( F ) HEK293 cells were transfected with IGF2BP1-directed (siI1-2) or control siRNAs (siC) for 72 h. The abundance of SNAI2 mRNA in response to IGF2BP1 knockdown was analyzed by qRT-PCR using the ΔΔC t -method and PPIA for normalization. ACTB served as control. ( G ) HEK293 cells transfected as in (F) were treated with ActD (5 µM) to block transcription for indicated times. SNAI2 mRNA turnover was analyzed by qRT-PCR using the ΔΔC t -method and PPIA for normalization. RNA decay is depicted in semi-logarithmic scale revealing no significant difference in mRNA turnover ( P -value not shown). ( H ) HEK293 cells were transfected with LEF1-directed (siL1-1) or control siRNAs (siC) for 72 h. The abundance of SNAI2 mRNA in response to LEF1 depletion was analyzed by qRT-PCR using the ΔΔC t -method and PPIA for normalization. RPLP0 served as control. ( I ) Schematic of Firefly luciferase reporters comprising the SNAI1 or SNAI2 promoter sequences, as previously reported ( , ). Indicated putative LEF1-binding sites within the SNAI1 or SNAI2 promoter were predicted [white boxes; as described in (A)] or as previously reported [gray boxes, only for SNAI2; ]. ( J ) The Firefly activity of SNAI1 or SNAI2 promoter fragments cloned in pGL4 as well as the activity of empty pGL4 vector was monitored in HEK293 cells on transient co-transfection with RFP or LEF1 for 30 h. RLUs were determined as described in (B). LEF1 only enhanced the activity of the SNAI2 promoter. ( K ) HEK293 cells were co-transfected with SNAI1 or SNAI2 promoter reporters and indicated shRNA-encoding vectors for 48 h. RLUs were determined as described in (B). SNAI2 promoter activity was reduced by IGF2BP1 as well as LEF1 knockdown, whereas the SNAI1 reporter activity remained largely unaffected and was barely elevated compared with the empty control reporter. Statistical significance was validated by Student’s t -testing: * P < 0.05; *** P < 0.0005. Error bars indicate SD of at least three independent analyses.

Article Snippet: The following plasmids were obtained from Addgene: SNAI2-directed shRNA lentiviral vector (ID: 10905); SNAI1 cDNA (ID: 36976), subcloned in pLVX-puro GFP; SNAI2 cDNA (ID: 36986), subcloned in pLVX-puro GFP; SNAI1 promoter (ID: 31694).

Techniques: Luciferase, In Silico, Binding Assay, Activity Assay, Plasmid Preparation, Cotransfection, Real-time Polymerase Chain Reaction, Positive Control, Negative Control, Transfection, shRNA, Quantitative RT-PCR, Blocking Assay, Clone 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