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: Developmental Biology

Article Title: Roles of ADAM13-regulated Wnt activity in early Xenopus eye development

doi: 10.1016/j.ydbio.2011.12.031

Figure Lengend Snippet: Snail2 rescues the eye phenotypes caused by ADAM13 MO. One dorsal-animal blastomere of 8-cell stage embryos was injected with the indicated MO (1.5 ng) together with or without Snail2 transcript (200 pg). Embryos were processed for in situ hybridization for pax6 at stage ~12.5 (A), or scored for eye defects at stage ~35 (B). The injected side is denoted with a red asterisk. See Fig. 1A and Materials and Methods for phenotype scoring. **, p = 0.008.

Article Snippet: X. tropicalis cDNA clones for snail2 (in pCS107; clone ID: Ttba075D05; from Geneservice) and cerberus (in pCS108; clone ID: 7579004; from OpenBiosystems) were identified by bioinformatics and used directly for expression (sequences of all clones were confirmed by DNA sequencing).

Techniques: Injection, In Situ Hybridization

Journal: Developmental Biology

Article Title: Roles of ADAM13-regulated Wnt activity in early Xenopus eye development

doi: 10.1016/j.ydbio.2011.12.031

Figure Lengend Snippet: ADAM13 controls cerberus expression through Snail2. One-cell stage embryos were injected with 12 ng of MO CT (A and B) or 13-1 (C and D), or MO 13-1 with 1 ng mRNA encoding Snail2 (E and F), and cultured to stage ~11. In situ hybridization was carried out for cerberus, and embryos were cleared with 2:1 benzyl benzoate/benzyl alcohol before photographed. One representative embryo of each injected group is shown in animal pole view (with dorsal at the top) in the left panels, and all embryos of each injected group are shown in the right panels.

Article Snippet: X. tropicalis cDNA clones for snail2 (in pCS107; clone ID: Ttba075D05; from Geneservice) and cerberus (in pCS108; clone ID: 7579004; from OpenBiosystems) were identified by bioinformatics and used directly for expression (sequences of all clones were confirmed by DNA sequencing).

Techniques: Expressing, Injection, Cell Culture, In Situ Hybridization

Journal: Cancer Science

Article Title: SNAIL2 contributes to tumorigenicity and chemotherapy resistance in pancreatic cancer by regulating IGFBP2

doi: 10.1111/cas.15162

Figure Lengend Snippet: SNAIL2 is highly expressed in tumorigenic pancreatic cancer cell lines. A, Microscopy images of 2D cultures and spheres (3D culture, day 7) in KLM1 and KMP5. Scale bars: 100 µm. B, qRT‐PCR (EMT‐TF) of KLM1 and KMP5. Relative expression levels normalized by HPDE cells were compared. n = 3, each. ND, not detected. Mean + SE. ** P < .01. C, qRT‐PCR ( SNAI2 ) of control vector versus shSNAI2 from KLM1 and KMP5. n = 3, each. Mean + SE. ** P < .01. D, Western blot (SNAIL2, CDH1, VIM, and ACTB) of control vector versus shSNAI2 from KLM1 and KMP5. The relative intensity (ratio) is shown below each band. shRNA was used shSNAI2_1

Article Snippet: The SNAIL2 and IGFBP2 overexpression vectors and control vector were purchased from Sino Biologicals.

Techniques: Microscopy, Quantitative RT-PCR, Expressing, Plasmid Preparation, Western Blot, shRNA

Journal: Cancer Science

Article Title: SNAIL2 contributes to tumorigenicity and chemotherapy resistance in pancreatic cancer by regulating IGFBP2

doi: 10.1111/cas.15162

Figure Lengend Snippet: SNAI2 is highly expressed in spheroids established from surgically resected human pancreatic cancer. A, Immunohistochemical SNAIL2 staining of human pancreatic cancer (2 samples). Scale bars: 100 µm. B, Microscope image of spheroids in 3D culture (left), H&E staining of spheroids (middle), H&E staining of primary tumor (right). Scale bars: 100 µm. C, qRT‐PCR (EMT‐TF) of Sphs. Relative expression levels normalized by HPDE cells were compared. n = 3. ND, not detected. Mean + SE. ** P < .01. D, Microscopic image of control vector and shSNAI2 from Sphs in 3D culture. Scale bars: 100 µm. E, qRT‐PCR ( SNAI2 ) of control vector versus shSNAI2 from spheroids. n = 3, each. Mean + SE. * P < .05, ** P < .01. F, Western blot (SNAIL2, CDH1, VIM, and ACTB) of control vector versus shSNAI2 from Sph. shRNA was used shSNAI2_1. The relative intensity (ratio) is shown below each band

Article Snippet: The SNAIL2 and IGFBP2 overexpression vectors and control vector were purchased from Sino Biologicals.

Techniques: Immunohistochemical staining, Staining, Microscopy, Quantitative RT-PCR, Expressing, Plasmid Preparation, Western Blot, shRNA

Journal: Cancer Science

Article Title: SNAIL2 contributes to tumorigenicity and chemotherapy resistance in pancreatic cancer by regulating IGFBP2

doi: 10.1111/cas.15162

Figure Lengend Snippet: Gene transduction of IGFBP2 and SNAI2 into shSNAI2 restores mRNA and protein expression and improves tumorigenicity and resistance to gemcitabine. A, qRT‐PCR ( SNAI2 and IGFBP2 ) in sh+Vector, sh+IGFBP2 and sh+SNAI2 from KLM1. n = 3, each. Mean + SE. ** P < .01. B, Western blot (SNAIL2, IGFBP2, and ACTB) of sh+Vector versus sh+IGFBP2 and sh+SNAI2 from KLM1 cells. The relative intensity (ratio) is shown below each band. C, D, Sphere formation assay of parental cells, sh+Vector, sh+IGFBP2 and sh+SNAI2 from KLM1 cell at day 7. C, Microscope images. Scale bars: 100 µm. D, Quantification of the number of spheres. n = 3, each. Mean + SE. ** P < .01. E‐H, sh+Vector, sh+IGFBP2, and sh+SNAI2 cells from KLM1 cells were subcutaneously transplanted into NOD‐SCID mice (10 5 cells, 10 4 cells per site), respectively. n = 3. E, Macroscope images of the formed tumor. F, Number of tumor‐forming mice per total transplanted mice at 12 wk after transplantation. G, Tumor growth curves. Mean + SE. * P < .05, ** P < .01. H, H&E staining of the formed tumor. The mesenchymal area (indicated by arrows) ratio is shown below each picture. Scale bars: 100 µm. I, The relative viability of parent cells, sh+Vector versus sh+IGFBP2, and sh+SNAI2 from KLM1 cells 96 h after treatment with gemcitabine. Viability curves at 0, 1, 10, 100 ng/mL gemcitabine. n = 4, each. Mean + SE. ** P < .01

Article Snippet: The SNAIL2 and IGFBP2 overexpression vectors and control vector were purchased from Sino Biologicals.

Techniques: Transduction, Expressing, Quantitative RT-PCR, Plasmid Preparation, Western Blot, Tube Formation Assay, Microscopy, Transplantation Assay, Staining

Journal: Birth defects research

Article Title: Alcohol-Mediated Calcium Signals Dysregulate Pro-Survival Snai2/PUMA/Bcl2 Networks to Promote p53-Mediated Apoptosis in Avian Neural Crest Progenitors

doi: 10.1002/bdr2.1508

Figure Lengend Snippet: (A) Snai2 protein content in 10–12 somite-stage dissected crania is significantly increased 10hr after exposure to 52 mM alcohol, as quantified by western blot analysis. Content is normalized against Gapdh. (B) Immunostain for Snai2 protein (green) in hindbrain sections shows this increase does not represent an ectopic Snai2 induction, but is confined to Snai2+ mesenchymal and ectodermal cells consistent with neural crest (compare green signal at arrows). (C) Snai2 mRNA expression, but not Snai1 mRNA, is significantly increased 10hr after exposure to 52 mM alcohol, as quantified by qPCR. Values are normalized against Gapdh. (D) Pretreatment with Bapta-AM prevented the induction of Snai2 by alcohol and did not affect its expression in controls, as measured by qPCR. Ionomycin treatment of otherwise normal cells was sufficient to induce Snai2. Values are mean ± SEM of three independent experiments having 7–10 crania per treatment. Data analysis used two-tailed Student’s t-test for (A, C), and one-way analysis of variance and Holm-Sidak post-hoc analysis for (D). C, control; C+B, control pretreated with Bapta-AM; C+Io, control treated with ionomycin; Et, ethanol-treated; Et+B, ethanol pretreated with Bapta-AM.

Article Snippet: Electroporation of in ovo embryos was performed as described ( Flentke et al. 2011 ). cDNA encoding Snai2 was from Addgene (#34584; Cambridge, MA); cDNA encoding enhanced green fluorescent protein (eGFP) was a kind gift of T. Suzuki.

Techniques: Western Blot, Expressing, Two Tailed Test, Control

Journal: Birth defects research

Article Title: Alcohol-Mediated Calcium Signals Dysregulate Pro-Survival Snai2/PUMA/Bcl2 Networks to Promote p53-Mediated Apoptosis in Avian Neural Crest Progenitors

doi: 10.1002/bdr2.1508

Figure Lengend Snippet: (A) CyclinD1/ protein content is significantly reduced 10hr after 52 mM alcohol exposure, as shown by western blot analysis and normalized against Gapdh; The CyclinD1 antibody detects a doublet in chick (Clark et al. 2000). Mean ± SD of three replicates using independent protein extracts, analysis using two-tailed Student’s t-test. (B) Enumeration of BrdU+ cells within control and alcohol-exposed Snai2+ populations and within the overlying ectoderm. Alcohol exposure significantly reduced the number of BrdU+Snai2+ cells, but did not alter BrdU+ cell numbers within the adjacent ectoderm. Mean ± SEM of triplicate experiments having 8–10 embryos per treatment. Analysis using two-tailed Student’s t-test. (C-H) Representative histochemical sections visualize Snai2 protein (green, C, F) and BrdU (red, D, G) in control (C-E) and alcohol-exposed (F-H) hindbrains in transverse cross-section. Alcohol-exposed crania contain fewer Snai2+BrdU+ cells (arrowheads) and more Snai2+ BrdU- cells (arrows). The boxed region in E and H indicates the merged region depicted in single channels.

Article Snippet: Electroporation of in ovo embryos was performed as described ( Flentke et al. 2011 ). cDNA encoding Snai2 was from Addgene (#34584; Cambridge, MA); cDNA encoding enhanced green fluorescent protein (eGFP) was a kind gift of T. Suzuki.

Techniques: Western Blot, Two Tailed Test, Control

Journal: Birth defects research

Article Title: Alcohol-Mediated Calcium Signals Dysregulate Pro-Survival Snai2/PUMA/Bcl2 Networks to Promote p53-Mediated Apoptosis in Avian Neural Crest Progenitors

doi: 10.1002/bdr2.1508

Figure Lengend Snippet: Neural folds were electroporated with eGFP-only or Snai2 plus eGFP at stage 9 (7–8 somites), exposed to alcohol or saline control 3hr later, and apoptosis was assessed 10hr thereafter using LysoTracker Red (LTR; A-D, G-J) or TUNEL (E, F, K, L). In all images, the transfected side is on the embryo’s and viewers’ right side, as indicated by the eGFP signal in intact embryos (A-D, G-J), and by an asterisk (*) on all images (E, F, K, L). The white line demarcates the transfected and non-transfected sides in the TUNEL-stained transverse sections, taken at the level of the hindbrain. (A-F) In controls, eGFP-only (A) does not increase apoptosis levels as assessed using LTR (B, white dots at arrows) or TUNEL (E, green signal at arrows). Ectopic Snai2 plus eGFP (C) does not increase cell death, as assessed by LTR (D) or TUNEL (F). (G-L) In contrast, alcohol causes significantly more apoptosis in neural crest and neural progenitors, revealed by LTR (H) or TUNEL (K). Ectopic Snai2 did not reduce the incidence of apoptosis following alcohol exposure, as compared with the apoptosis levels in the hindbrain’s untransfected left side, as assessed using LTR (H versus J, compare white signal between left and right sides at arrows) or using TUNEL (K versus L, compare green signal on right sides with asterisk). (M) Quantitation of TUNEL+ neural crest in sections of alcohol-treated (Et) and control (C) right hindbrain halves transfected with eGFP-only or Snai2 + eGFP. Values are mean ± SEM of triplicate experiments having N=7–9 embryos per treatment. * indicates p<0.001 compared with its electroporation control, analyzed using Kruskal-Wallis one-way analysis of variance on ranks, followed by pairwise multiple comparison procedures using Dunn’s Method for post-hoc analysis.

Article Snippet: Electroporation of in ovo embryos was performed as described ( Flentke et al. 2011 ). cDNA encoding Snai2 was from Addgene (#34584; Cambridge, MA); cDNA encoding enhanced green fluorescent protein (eGFP) was a kind gift of T. Suzuki.

Techniques: Saline, Control, TUNEL Assay, Transfection, Staining, Quantitation Assay, Electroporation, Comparison