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: 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 are downregulated in OC tissues and decreased expressions of miR-137 and miR-34a are associated with poor survival in OC patients. a Venn diagram showing the overlap of miRNAs that were predicted to bind to the Snail 3′-UTR by alternative algorithms (TargetScan, miRSystem and DIANA-MicroT-CDS). The 6 predicted miRNAs were common to these three algorithms. b , c qPCR analysis of miR-137 ( b ) and miR-34a ( c ) levels in 50 paired cancerous and normal tissue samples from OC patients. d , e Kaplan-Meier analysis of overall survival in 50 OC patients with high median ( n = 25) or low median ( n = 25) expression levels of miR-137 ( d ) or miR-34a ( e )

Article Snippet: The Snail 3′-UTR luciferase vector was purchased from OriGene.

Techniques: Expressing

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: Snail is a direct target of miR-137 and miR-34a in OC cells. a Relative miR-34a expression in OC cell lines (SKOV-3 and ES-2) and normal ovarian epithelial NOEC cells. b , c ES-2 cells were transfected with reporter constructs containing either wild-type (WT) Snail , or Snail 3′-UTR with mutation (MUT), along with miR-137 mimic ( b ), miR-34a mimic ( c ), or negative control mimic (Neg mimic), respectively. Relative luciferase activity was measured. d , e qPCR analysis of Snail expression in OC cells after overexpression ( d ) or knockdown ( e ) of miR-137 and miR-34a. f Western blotting analysis of Snail expression in OC cells after overexpression or knockdown of miR-137 and miR-34a. g , h qPCR analysis of indicated mRNAs in OC cells after transient overexpression or knockdown of miR-137 ( g ) and miR-34a ( h ). i Relative mRNA expression of Snail in OC tissues and matched normal tissues. j Analysis of Snail mRNA expression using microarray (Oncomine) on normal ovary versus OC tissue. ** P < 0.01

Article Snippet: The Snail 3′-UTR luciferase vector was purchased from OriGene.

Techniques: Expressing, Transfection, Construct, Mutagenesis, Negative Control, Luciferase, Activity Assay, Over Expression, Western Blot, Microarray

Journal: The FASEB Journal

Article Title: SNAI1 is upregulated during muscle regeneration and represses FGF21 and ATF3 expression by directly binding their promoters

doi: 10.1096/fj.202200215r

Figure Lengend Snippet: FIGURE 1 Expression of SNAI1 protein and mRNA during muscle regeneration and myoblasts differentiation. (A) Eosin–hematoxylin staining of cross-sections from tibialis anterior muscles at day 0 (untreated), 3, 6, and 10 days after bupivacaine treatment (abt). Bar, 50 μm. (B) Snai1 protein expression was detected by western blot analysis of whole extracts prepared from regenerating muscles at the indicated days abt. Histone H3 expression was used to confirm equal loading. PAX7, MyoD, and MYH3 expression was detected as control of satellite cell-derived myoblasts proliferation (PAX7 and MyoD) and differentiation (MYH3). (C) Quantitative evaluation of Snai1 transcript in regenerating muscle by RT-qPCR. MyoD and Pax7 transcripts were measured as muscle regeneration control. The values normalized to the glyceraldehyde-3-phosphate dehydrogenase (Gapdh) mRNA levels are expressed as the mean ± SD of 3 independent experiments. Asterisks indicate significant differences (p-values) between expression in untreated muscle (0) and each other time point. Snai1 protein expression was detected by Western blot analysis of whole-cell extracts prepared from proliferating and differentiating C2C12 cells (D) and primary mouse myoblasts (E). Time (d) indicates the days after switching to differentiation medium (DM). Histone H3 expression was used to confirm equal loading. MyoD, MYF4, and MYH3 expressions were detected as control of myoblasts differentiation. p21WAF1/Cip (p21) was used as a marker of cell cycle arrest. (F) Quantitative evaluation of Snai1 transcript by RT-qPCR during C2C12 differentiation. The values normalized to Gapdh mRNA levels are expressed as the mean ± SD of 3 independent experiments. Asterisks indicate significant differences (p-values) between expression at day 0 and each other time point after switching to DM.

Article Snippet: The pCMV6 expression vectors for murine Flag- tagged SNAI1 (OriGene Technologies, Inc., Rockville, MD, US) were used in experiments of co- transfection, and the pCMV6- Entry vector was used as control.

Techniques: Expressing, Staining, Muscles, Western Blot, Control, Derivative Assay, Quantitative RT-PCR, Marker

Journal: The FASEB Journal

Article Title: SNAI1 is upregulated during muscle regeneration and represses FGF21 and ATF3 expression by directly binding their promoters

doi: 10.1096/fj.202200215r

Figure Lengend Snippet: FIGURE 2 Transcriptome analysis of C2C12 myoblasts silenced for SNAI1 knockdown in proliferating C2C12 myoblasts. (A) SNAI1 expression was silenced in C2C12 cells by transduction

Article Snippet: The pCMV6 expression vectors for murine Flag- tagged SNAI1 (OriGene Technologies, Inc., Rockville, MD, US) were used in experiments of co- transfection, and the pCMV6- Entry vector was used as control.

Techniques: Knockdown, Expressing, Transduction

Journal: The FASEB Journal

Article Title: SNAI1 is upregulated during muscle regeneration and represses FGF21 and ATF3 expression by directly binding their promoters

doi: 10.1096/fj.202200215r

Figure Lengend Snippet: FIGURE 4 FGF21 and ATF3 expression was repressed by overexpression of SNAI1 in differentiating myoblasts. (A) C2C12 myoblasts were transiently transfected with the pCMV6-emty plasmid or with the pCMV6-SNAI1 plasmid and exogenous SNAI1 protein expression was detected by Western blot analysis by means of anti-FLAG antibody. Myoblasts differentiation was induced 48 h post-transfection by the medium switch, which corresponds to day 0 of differentiation. Histone H3 expression was used to confirm equal loading. Fgf21 (B) and Atf3 (C) expression was analyzed by RT-qPCR at 24, 48, and 72 h after transfection corresponding to differentiation day −1, day 0, and day 1, respectively. The values were normalized to the Gapdh mRNA levels. Asterisks indicate significant differences (p-values) between expression in control (pCMV6) and SNAI1 overexpressing (pcMV6-SNAI1) C2C12 cells at the indicated days after the switch to differentiation medium. Data were evaluated from three independent experiments using the one-way analysis of variance ANOVA.

Article Snippet: The pCMV6 expression vectors for murine Flag- tagged SNAI1 (OriGene Technologies, Inc., Rockville, MD, US) were used in experiments of co- transfection, and the pCMV6- Entry vector was used as control.

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

Journal: The FASEB Journal

Article Title: SNAI1 is upregulated during muscle regeneration and represses FGF21 and ATF3 expression by directly binding their promoters

doi: 10.1096/fj.202200215r

Figure Lengend Snippet: FIGURE 7 SNAI1 represses Fgf21 promoter activity induced by ER stress. (A) The ER stressor thapsigargin induces the expression of Fgf21 mRNA in myoblasts. RT-qPCR was performed on RNA extracted from C2C12 myoblasts treated with DMSO (day 0) or with 0.2 μM thapsigargin at different time points. Asterisks indicate significant differences (p-values) between expression at day 0 and each time point. (B) Thapsigargin-induced Fgf21 promoter activity is repressed by overexpression of SNAI1. C2C12 cells were co-transfected with the pCMV6-empty or pCMV6-SNAI1 vectors and the reporter plasmid carrying the full-length Fgf21 (−1764) promoter and stimulated with 0.2 μM thapsigargin for 24 h. Luciferase activity was normalized to the Renilla luciferase internal control and expressed as fold change relative to cells co-transfected with the pCMV6-emty vector. Data are the mean and standard deviation from three to six independent experiments and were evaluated using one-way or two-way ANOVA. (C, D) In differentiating myoblasts, IRE1 and PERK inhibition reduce the expression of Fgf21 and Atf3 mRNA, respectively. RT-qPCR was performed on RNA extracted from C2C12 cells induced to differentiate for 24 h in presence of 10 μM of the ER stress inhibitors, GSK2656157 (PERK inhibitor), Melatonin (ATF6 inhibitor), and 4μ8C (IRE1 inhibitor). (E) Inhibition of the PERK pathway reduced DDIT3/CHOP expression. C2C12 cells were differentiated for 24 h in DM and whole-cell extracts were used in western blot analysis. (F) Inhibition of IRE1 and ATF6 pathways reduced MYH3 expression. C2C12 cells were differentiated for 48 h in DM and whole-cell extracts were used in western blot analysis.

Article Snippet: The pCMV6 expression vectors for murine Flag- tagged SNAI1 (OriGene Technologies, Inc., Rockville, MD, US) were used in experiments of co- transfection, and the pCMV6- Entry vector was used as control.

Techniques: Activity Assay, Expressing, Quantitative RT-PCR, Over Expression, Transfection, Plasmid Preparation, Luciferase, Control, Standard Deviation, Inhibition, Western Blot

Journal: The FASEB Journal

Article Title: SNAI1 is upregulated during muscle regeneration and represses FGF21 and ATF3 expression by directly binding their promoters

doi: 10.1096/fj.202200215r

Figure Lengend Snippet: FIGURE 8 Scheme of the regulatory network connecting SNAI1, FGF21, and ER stress in the myoblasts. SNAI1 directly represses Fgf21 expression that in turn promotes myoblast cell cycle exit and terminal differentiation. SNAI1 also represses the expression of ER stress target genes downstream of the IRE1 sensor and the terminal differentiation program driven by MyoD. HGF expression, consistently with its role in promoting myoblast proliferation, is indirectly activated by SNAI1.

Article Snippet: The pCMV6 expression vectors for murine Flag- tagged SNAI1 (OriGene Technologies, Inc., Rockville, MD, US) were used in experiments of co- transfection, and the pCMV6- Entry vector was used as control.

Techniques: Expressing

Journal: PLoS ONE

Article Title: Neuropilin-2 Expression Promotes TGF-β1-Mediated Epithelial to Mesenchymal Transition in Colorectal Cancer Cells

doi: 10.1371/journal.pone.0020444

Figure Lengend Snippet: A, HT29 ctrl , HT29 NRP2 , Colo320 siRNA-NRP2 and Colo320 siRNA-ctrl were analyzed for expression of epithelial and mesenchymal markers (respectively E-Cadherin and vimentin) by western-blotting (whole cell lysate). β-actin was used as a control of protein loading. The presence of NRP2 decreases E-cadherin expression and induces vimentin in HT29 colorectal cancer cells. Specific disruption of NRP2 using siRNA down-regulated vimentin in Colo320 cancer cells. Blotted proteins have been quantified with the BIO-1D advanced software and reported to the β-actin level. B, Frozen sections of the isolated tumors from HT29 ctrl and HT29 NRP2 xenografts were subjected to immunohistochemical staining with anti-cytokeratin-20, anti-Ecadherin antibodies and HES staining (magnification x100). HES (Hematoxiline Eosine Safran) staining was used to assess morphology. HT29 NRP2 invade smooth muscle (sm = smooth muscle) as indicated by arrows unlike HT29 ctrl which showed a local invasion. HT29 NRP2 xenografts lacked epithelial markers such as cytokeratin-20 and E-cadherin. C , Pancreatic and renal cancer cell lines were analyzed for NRP2, E-cadherin and vimentin expression by western-blotting. β-actin was used as a control of protein loading. The presence of NRP2 is positively correlated with mesenchymal marker vimentin and inversely correlated with epithelial marker E-cadherin. D , Transcriptional factors Snail1 and Twist1 were analyzed in HT29 ctrl , HT29 NRP2 , Colo320 siRNA-NRP2 and Colo320 siRNA-ctrl whole cell lysates by western-blotting. NRP2 expressing cells (Colo320 siRNA-ctrl and HT29 NRP2 ) have increased levels of Snail1 and Twist1 compared to NRP2 lacking cells (HT29 ctrl and Colo320 siRNA-NRP2 ). β-actin was used as a control of protein loading. Blotted proteins have been quantified with the BIO-1D advanced software and reported to the β-actin level. E, Evaluation of NRP2, Snail, Twist1, Gli1 and TGFβ1 expression by QRT-PCR analysis in Colo320 siRNA-ctrl and Colo320 siRNA-NRP2 . Decreased NRP2 expression is indeed associated with a down-regulation of transcriptional regulators Snail, Gli1 and twist1 and with a decreased TGF-β1 production. F, Confocal microscopy analysis of localization of β-catenin. Cells (HT29 ctrl and HT29 NRP2 ) were stained with DAPI (blue) for nuclear staining and also with anti-β-catenin antibody (green). Whereas β-catenin (green) is localized at the membrane in HT29 ctrl cells, HT29 NRP2 cells show a nuclear localization of this transcriptional activator.

Article Snippet: Duplicate samples were subjected to RT-qPCR. mRNA were quantified using primers listed below: NRP2 (Hs00187290_m1), Snail1 (Hs001955991_m1), TGF-β1 (Hs00171257_m1), Gli1 (Hs00171790_m1), Twist1 (Hs00361186_m1) (Applied Biosystems).

Techniques: Expressing, Western Blot, Software, Isolation, Immunohistochemical staining, Staining, Marker, Quantitative RT-PCR, Confocal Microscopy

Journal: PLoS ONE

Article Title: Neuropilin-2 Expression Promotes TGF-β1-Mediated Epithelial to Mesenchymal Transition in Colorectal Cancer Cells

doi: 10.1371/journal.pone.0020444

Figure Lengend Snippet: A, Western-blotting experiments were performed on nuclear extracts of HT29 NRP2 and Colo320 for the detection of nuclear smad proteins after treatment with increased doses of TGFRI kinase-inhibitor SB-431542 (10 µM, 50 µM). DMSO is the control medium. Histone-H1 was used as a control of protein loading. Whereas only 10 µM of SB-431542 decreases the nuclear level of phosphorylated Smad2 in Colo320 cancer cells, doses of 50 µM should be used to decrease the nuclear level of phosphorylated Smad2 in HT29 NRP2 cancer cells. Smad proteins have been quantified with the BIO-1D advanced software and reported to the Histone H1 level. B , Overnight treatment with increased doses of TGFRI kinase-inhibitor SB431542 (10 µM, 50 µM, 100 µM) inhibited dose-dependently vimentin expression in Colo320 and in HT29 NRP2 cells. TGF-β1 signaling inhibition restored in part E-cadherin in HT29 NRP2 cells. Proteins have been quantified with the BIO-1D advanced software and reported to the β-actin level. Data represent results of a representative experiment out of 3. C, Western-blotting experiment realized with Colo320 and HT29 NRP2 whole cell lysates overnight treated with increased doses of TGFRI kinase-inhibitor SB431542 (10 µM, 50 µM, 100 µM). SB431542 treatment inhibited dose-dependently snail1 and twist1 expression in Colo320 and in HT29 NRP2 cells. Proteins have been quantified with the BIO-1D advanced software and reported to the β-actin level. D , Confocal microscopy analysis of localization of β-catenin in HT29 NRP2 cells after overnight treatment with increased doses of TGFRI kinase-inhibitor SB431542 (10 µM, 50 µM). Cells (HT29 NRP2 ) were stained with DAPI (blue) for nuclear staining, with mitotracker (purple) for cytotoxicity dectection and also with an anti-β-catenin antibody (green). Mitotracker staining (purple) was realized in order to assess potential cytotoxicity of the SB-431542 treatment. Loss of staining of this marker correlates with increased cytotoxicity. 16 hours treatment with 50 µM of SB-431542 induces relocalization of beta-catenin at the membrane of the cells, indicating that TGFRI activity neutralization by SB-431542 can reverse EMT in HT29 NRP2 colorectal cancer cells. No cytotoxicity was observed at any dose of SB-431542 used. For fluorescence quantification, ratio of fluorescence intensity was calculated in each condition. A decreased ratio of nuclear fluorescence/cytoplasm-membrane fluorescence intensity after treatment with 50 µM of SB-431542 in HT29 NRP2 cells was observed indicating that beta-catenin relocalizes at the membrane of the cells.

Article Snippet: Duplicate samples were subjected to RT-qPCR. mRNA were quantified using primers listed below: NRP2 (Hs00187290_m1), Snail1 (Hs001955991_m1), TGF-β1 (Hs00171257_m1), Gli1 (Hs00171790_m1), Twist1 (Hs00361186_m1) (Applied Biosystems).

Techniques: Western Blot, Software, Expressing, Inhibition, Confocal Microscopy, Staining, Marker, Activity Assay, Neutralization, Fluorescence

Journal: Cell Death & Disease

Article Title: Partitioning defective 6 homolog alpha (PARD6A) promotes epithelial–mesenchymal transition via integrin β1-ILK-SNAIL1 pathway in ovarian cancer

doi: 10.1038/s41419-022-04756-2

Figure Lengend Snippet: Representative western blot of EMT-associated genes in SKOV3 ( A ) and A2780 cells ( B ) with PARD6A silenced. Total protein lysates were subjected to immunoblot analysis with the indicated antibodies, except that nuclear lysates were subjected to immunoblot analysis with SNAIL1 or Histone H3 antibodies. Representative western blot of EMT-associated genes in HO8910 ( C ) and OVCAR8 cells ( D ) with PARD6A overexpressed. Total protein lysates were subjected to immunoblot analysis with the indicated antibodies, except that nuclear lysates were subjected to immunoblot analysis with SNAIL1 or Histone H3 antibodies.

Article Snippet: The proximal promoter region of SNAIL1 (−1123 to +92) [ ] was synthesized by GenScript.

Techniques: Western Blot

Journal: Cell Death & Disease

Article Title: Partitioning defective 6 homolog alpha (PARD6A) promotes epithelial–mesenchymal transition via integrin β1-ILK-SNAIL1 pathway in ovarian cancer

doi: 10.1038/s41419-022-04756-2

Figure Lengend Snippet: A , B Overexpression of SNAIL1 reversed the effects of PARD6A silencing on EMT in SKOV3 cells. SKOV3 cells were first transfected with siPARD6As for 24 h, and then transfected with SNAIL1 overexpression plasmids. Then the cells were incubated for 6 h, and replaced with fresh medium and incubated for 24 h before experiments. Representative images ( A ) and statistical analysis ( B ) of transwell migration and invasion assays were shown here. Scale bar = 200 µm. C SNAIL1 overexpression reversed the change of protein levels of VIMENTIN and E-cadherin by PARD6A silencing in SKOV3 cells. Total protein lysates were subjected to immunoblot analysis with the indicated antibodies, except that nuclear lysates were subjected to immunoblot analysis with SNAIL1 or Histone H3 antibodies. D , E Silencing of SNAIL1 reversed the effects of PARD6A overexpression on EMT in HO8910 cells. HO8910 cells were first infected with lentivirus to overexpress PARD6A, and then treated with siSNAIL1s to silence SNAIL1. Representative images ( D ) and statistical analysis ( E ) of transwell migration and invasion assays were shown here. Scale bar = 200 µm. F Silencing of SNAIL1 reversed the change of protein levels of VIMENTIN and E-cadherin by PARD6A overexpression in HO8910 cells. Total protein lysates were subjected to immunoblot analysis with the indicated antibodies, except that nuclear lysates were subjected to immunoblot analysis with SNAIL1 or Histone H3 antibodies. Data shown in B and E are the mean values (±SD) from three independent experiments. Statistically significant differences with P < 0.05 were considered significant (* P < 0.05; ** P < 0.01; *** P < 0.001).

Article Snippet: The proximal promoter region of SNAIL1 (−1123 to +92) [ ] was synthesized by GenScript.

Techniques: Over Expression, Transfection, Incubation, Migration, Western Blot, Infection

Journal: Cell Death & Disease

Article Title: Partitioning defective 6 homolog alpha (PARD6A) promotes epithelial–mesenchymal transition via integrin β1-ILK-SNAIL1 pathway in ovarian cancer

doi: 10.1038/s41419-022-04756-2

Figure Lengend Snippet: A Expression of PARD6A didn’t significantly affect protein levels of RhoA. a , b Protein levels of RhoA in SKOV3 ( a ) and A2780 cells ( b ) with PARD6A silenced. c , d Protein levels of RhoA in HO8910 ( c ) and OVAR8 cells ( d ) with PARD6A overexpressed. B Expression of PARD6A significantly affected cell adhesion. a , b Relative cell adhesion values of SKOV3 ( a ) and A2780 cells ( b ) with PARD6A silenced. c , d Relative cell adhesion values of HO8910 ( c ) and OVAR8 cells ( d ) with PARD6A overexpressed. Data shown are the mean values (±SD) from three independent experiments. Statistically significant differences with P < 0.05 were considered significant (***P < 0.001). C Expression of PARD6A regulates the protein levels of integrin β1. a , b Protein levels of Integrin β1 in SKOV3 ( a ) and A2780 cells ( b ) with PARD6A silenced. c , d Protein levels of Integrin β1 in HO8910 ( c ) and OVAR8 cells ( d ) with PARD6A overexpressed. D Expression of PARD6A regulates the protein levels of integrin β1. a , b Protein levels of ILK, p-GSK3β, and GSK3β in SKOV3 ( a ) and A2780 cells ( b ) with PARD6A silenced. c , d Protein levels of ILK, p-GSK3β, and GSK3β in HO8910 ( c ) and OVAR8 cells ( d ) with PARD6A overexpressed. E Representative images of immunohistochemistry detections of protein levels of ILK in serous, mucinous, and clear cell subtypes of ovarian cancer tissues versus normal ovarian epithial tissues. Scale bar = 50 µm. F , H Expression of ILK regulates expression of SNAIL1. F Protein levels of SNAIL1 in nuclear lysates and protein levels of ILK, p-GSK3β, and GSK3β in total protein lysates of SKOV3 and A2780 cells with ILK silenced and those levels of the indicated proteins in HO8910 and OVAR8 cells with ILK overexpression, respectively. G Relative mRNA expression of SNAIL1 in SKOV3 and A2780 cells with ILK silenced and that of SNAIL1 in HO8910 and OVCAR8 cells with ILK overexpression assessed by qRT-PCR. H Relative luciferase activity of pGL3-SNAIL1-promotor with/without ILK overexpression. I Scheme of the molecular pathways of EMT mediated by PAR6α-Integrin β1-ILK-SNAIL1 and finally implemented by E-cadherin and VIMENTIN.

Article Snippet: The proximal promoter region of SNAIL1 (−1123 to +92) [ ] was synthesized by GenScript.

Techniques: Expressing, Immunohistochemistry, Over Expression, Quantitative RT-PCR, Luciferase, Activity Assay