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: 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: 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: Viruses

Article Title: Human Cytomegalovirus Induces Vitamin-D Resistance In Vitro by Dysregulating the Transcriptional Repressor Snail

doi: 10.3390/v14092004

Figure Lengend Snippet: HCMV regulates the VDR repressors Snail1 and Snail2. ( A ) RT-qPCR analysis of VDR, Snail1 and Snail2 mRNA expression in mock- and CMV-infected HFF is shown. Asterisks indicate significant differences to the mock control sample (unpaired Student’s t test). ( B ) Immunoblots showing VDR, Snail1, Snail2 and IE1/2 expression compared to GAPDH loading control in HFF infected with CMV and harvested at the indicated time point p.i. Representative blot and quantification from multiple experiments with significant differences to the mock (0 h) control sample as determined by ANOVA Dunnett’s multiple comparison post-hoc tests.

Article Snippet: Single IE1/2, DNA-PKcs and Snail1-targeting siRNAs as well as non-targeting control siRNA siCTRL were produced by Microsynth (Austria) with 3’ dTdT overhangs (see ).

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

Journal: Viruses

Article Title: Human Cytomegalovirus Induces Vitamin-D Resistance In Vitro by Dysregulating the Transcriptional Repressor Snail

doi: 10.3390/v14092004

Figure Lengend Snippet: VDR holoenzyme downregulation is dependent on immediate early gene transcription. ( A ) Vitamin-D-receptor expression was measured in mock- or HCMV-infected HFF at the indicated time points, showing fold change (2 − Δ Δ Ct) compared to mock-infected cells (mean ± SEM). Asterisks indicate significant differences to the mock control sample as determined by ANOVA Dunnett’s multiple comparison post-hoc test. ( B ) VDR, IE1 (pp72) and GAPDH protein expression were determined by Western blot in HFF that were infected with mock (M), CMV (C) and UV-inactivated CMV (U) inocula and harvested at the indicated times post infection. ( C ) VDR and HCMV immediate early protein expression was determined in lysates of HFF transfected with a non-targeting control siRNA or IE1/2 specific siRNA harvested at 24 h p.i. Representative blot (left) and quantification of bands from multiple experiments relative to the GAPDH loading control (right). Asterisks indicate significant differences to the CMV siCTRL sample as determined by ANOVA Dunnett’s multiple comparison post-hoc test. ( D ) HEK293 cells were transfected with pcDNA empty vector control or pcDNA containing the IE2 open reading frame. Lysates were analyzed by Western blot at 48 h post transfection. Asterisks indicate significant differences to empty vector control (unpaired t-test). ( E ) VDR and Snail1 expression was determined at 96 h p.i. in lysates of HFF treated with vehicle (DMSO) or 10 μg/mL Ganciclovir (GCV). HCMV immediate early and pp150 late antigens were stained as a control. ( F ) RXR α protein expression in HFF was determined by specific immunoblots at the indicated time points p.i. Representative blot (left) and quantification of bands from multiple experiments relative to the GAPDH loading control (right, one-way ANOVA and Dunnett’s post-hoc test). ( G ) ARPE-19 cells were infected with mock supernatant or the pentamer-positive (TS15-rN) variant of the Towne strain and harvested 14 d p.i. VDR, Snail1, pp150 late antigen and a GAPDH loading control detection by Western blot is shown.

Article Snippet: Single IE1/2, DNA-PKcs and Snail1-targeting siRNAs as well as non-targeting control siRNA siCTRL were produced by Microsynth (Austria) with 3’ dTdT overhangs (see ).

Techniques: Expressing, Infection, Western Blot, Transfection, Plasmid Preparation, Staining, Variant Assay

Journal: Viruses

Article Title: Human Cytomegalovirus Induces Vitamin-D Resistance In Vitro by Dysregulating the Transcriptional Repressor Snail

doi: 10.3390/v14092004

Figure Lengend Snippet: Snail phosphorylation and regulation by DNA-Protein kinase. ( A ) Lysates from mock- and HCMV-infected cells were harvested at 24 h p.i. and subjected to Phos-Tag SDS-PAGE. As additional controls, CMV-infected HFF were treated with a phosphatase inhibitor for 2 h before lysis (calyculin A), or with lambda phosphatase for 30 min λ ). A representative Snail1-specific immunoblot shows the ratio between unphosphorylated (black arrows) and phosphorylated Snail1 (red circles). The same blot is shown with short (left) and long exposure (right). Height of phosphorylated bands also present in the CMV condition is marked with full red circles, open red circles indicate further (weak) phosphorylation bands in the calyculin A treatment control. ( B ) Representative Western blot showing DNA-PK and Snail protein expression determined in lysates of HCMV-infected HFF transfected with a non-targeting control siRNA or DNA-PK specific siRNAs and harvested at 24 h p.i. (representative Western blot on the left and quantification relative Snail1 band intensity of multiple independent experiments on the right; asterisks show significant differences to the CMV siCTRL sample as determined by ANOVA Dunnett’s multiple comparison post-hoc test).

Article Snippet: Single IE1/2, DNA-PKcs and Snail1-targeting siRNAs as well as non-targeting control siRNA siCTRL were produced by Microsynth (Austria) with 3’ dTdT overhangs (see ).

Techniques: Infection, SDS Page, Lysis, Western Blot, Expressing, Transfection

Journal: Viruses

Article Title: Human Cytomegalovirus Induces Vitamin-D Resistance In Vitro by Dysregulating the Transcriptional Repressor Snail

doi: 10.3390/v14092004

Figure Lengend Snippet: Snail ablation restores VDR expression. ( A ) VDR and Snail protein expression was determined in lysates of HFF transfected with a non-targeting control siRNA or Snail1 specific siRNA and harvested at 24 h p.i. Representative blot (left) and quantification of bands from multiple experiments relative to the GAPDH loading control (right). Asterisks show significant differences to the CMV siCTRL control sample as determined by ANOVA Dunnett’s multiple comparison post-hoc test. ( B ) Plaque assay showing the percentage of plaques in siRNA-transfected cells compared to an untransfected control. Asterisks show significant differences in two different Snail-targeting siRNAs to the CMV siCTRL control sample as determined by ANOVA Dunnett’s multiple comparison post-hoc test. Light microscope pictures of representative areas are shown on the right.

Article Snippet: Single IE1/2, DNA-PKcs and Snail1-targeting siRNAs as well as non-targeting control siRNA siCTRL were produced by Microsynth (Austria) with 3’ dTdT overhangs (see ).

Techniques: Expressing, Transfection, Plaque Assay, Light Microscopy