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: Snail1 siRNA mix sc-38398 (containing 3–5 different siRNAs targeting the same mRNA) was obtained from Santa Cruz Biotechnology.

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

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: Snail1 siRNA mix sc-38398 (containing 3–5 different siRNAs targeting the same mRNA) was obtained from Santa Cruz Biotechnology.

Techniques: Expressing, Infection, Control, Comparison, 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: Snail1 siRNA mix sc-38398 (containing 3–5 different siRNAs targeting the same mRNA) was obtained from Santa Cruz Biotechnology.

Techniques: Phospho-proteomics, Infection, SDS Page, Lysis, Western Blot, Control, Expressing, Transfection, Comparison

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: Snail1 siRNA mix sc-38398 (containing 3–5 different siRNAs targeting the same mRNA) was obtained from Santa Cruz Biotechnology.

Techniques: Expressing, Transfection, Control, Comparison, Plaque Assay, Light Microscopy

Journal: Journal of experimental & clinical cancer research : CR

Article Title: High expression of Collagen Triple Helix Repeat Containing 1 (CTHRC1) facilitates progression of oesophageal squamous cell carcinoma through MAPK/MEK/ERK/FRA-1 activation.

doi: 10.1186/s13046-017-0555-8

Figure Lengend Snippet: Fig. 6 CTHRC1 upregulates cyclin D1 and snail1/MMP14 by activating the MAPK/MEK/ERK/FRA-1 cascade. a The relative mRNA levels of FOSL1, CCND1, SNAI1 and MMP14 in CTHRC1-knockdown cells or CTHRC1-overexpressing cells and control cells were detected by RT-PCR. b Western blot was conducted to detect the protein levels of CTHRC1, p-FRA-1, FRA-1, cyclin D1, snail1 and MMP14 in CTHRC1-knockdown KYSE510 and KYSE30 cells and corresponding vector control cells. c CTHRC1-overexpressing KYSE450 cells and control cells were treated with U0126 (10 μM) or DMSO. The protein levels of CTHRC1, p-FRA-1, FRA-1, cyclin D1, snail1 and MMP14 were detected by western blot. d, e CTHRC1-overexpressing KYSE450 cells and control cells were transfected with FRA-1 siRNA or negative control (NC) siRNA. d Cell viability was measured using the CCK8 assay. e Migration and invasion of cells were investigated using transwell assays. The results are shown as the mean ± SD. The difference between groups was analysed using two-tailed Student’s t-test. *P < 0.05 and **P < 0.01. f CTHRC1-overexpressing KYSE450 cells were transfected with FRA-1 siRNA or snail1 siRNA. The protein levels of CTHRC1, FRA-1, cyclin D1, snail1 and MMP14 in KYSE450 cells with different treatment were detected. g Schematic diagram illustrating the proposed CTHRC1-mediated activation of ERK1/2 signalling pathway and its role in ESCC cells

Article Snippet: Immunohistochemistry (IHC) was performed as previously described [16], using anti-CTHRC1 (ab192778, Abcam, USA), anti-FRA-1 (TA500624S, Origene, USA), anti-cyclin D1 (2978, CST, USA), anti-snail1 (TA500316S, Origene, USA) and anti-MMP14 (ab51047, Abcam, USA) antibodies.

Techniques: Knockdown, Control, Reverse Transcription Polymerase Chain Reaction, Western Blot, Plasmid Preparation, Transfection, Negative Control, CCK-8 Assay, Migration, Two Tailed Test, Activation Assay

Journal: Journal of experimental & clinical cancer research : CR

Article Title: High expression of Collagen Triple Helix Repeat Containing 1 (CTHRC1) facilitates progression of oesophageal squamous cell carcinoma through MAPK/MEK/ERK/FRA-1 activation.

doi: 10.1186/s13046-017-0555-8

Figure Lengend Snippet: Fig. 7 mRNA and protein level of CTHRC1, FRA-1, cyclin D1, snail1 and MMP14 in ESCC tissues. a Correlation between CTHRC1 and CCND1 accord- ing to transcriptome-wide microarray profiling data (n = 119). Pearson’s correlation coefficient. b Correlation between CTHRC1 and SNAI1 accord- ing to transcriptome-wide microarray profiling data (n = 119). Pearson’s correlation coefficient. c Correlation between SNAI1 and MMP14 according to transcriptome-wide microarray profiling data (n = 119). Pearson’s correlation coefficient. d Representative IHC images of FRA-1, snail1, cyclin D1 and MMP14 staining in ESCC tumour tissues. e, f Overall survival analysis based on the level of cyclin D1 and MMP14 expression, as measured by IHC, in ESCC patients. Survival rates were determined using Kaplan-Meier survival analysis

Article Snippet: Immunohistochemistry (IHC) was performed as previously described [16], using anti-CTHRC1 (ab192778, Abcam, USA), anti-FRA-1 (TA500624S, Origene, USA), anti-cyclin D1 (2978, CST, USA), anti-snail1 (TA500316S, Origene, USA) and anti-MMP14 (ab51047, Abcam, USA) antibodies.

Techniques: Microarray, Staining, Expressing

Journal: Journal of Biological Chemistry

Article Title: Lung Tumor-associated Osteoblast-derived Bone Morphogenetic Protein-2 Increased Epithelial-to-Mesenchymal Transition of Cancer by Runx2/Snail Signaling Pathway

doi: 10.1074/jbc.m111.256156

Figure Lengend Snippet: FIGURE 7. Runx2 is the upstream regulatory factor of Snail. A and B, inhibition of Runx2 decreases BMP-2-mediated Snail up-regulation and E-cadherin down-regulation (A), as well as cell migration (B). Cells were transfected with pLKO-AS2 or pLKO-AS2-RUNX2 shRNA. Stable clones were created by puromycin selection, and the efficacy of shRNA was assessed by RT-PCR. Cells were treated with BMP-2 (20 ng/ml) for the specified times (cell migration, 24 h; Runx2 and Snail, 6 h; E-cadherin, 24 h). Then the expression of various proteins was then assessed by immunoblot assay. C, overexpression of Snail reversed the inhibitory effect of Runx2 shRNA on BMP-2-mediated cell migration. Runx2-transfected A549 and CL1–0 cells were transected with pCMV or pSnail plasmid, and stable clones were established by G418 and puromycin. The asterisk indicates a significant difference between control and test groups, as analyzed by Dunnett’s test (p 0.05). The data shown are representative of three independent experiments.

Article Snippet: Runx2-transfectedA549 andCL1–0 cell were transected with pCMV or pSnail plasmid (Origene, FIGURE 1.

Techniques: Inhibition, Migration, Transfection, shRNA, Clone Assay, Selection, Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, Over Expression, Plasmid Preparation, Control

Journal: Nucleic Acids Research

Article Title: Transcription factor 4 maintains endothelial cell identity by inhibiting endothelial to mesenchymal transition

doi: 10.1093/nar/gkaf931

Figure Lengend Snippet: TCF4 modulates the TGFβ signaling pathway in EndoMT. ( A ) qPCR results showing gene expression changes of TGFβ1 after TCF4 KD in HUVECs. ( B ) ELISA results showing protein expression changes of TGFβ after TCF4 KD in HUVECs. ( C ) qPCR results showing gene expression changes of SNAI1 after TCF4 KD in HUVECs. ( D and E ) GSEA of the gene signature on differential gene expression data from TCF4 KD versus control RNA-seq. Results for gene sets of TGFβ signaling pathways from two different sources are shown. ( F and G ) Genomic tracks showing TCF4 peaks at TGFβ1 gene loci (F) and at SNAI1 gene loci (G) (highlighted in orange rectangles), along with other epigenetic signals, including DNase-seq, H3K4me1, H3K4me1, and H3K27ac. ( H–K ) qPCR results showing mRNA expression changes of TCF4 and mesenchymal markers (α-SMA, SNAI1, and Col1a1) in control, TCF4 KD, and TCF4 KD + SB431542 HUVECs. ( L ) Western blot showing changes in protein expression levels after TCF4 KD and SB431542 treatment. ( M–P ) qPCR results showing mRNA expression changes of TCF4 and mesenchymal markers (α-SMA, SNAI1, and Col1a1) in control, TCF4 KD, and TCF4 KD + SNAI1 KD HUVECs. ( Q ) Western blot showing protein expression level change after TCF4 KD and SNAI1 KD. ( R ) Genomic tracks of CUT&RUN showing the binding of TCF4 and SNAI1 at the CDH5 gene region (highlighted in orange rectangles), along with other tracks of DNase-seq and related histone modification. ( S ) SNAI1 ChIP-qPCR in both control and TCF4 KD HUVECs for the SNAI1–CDH5 binding site. ( T ) SNAI1 ChIP-qPCR in both SNAI1 OE and TCF4 OE HUVECs for the SNAI1–CDH5 binding site * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. P -values were determined by two-tailed Student's t -test.

Article Snippet: For the SNAI1 overexpression plasmid (Addgene, #115446), transfection was performed using LipofectamineTM 3000 reagent (Thermo Fisher Scientific) according to the manufacturer's instructions.

Techniques: Gene Expression, Enzyme-linked Immunosorbent Assay, Expressing, Control, RNA Sequencing, Protein-Protein interactions, Western Blot, Binding Assay, Modification, ChIP-qPCR, Two Tailed Test