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: 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

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: The Journal of steroid biochemistry and molecular biology

Article Title: Epithelial-to-mesenchymal transition contributes to the downregulation of progesterone receptor expression in endometriosis lesions.

doi: 10.1016/j.jsbmb.2021.105943

Figure Lengend Snippet: Fig. 3. TGF-β1 dose-dependent changes of gene expression in endometriotic epithelial cell lines (A) EM’osis cells and (B) 12Z cells were treated in triplicates with TGF-β1 (0.125, 0.25, 0.5, 0.75, 1 ng/mL) for 24 h. Values are presented by mean ± SEM of one representative experiment. (C) Expression of CDH2, PGR, and EMT related TFs (ZEB1, SNAI1 and SNAI2) in EM’osis cells treated in four independent experiments with TGF-β1 (1 ng/mL) and TGF-β1 inhibitor (100 nM) for 24 h. Control group were under the equivalent volume of vehicles. (D) Expression of CDH2, PGR, and EMT related TFs (ZEB1, SNAI1 and SNAI2) in 12Z cells treated in four independent experiments with TGF-β1 (0.5 ng/mL) and TGF-β1 inhibitor (100 nM) for 24 h. Values are presented as mean ± SEM and P values were calculated by one-way ANOVA test. * P < 0.05, **P < 0.001, “ns” stands for non-significant.

Article Snippet: 12Z cells were transfected with 20 nM of a pool of equal proportion of 3 siRNA against SNAI1 (SR304489, Human siRNA Oligo Duplex, Locus ID 6615, Origene, USA), as well as SNAI2 (Silencer siRNA ID 106954, Ambion Inc., USA) or scrambled control from the same set of si-SNAI1 (SR304489, Human siRNA Oligo Duplex, Locus ID 6615, Origene USA).

Techniques: Gene Expression, Expressing, Control

Journal: The Journal of steroid biochemistry and molecular biology

Article Title: Epithelial-to-mesenchymal transition contributes to the downregulation of progesterone receptor expression in endometriosis lesions.

doi: 10.1016/j.jsbmb.2021.105943

Figure Lengend Snippet: Fig. 4. EMT related PR repression is SNAI- dependent (A) Silencing SNAI1 in EM’osis cells was done by transfection of siRNA against SNAI1 (20 nM) in triplicates in two indepen dent experiments. Transfections with the same amount of scrambled siRNA, and untreated cells were used as controls. (B) Silencing SNAI1/2 in 12Z cells was performed in triplicates in two independent experiments, using the pooled SNAI1/2 with 2:1 ratio of si-SNAI1 and si- SNAI2. Controls were the same as for EM’osis cells. (A, B) Values are presented as mean ± SEM and P values were calculated by one-way ANOVA test. * P < 0.05, **P < 0.001, “ns” stands for non-significant. (C–F) Representative staining of SNAI1/2 (green) was shown in part of epithelial component, and the representative staining of PR (red) in ectopic epithelial glands (magnification 400x). The grey scale bars represent 50 μm. (G) Correlation analysis on the intensity of fluorescence for SNAI1/2 and PR in epithelial cells. DIE lesions were co-stained with PR and SNAI1/2, and the intensities of fluorescence were quantified by Qupath in each epithelial cells. P value was calculated by Pearson r.

Article Snippet: 12Z cells were transfected with 20 nM of a pool of equal proportion of 3 siRNA against SNAI1 (SR304489, Human siRNA Oligo Duplex, Locus ID 6615, Origene, USA), as well as SNAI2 (Silencer siRNA ID 106954, Ambion Inc., USA) or scrambled control from the same set of si-SNAI1 (SR304489, Human siRNA Oligo Duplex, Locus ID 6615, Origene USA).

Techniques: Transfection, Staining, Fluorescence

Journal: Oncogene

Article Title: Aurora-A kinase oncogenic signaling mediates TGF-β-induced triple-negative breast cancer plasticity and chemoresistance.

doi: 10.1038/s41388-021-01711-x

Figure Lengend Snippet: Fig. 4 Transcriptomic analysis of MDA-MB 231 TNBC cells. a RNA-Seq analysis was performed on MDA-MB 231 cells treated with 10 ng/ml TGF-β1 for 48 h. 14,239 genes were differentially expressed between control and TGF-β1 groups. b STRINGdb software was used to identify a SNAI1/ MMP9/FN1 Network. c Quantification analysis showing FN1, MMP9, and SNAI1 expression before and after TGF-β1 treatment. Experiments were performed in triplicate with a p-value < 0.05. d Real-time quantitative RT- PCR to detect SNA1, FN1, and MMP9 gene expression using MDA-MB 231 cells treated with 10 ng/ml TGF-β1 and infected with scrambled Lenti-shRNA or Lenti-shRNA targeting AURKA (Origene) for 48 h. Three independent experiments were performed in triplicate (±S.D. and P-value < 0.05).

Article Snippet: Scrambled (control), AURKA (Origene, TL320538), SMAD3 (Origene, TL309254), and SNAI1 shRNA lentivectors (Origene, TL309226) were purchased by OriGene Technologies (Rockville, MD, USA) and used according to manufacturer’s instructions.

Techniques: RNA Sequencing, Control, Software, Expressing, Quantitative RT-PCR, Gene Expression, Infection, shRNA