Journal: Nature cell biology

Article Title: Snail/Slug-YAP/TAZ Complexes Control Skeletal Stem Cell Self-Renewal and Differentiation

doi: 10.1038/ncb3394

Figure Lengend Snippet: (a) Western blot of p-YAP, p-TAZ, p-Lats1 and p-Mst1 in SSCs isolated from Snail f/f /Slug +/+ and Snail f/f /Slug −/− mice and transduced with adeno-GFP or Cre. Results are representative of 3 experiments performed. (b) Association of YAP/TAZ with Snail was detected by immunoprecipitation in Cos-1 cells transfected with Flag-YAP, Flag-TAZ and Snail-HA, respectively, as indicated. Results are representative of 3 experiments performed. (c) Association of YAP/TAZ with Slug was detected by immunoprecipitation in Cos-1 cells transfected with Flag-YAP, Flag-TAZ and Slug-Myc, respectively, as indicated. Results are representative of 3 experiments performed. (d) Associations between endogenous YAP/TAZ and Snail/Slug were detected in SSCs cultured in the absence or presence of osteogenic medium. Results are representative of 3 experiments performed. (e) BMP2 increases Snail/Slug-YAP/TAZ complex formation. Calvarial osteoblast progenitors isolated from E17.5 mice were treated with 100 ng/ml BMP2 for 2 h. Endogenous Snail/Slug-YAP/TAZ complexes were detected by immunoprecipitation. Results are representative of 3 experiments performed. (f) In situ proximity ligation assay detection of endogenous Snail/YAP/TAZ and Slug/YAP/TAZ interactions in human SSCs. siRNAs: siGFP, siSNAIL, siSLUG or siTEADs(1–4) were transfected, respectively. Nuclei were counterstained with DAPI (blue). The detected interaction sites are marked by fluorescent dots (red). Scale bar: 5μm. Results are representative of 3 experiments performed.

Article Snippet: Human or mouse TEF-1, TEF-3, TEF-4 and TEF-5 (TEAD1–4) siRNAs, and human SNAIL or SLUG siRNAs were obtained from Santa Cruz Technology.

Techniques: Western Blot, Isolation, Transduction, Immunoprecipitation, Transfection, Cell Culture, In Situ, Proximity Ligation Assay

Journal: Cell Death and Differentiation

Article Title: A novel microRNA regulator of prostate cancer epithelial–mesenchymal transition

doi: 10.1038/cdd.2017.69

Figure Lengend Snippet: miR-3622a inhibits EMT by direct targeting of EMT effectors in PCa. (a) Relative ZEB1, SNAI2 and CTNNB1 mRNA expression upon indicated treatments in PPEC, BPH1 cells and PCa cell lines (PC3, LNCaP) as assessed by real-time PCR. The data were normalized to GAPDH control. (b)Immunoblots for endogenous ZEB1, SNAI2, CTNNB1 protein in anti-miR-CON/ anti-miR-3622a-transduced BPH1 cells (left panels) and miR-CON/ miR-3622a transfected PC3 cells (middle panels). Immunoblots for LNCaP cells are represented in right panels. GAPDH was used a loading control.(c) Schematic representation of ZEB1, CTNNB1, SNAI2 3′-UTRs showing putative miR-3622a target site/sites. For luciferase reporter assays, the potential miR-3622a binding sites were mutated to the sequences shown below. (d) Luciferase reporter assays with the indicated wild type and mutated 3′-UTR constructs or control luciferase construct co-transfected with anti-miR-CON/anti-miR-3622a-transfected BPH1 cells (left panel) and miR-CON/miR-3622a-transfected PC3 and LNCaP cells (middle and right panels, respectively). Firefly luciferase values were normalized to Renilla luciferase activity and plotted as relative luciferase activity

Article Snippet: Trilencer-27 predesigned siRNA (Origene) was used for siRNA-mediated knockdowns of ZEB1 (SR304746), SNAI2 (SR304468), and CTNNB1 (SR301063).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Transfection, Luciferase, Binding Assay, Construct, Activity Assay

Journal: Cell Death and Differentiation

Article Title: A novel microRNA regulator of prostate cancer epithelial–mesenchymal transition

doi: 10.1038/cdd.2017.69

Figure Lengend Snippet: ZEB1 and SNAI2 are functionally relevant targets of miR-3622a in prostate cancer. PC3 cells were transfected with siRNA specific to ZEB1/SNAI2/CTNNB1 or a nonspecific (NS) control siRNA for 72 h followed by functional assays (a,b). (a) Transwell invasion assay and (b) migration assay in NS/ZEB1/SNAI2/CTNNB1 siRNA-transfected PC3 cells. Scale bar: 100 μm. To determine whether ZEB1 and SNAI2 are the key mediators of EMT induced by miR-3622a inhibition in the non-transformed BPH1 cell line, we performed siRNA-mediated inhibition of ZEB1 and SNAI2 in miR-3622a-inhibited BPHI cells (Anti-miR-3622a BPHI.) (Supplementary Figure S4B) followed by functional assays (c–e). (c) Transwell invasion assay and (d) migration assay in NS/ZEB1/SNAI2 siRNA-transfected BPH1 cells. Scale bar: 200 μm. (e) Relative mRNA expression of CDH1 in NS/ZEB1/SNAI2 siRNA-transfected BPH1 cells as assessed by real time PCR. The data were normalized to GAPDH control. (f) Relative expression levels of ZEB1 and SNAI2 (each normalized to GAPDH) and miR-3622a (normalized to RNU48) as assessed by real-time PCR in prostate cancer clinical tissues

Article Snippet: Trilencer-27 predesigned siRNA (Origene) was used for siRNA-mediated knockdowns of ZEB1 (SR304746), SNAI2 (SR304468), and CTNNB1 (SR301063).

Techniques: Transfection, Functional Assay, Transwell Invasion Assay, Migration, Inhibition, Transformation Assay, Expressing, Real-time Polymerase Chain Reaction

Journal: International Journal of Clinical and Experimental Pathology

Article Title: YAP promotes epithelial mesenchymal transition by upregulating Slug expression in human colorectal cancer cells

doi:

Figure Lengend Snippet: YAP inhibits E-cadherin expression by up-regulation of Slug. A. The primary SW620 cells were respectively transfected with YAP and its control plasmids, ShYAP and its control shRNA for 40 h by western blotting assays. Western blot assays show that YAP promotes Slug expression and reduces E-cadherin expression. B. Western blot is used to detect the changes of E-cadherin expression by transiently transfected interfering Slug siRNA in stable SW620 cell line overexpressing YAP. Western blot assays show that knockdown of Slug promotes the expression of E-cadherin. C. Western blot is used to detect the expression changes of E-cadherin by transiently transfected overexpressing Slug plasmid in stable SW620 cell line knocking down YAP. Western blot assays show that overexpressing Slug inhibits the expression of E-cadherin. GAPDH was used as an internal control. The above experiments were performed at least three times. *P<0.05, **P<0.01, ***P<0.001 vs. CTRL.

Article Snippet: Two different double-strand SLUG-targeting siRNA oligonucleotides (GenePharma, China) were used together; the Slug siRNA1 sequences were as follows: 5’-GGACCACAGTGGCTCAGAA-3’, and the Slug siRNA2 sequences were as follows: 5’-CCTCACTGCAACAGAGCATTT-3’.

Techniques: Expressing, Transfection, Control, shRNA, Western Blot, Knockdown, Plasmid Preparation

Journal: BioMed Research International

Article Title: Emodin Inhibits the Epithelial to Mesenchymal Transition of Epithelial Ovarian Cancer Cells via ILK/GSK-3 β /Slug Signaling Pathway

doi: 10.1155/2016/6253280

Figure Lengend Snippet: Emodin inhibited EMT in A2780 and SK-OV-3 cells through targeting Slug. (a) Different concentration of emodin exposure for 48 hours upregulated the protein expression of E-cadherin and Claudin and downregulated protein expression of N-cadherin, Vimentin, and Slug in A2780 and SK-OV-3 cells. (b) Quantitative analyses of E-cadherin, N-cadherin, and Slug in SK-OV-3 cells shown in (a). ∗ P < 0.05, ∗∗ P < 0.005, and ∗∗∗ P < 0.001. (c) A2780 and SK-OV-3 cells were transfected with siRNA of Slug and cultured for an additional 48 h treated with emodin 20 μ M or without emodin. Western blot analysis results of Slug, E-cadherin, and N-cadherin protein levels were shown. (d) Quantitative analyses of markers in SK-OV-3 cells shown in (c). ∗ P < 0.05, ∗∗ P < 0.005, and ∗∗∗ P < 0.001. # P < 0.05. (e) Representative transwell migration and invasion assay of A2780 and SK-OV-3 cells after transfection with siRNA of Slug with or without emodin treatment. (f) Quantification of migration and invasion capacity of SK-OV-3 cells in (e). ∗∗ P < 0.005 and ∗∗∗ P < 0.001. # P < 0.05 and ## P < 0.005. N.C. represents transfection of siRNA-control.

Article Snippet: Slug and ILK specific siRNA and their control were purchased from GenePharma Company (Shanghai, China).

Techniques: Concentration Assay, Expressing, Transfection, Cell Culture, Western Blot, Migration, Invasion Assay, Control

Journal: BioMed Research International

Article Title: Emodin Inhibits the Epithelial to Mesenchymal Transition of Epithelial Ovarian Cancer Cells via ILK/GSK-3 β /Slug Signaling Pathway

doi: 10.1155/2016/6253280

Figure Lengend Snippet: Emodin repressed EMT by targeting ILK in A2780 and SK-OV-3 cells. (a, c) A2780 and SK-OV-3 cells were transfected with siRNA-ILK and treated with or without emodin 20 μ M for 48 hours. Protein levels of ILK, p-GSK-3 β , GSK-3 β , β -catenin, Slug, E-cadherin, and N-cadherin were shown. (b, d) Quantitative analyses of factors in SK-OV-3 cells shown in (a) and (c). ∗ P < 0.05, ∗∗ P < 0.005, and ∗∗∗ P < 0.001. # P < 0.05, ## P < 0.005. (e) Representative transwell migration and invasion assay of A2780 and SK-OV-3 cells after transfection with siRNA of ILK with or without emodin treatment. (f) Quantification of migration and invasion capacity of SK-OV-3 cells in (e). ∗ P < 0.05, ∗∗ P < 0.005, and ∗∗∗ P < 0.001. # P < 0.05, ## P < 0.005. N.C. represents transfection of siRNA-control.

Article Snippet: Slug and ILK specific siRNA and their control were purchased from GenePharma Company (Shanghai, China).

Techniques: Transfection, Migration, Invasion Assay, Control

Journal: Cancer Cell International

Article Title: Mutual regulation of JAG2 and PRAF2 promotes migration and invasion of colorectal cancer cells uncoupled from epithelial–mesenchymal transition

doi: 10.1186/s12935-019-0871-5

Figure Lengend Snippet: Comparison of the TGF-β-induced EMT model and the JAG2-overexpressing cell model. a Western blot analysis of the expression of EMT-related markers in HT29 cells after TGF-β induction and JAG2 overexpression. b Relative cell migration or invasion abilities were determined in HT29 cells treated with TGF-β (5 ng/mL), LY2157299 (100 nm) or Slug siRNA (20 nm) for 24 h. LY2157299 and Slug siRNA could inhibit TGF-β induced migration and invasion of colorectal cells. c Relative cell migration or invasion abilities were determined in JAG2-overexpressed HT29 cells treated with LY2157299 or Slug siRNA. LY2157299 and Slug siRNA could not inhibit JAG2-induced cell migration and invasion. Data are the mean ± SD. of three independent experiments. * P < 0.05 versus control

Article Snippet: In TGF-β induced EMT model, cells were treated with 5 ng/mL TGF-β1 combined with 100 nm LY2157299 (Selleck, Houston, USA) or 20 nm Slug siRNA for 24 h. The siRNAs were purchased from Shanghai GenePharma Co., Ltd (China), and all of them were verified by off-target and validity.

Techniques: Western Blot, Expressing, Over Expression, Migration

Journal: Experimental and Therapeutic Medicine

Article Title: Trichostatin A reverses epithelial-mesenchymal transition and attenuates invasion and migration in MCF-7 breast cancer cells

doi: 10.3892/etm.2020.8422

Figure Lengend Snippet: TSA-mediated suppression of SLUG is involved in reversing EMT. (A) pcDNA-3.1 and pcDNA-SLUG were expressed in MCF-7 cells, and cells with pcDNA-SLUG were treated with or without TSA. Subsequently, the mRNA levels of SLUG, E-cadherin and vimentin were examined by reverse transcription-quantitative polymerase chain reaction. ## P<0.01 and ### P<0.001 vs. pcDNA-3.1. **P<0.01 and ***P<0.001 vs. pcDNA-SLUG. (B) pcDNA-3.1 and pcDNA-SLUG were expressed in MCF-7 cells, and cells with pcDNA-SLUG were incubated with or without TSA. Subsequently, the protein levels of SLUG, E-cadherin and vimentin were examined by western blot analysis. (C) SLUG siRNA or control siRNA was transfected in MCF-7 cells, and images were captured of the invasive and migrated cells. Scale bar=200 µm. *P<0.05 and ***P<0.001 vs. control. (D) SLUG siRNA or control siRNA were transfected in MCF-7 cells, and subsequently the expression levels of SLUG, E-cadherin, and vimentin protein were examined by western blot analysis. TSA, trichostatin A; EMT, epithelial-mesenchymal transition; SLUG, zinc finger protein SNAI2; E-cadherin, epithelial cadherin; siRNA, small interfering RNA.

Article Snippet: A validated negative control oligonucleotide (5′-GCAACGUACAGUGGUUCAA-3′/5′-UUGAACCACUGUACGUUGC-3′, Guangzhou RiboBio Co., Ltd.) and the siRNA oligonucleotides targeting SLUG (5′-GCAUUUGCAGACAGGUCAA-3′/5′-UUGAACUGUCUGCAAAUGC-3′, Guangzhou RiboBio Co., Ltd, Guangzhou, China) were used for transfection.

Techniques: Real-time Polymerase Chain Reaction, Incubation, Western Blot, Transfection, Expressing, Small Interfering RNA

Journal: Experimental and Therapeutic Medicine

Article Title: Trichostatin A reverses epithelial-mesenchymal transition and attenuates invasion and migration in MCF-7 breast cancer cells

doi: 10.3892/etm.2020.8422

Figure Lengend Snippet: Protein levels of SLUG, E-Cadherin and vimentin in cells transfected with control siRNA or SLUG siRNA.

Article Snippet: A validated negative control oligonucleotide (5′-GCAACGUACAGUGGUUCAA-3′/5′-UUGAACCACUGUACGUUGC-3′, Guangzhou RiboBio Co., Ltd.) and the siRNA oligonucleotides targeting SLUG (5′-GCAUUUGCAGACAGGUCAA-3′/5′-UUGAACUGUCUGCAAAUGC-3′, Guangzhou RiboBio Co., Ltd, Guangzhou, China) were used for transfection.

Techniques: Transfection, Expressing

Journal: Cancer Science

Article Title: Radiation promotes malignant phenotypes through SRC in breast cancer cells

doi: 10.1111/cas.12574

Figure Lengend Snippet: Radiation-activated SRC transduces intracellular signaling of phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) and p38 MAPK to increase migratory and invasive behavior. (a) PI3 kinase assay and Western blot analysis for phosphorylation status of AKT and p38 MAPK in MCF7 cancer cells transfected with siRNA targeting SRC or scrambled control siRNA (si-Cont) prior to irradiation. (b) Western blot analysis for phosphorylation status of AKT in MCF7 cancer cells transfected by dominant negative (DN)-p38 or control pCMV5 prior to irradiation. (c) Migration and invasion assay in MCF7 breast cancer cells transfected with siRNA targeting AKT (si-AKT) or scrambled control siRNA prior to irradiation. β-actin was used as a loading control. Error bars represent mean ± SD of triplicate samples. ** P < 0.01. PTEN, phosphatase and tensin homolog. Cont, control.

Article Snippet: All siRNAs targeting c-Src, AKT, and SLUG and a negative control siRNA were purchased from Genolution Pharmaceuticals (Seoul, Korea).

Techniques: Kinase Assay, Western Blot, Phospho-proteomics, Transfection, Control, Irradiation, Dominant Negative Mutation, Migration, Invasion Assay

Journal: Cancer Science

Article Title: Radiation promotes malignant phenotypes through SRC in breast cancer cells

doi: 10.1111/cas.12574

Figure Lengend Snippet: Irradiation promotes breast cancer stem cell populations through SRC signaling. (a) Quantification of CD44 + /CD24 − cell population by FACS analysis in MCF7 and SKBR3 cancer cells after irradiation. (b) Quantification of CD44 + cell population by FACS analysis in MCF7 cancer cells transfected with siRNA targeting SRC (si-SRC) or scrambled control siRNA (si-Con) prior to irradiation. (c, d) Western blot analysis for CD44, SOX2, Notch-1, and Notch-2 (c), and immunocytochemistry for CD44 (d) in MCF7 cancer cells transfected with siRNA targeting SRC or scrambled control siRNA prior to irradiation. (e, f) Quantification of the CD44 + cell population by FACS analysis in MCF7 cancer cells transfected with siRNA targeting AKT (si-AKT) (e) or p38 MAPK (si-p38) (f) prior to irradiation. (g, h) Quantification of the CD44 + cell population by FACS analysis in MCF7 (g) and SKBR3 (h) cancer cells 48 h after transfection with SRC WT, mutant form SRC Y527F, or control vector pcDNA3.1. (i) Western blot analysis for SOX2 in MCF7 cells 48 h after transfection with SRC WT, mutant form SRC Y527F, or control vector pcDNA3.1. β-actin was used as a loading control. Error bars represent mean ± SD of triplicate samples. * P < 0.05; ** P < 0.01. Cont, control.

Article Snippet: All siRNAs targeting c-Src, AKT, and SLUG and a negative control siRNA were purchased from Genolution Pharmaceuticals (Seoul, Korea).

Techniques: Irradiation, Transfection, Control, Western Blot, Immunocytochemistry, Mutagenesis, Plasmid Preparation