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: Journal of Cell Communication and Signaling

Article Title: TGF-β1 and TNF-α synergistically induce epithelial to mesenchymal transition of breast cancer cells by enhancing TAK1 activation

doi: 10.1007/s12079-019-00508-8

Figure Lengend Snippet: The up-regulation of TGF-β receptors contributes to the gradually enhanced activation of TAK1 during long-lasting co-stimulation. a–c MCF-7 cells were cultured in absence or presence of TGF-β1/TNF-α (left) for the indicated time. Or the cells were cultured for 6 days in presence of TGF-β1 and or TNF-α. The expression of TGF-βRI (a), and TGF-βRII (b) was detected by real-time RT-PCR. c The expression of TβRI and TβRII was detected by Western blot after 6-d culture (left). Relative expression of TβRI and TβRII were calculated after densitometry assay as standardized by β-actin (right). d MCF-7 cells were unstimulated or stimulated with TGF-β1/TNF-α in absence or presence of SIS3 (10 μM), QNZ (40 nM), SB203580 (SB, 10 μM), PD98059 (PD, 10 μM) and SP600125 (SP, 10 μM) for 6 days. The expression of TGF-βRI (left) and TGF-βRII (right) was detected by real-time RT-PCR. e–h MCF-7 cells were transducted with control, TβRI or TβRII shRNA lentivirus. And then the cells were selected for stable expression using puromycin. e The expression of TβRI and TβRII was detected by Western blot (left). Relative expression of TβRI and TβRII was calculated after densitometry assay as standardized by β-actin (right). f The phospho-TAK1, TAK1, phospho-Smad2, Smad2, phospho-Smad3 and Smad3 were detected by Western blot. g The activity of NF-κB was assayed as described in Methods. h The ratio of phosphorylated protein to total protein of p38 MAPK, ERK1/2 and JNK was calculated after densitometric analysis of the blots. Data are representative of three independent experiments, or pooled from three independent experiments. P values, * P < 0.05, **P < 0.01

Article Snippet: Cell transduction Control shRNA lentiviral particles (sc-108,080), TβRI lentiviral particles (sc-40,222-V), TβRII shRNA lentiviral particles (sc-36,658-V) and SLUG shRNA lentiviral particles (sc-38,393-V) were all purchased from Santa Cruz Biotechnology (sc-).

Techniques: Activation Assay, Cell Culture, Expressing, Quantitative RT-PCR, Western Blot, Control, shRNA, Activity Assay

Journal: Journal of Cell Communication and Signaling

Article Title: TGF-β1 and TNF-α synergistically induce epithelial to mesenchymal transition of breast cancer cells by enhancing TAK1 activation

doi: 10.1007/s12079-019-00508-8

Figure Lengend Snippet: The sustained activation of signaling pathways and up-regulation of SLUG are required for inducing EMT and invasiveness in breast cancer cells. a and b MCF-7 cells were stimulated or unstimulated with TGF-β1 and TNF-α for 6 days in absence or presence of (5Z)-7-oxozeaenol. a The expression of E-cadherin and vimentin was detected by Western blot. b The cells were used for matrigel invasion assay. c and d MCF-7 cells were stimulated or unstimulated with TGF-β1 and TNF-α for 6 days in absence or presence of SIS3, QNZ, SB203580, PD98059 and SP600125 for 6 days. c The expression of E-cadherin and vimentin was detected by Western blot (left). Relative expression of E-cadherin and vimentin were calculated after densitometry assay as standardized by β-actin (right). d The cells were used for matrigel invasion assay. e MCF-7 cells were cultured in presence of TGF-β1 and/or TNF-α for 6 days. The expression of SLUG, SNAIL, ZEB1 and TWIST1 was detected by real-time RT-PCR. f–h MCF-7 cells were transducted with control or SLUG shRNA lentivirus, and then selected for stable expression using puromycin. f The expression of SLUG, E-cadherin, vimentin and β-actin was detected by Western blot. g Relative expression of SLUG, E-cadherin and vimentin were calculated after densitometry assay as standardized by β-actin. h The cells were used for matrigel invasion assay. Data are representative of three independent experiments, or pooled from three independent experiments. P values, * P < 0.05, **P < 0.01

Article Snippet: Cell transduction Control shRNA lentiviral particles (sc-108,080), TβRI lentiviral particles (sc-40,222-V), TβRII shRNA lentiviral particles (sc-36,658-V) and SLUG shRNA lentiviral particles (sc-38,393-V) were all purchased from Santa Cruz Biotechnology (sc-).

Techniques: Activation Assay, Protein-Protein interactions, Expressing, Western Blot, Invasion Assay, Cell Culture, Quantitative RT-PCR, Control, shRNA

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 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