Journal: International Journal of Molecular Sciences

Article Title: Mechanism of THBS1 Regulation of MDCK Cell Proliferation and Apoptosis Through TGF-β/Smad Signalling

doi: 10.3390/ijms26010395

Figure Lengend Snippet: Determination of optimal concentration of TGF-β activator SRI-011381 on THBS1 -sh-122484 cells and inhibitor LY2109761 on THBS1 -OE cells, and its effect on the expression of target genes, THBS1 , downstream of TGF-β/Smad signalling. ( a ) Effect of different concentrations of SRI-011381 on the growth status of THBS1 -sh-122484 cells. ( b ) Effects of different concentrations of LY2109761 on the growth status of THBS1 -OE cells. ( c ) Effects of different concentrations of SRI-011381 on the differential expression of target genes downstream of TGF-β/Smad signalling and THBS1 mRNA levels in THBS1 -sh-122484 cells. ( d ) Effects of different concentrations of LY2109761 on the expression of target genes downstream of TGF-β/Smad signalling and THBS1 , and effects of different concentrations of LY2109761 on the expression of target genes downstream of THBS1 -Sh-122484 cells. LY2109761 on THBS1 -OE cells TGF-β/Smad signalling downstream target genes and THBS1 mRNA level expression differences. ( e ) Effect of different concentrations of SRI-011381 on THBS1 -sh-122484 cells and different concentrations of LY2109761 on THBS1 -OE cells TGF-β/Smad signalling downstream target genes and THBS1 protein level expression differences. ( f ) Grey scale analysis of the effects of different concentrations of SRI-011381 on the expression of target genes and THBS1 protein level downstream of TGF-β/Smad signalling in THBS1 -sh-122484 cells. ( g ) The effects of different concentrations of LY2109761 on the expression of TGF-β/Smad signalling downstream of TGF-β/Smad protein in THBS1 -OE cells target gene and THBS1 protein level expression grey value analysis. * indicates statistically significant difference (* p < 0.05; ** p < 0.01; *** p < 0.001) and no * indicates no difference.

Article Snippet: To further evaluate the mechanism by which THBS1 regulates MDCK cell proliferation and apoptosis through TGF-β/Smad signalling, we observed the cell growth status with different concentration gradients of the TGF-β/Smad signalling pathway activator SRI-011381 (MCE, Shanghai, China, HY-12075) of THBS1-knockdown cells ( THBS1 -sh122484) and the inhibitor LY2109761 (MCE, Shanghai, China, HY-100347) of THBS1 -overexpressing cells ( THBS1 -OE).

Techniques: Concentration Assay, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Mechanism of THBS1 Regulation of MDCK Cell Proliferation and Apoptosis Through TGF-β/Smad Signalling

doi: 10.3390/ijms26010395

Figure Lengend Snippet: Effect of SRI-011381 (5 μg/mL) on the expression of PI3K/Akt , target genes downstream of P53 signalling pathway, SCARB2 in knockdown THBS1 cells ( THBS1 -sh-122484), and LY2109761 (10 μg/mL) in overexpressing THBS1 ( THBS1 -OE) cells. ( a ) SRI-011381 (5 μg/mL) intervention in THBS1 -sh-122484 cells showed differential expression of PI3K/Akt , target genes downstream of P53 signalling, and SCARB2 at the mRNA level. ( b ) LY2109761 (10 μg/mL) intervention in THBS1 -OE cells showed differential expression of PI3K/Akt , target genes downstream of P53 signalling, and SCARB2 expression differences at the mRNA level. ( c ) PI3K/Akt , P53 signalling downstream target genes, and SCARB2 expression differences at the protein level after SRI-011381 (5 μg/mL) intervention in THBS1 -sh-122484 cells and LY2109761 (10 μg/mL) intervention in THBS1 -OE cells. ( d ) SRI-011381 (5 μg/mL) intervention in THBS1 -sh-122484 cells after PI3K/Akt , P53 signalling downstream target genes, and SCARB2 expression differences at protein level grey value analysis. ( e ) LY2109761 (10 μg/mL) intervention in THBS1 -OE cells after PI3K/Akt , P53 signalling downstream target genes, and SCARB2 expression difference at protein level grey value analysis. * indicates statistically significant difference (* p < 0.05; *** p < 0.001) and no * indicates no difference.

Article Snippet: To further evaluate the mechanism by which THBS1 regulates MDCK cell proliferation and apoptosis through TGF-β/Smad signalling, we observed the cell growth status with different concentration gradients of the TGF-β/Smad signalling pathway activator SRI-011381 (MCE, Shanghai, China, HY-12075) of THBS1-knockdown cells ( THBS1 -sh122484) and the inhibitor LY2109761 (MCE, Shanghai, China, HY-100347) of THBS1 -overexpressing cells ( THBS1 -OE).

Techniques: Expressing, Knockdown

Journal: International Journal of Molecular Sciences

Article Title: Mechanism of THBS1 Regulation of MDCK Cell Proliferation and Apoptosis Through TGF-β/Smad Signalling

doi: 10.3390/ijms26010395

Figure Lengend Snippet: Effects of SRI-011381 on knockdown THBS1 cells ( THBS1 -sh-122484) and LY2109761 on proliferation and migration ability of overexpressing THBS1 cells ( THBS1 -OE). ( a ) Growth curve of SRI-011381 intervention knockdown THBS1 cells ( THBS1 -sh-122484). ( b , c ) SRI-011381 intervention knockdown THBS1 cells ( THBS1 -sh-122484) migration ability assay. ( d ) SRI-011381 intervention knockdown THBS1 cells ( THBS1 -sh-122484) apoptosis ability assay. ( e ) LY2109761 intervention overexpression THBS1 cells (THBS1-OE) growth curve. ( f , g ) LY2109761 intervention overexpression THBS1 cells ( THBS1 -OE) migration ability assay. ( h ) LY2109761 intervention overexpression THBS1 cells (THBS1-OE) apoptosis ability assay. * indicates statistically significant difference (** p < 0.01; *** p < 0.001) and no * indicates no difference.

Article Snippet: To further evaluate the mechanism by which THBS1 regulates MDCK cell proliferation and apoptosis through TGF-β/Smad signalling, we observed the cell growth status with different concentration gradients of the TGF-β/Smad signalling pathway activator SRI-011381 (MCE, Shanghai, China, HY-12075) of THBS1-knockdown cells ( THBS1 -sh122484) and the inhibitor LY2109761 (MCE, Shanghai, China, HY-100347) of THBS1 -overexpressing cells ( THBS1 -OE).

Techniques: Knockdown, Migration, Over Expression

Journal: Cell Death & Disease

Article Title: Dexamethasone mediates pancreatic cancer progression by glucocorticoid receptor, TGF β and JNK/AP-1

doi: 10.1038/cddis.2017.455

Figure Lengend Snippet: Dexamethasone induces TGF β -associated EMT and metastasis in vitro . ( a ) The cells were treated with 1 μ M dexamethasone (DEX) for 0.5 to 48 h as indicated, and the expression of TGF β was detected by western blot analysis as described above. ( b ) The amount of soluble TGF β 1 ligand (pg/ml) in AsPC-1 cell culture supernatant was examined by ELISA assay after dexamethasone (DEX, 1 μ M) treatment as described above. The glucocorticoid receptor antagonist mifepristone-RU486 (RU, 1 μ M) was added 30 min before dexamethasone treatment. Vehicle controls were CO1 (EtOH 1 : 25 000 for DEX) and CO2 (EtOH 1 : 12 500 for DEX and RU486). Error bars are shown together with the significance between DEX and CO1 and between DEX+RU486 and CO2. * P <0.05 and ** P <0.01. The values were calibrated to the NIBSC/WHO TGF- β 1 International Reference Reagent 87/514. ( c ) AsPC-1 cells were left untreated (CO) or were precultured in 1 μ M dexamethasone (DEX) for 48 h. Then, 50 nM gemcitabine was added to untreated cells (GEM), and DEX-treated cells (D&G) for another 48 h in the presence or absence of the TGF β R-I/II kinase inhibitor LY2109761 (TGF-Inh. LY, 10 μ M) that was administered together with dexamethasone to the groups indicated. The viability was measured by MTT assay. The percentage of viable cells in the control groups was set to 100%, ** P <0.01. Representative photographs of morphology are shown below the diagrams in the mentioned five groups. ( d ) ASPC-1 cells were treated as described above. At 48 h after gemcitabine treatment, 400 viable cells/well were seeded at clonal density into 6-well plates. The cells were grown without a change of medium for 2 weeks, followed by the evaluation of fixed and Coomassie-stained colonies consisting of 50 cells at least. The plating efficiency, expressed as a percentage, was calculated by the following formula: 100 × number of colonies/number of seeded cells, * P <0.05, ** P <0.01. Representative photographs of colonies 2 weeks after the incubation are shown below the diagrams in the mentioned five groups. ( e ) AsPC-1 cells were treated as described above and then seeded in ultra-low attachment plates at a low density of 1 × 10 4 cells/ml in serum-free, growth factor-containing medium for spheroid formation. After 10 days, photographs were obtained and photographed under × 100 magnification. To detect the number of spheroidal-growing cells, the spheroids were dissolved, and the single cells were counted. The percentage of spheroidal-growing cells relative to the seeded cells was evaluated, * P <0.05, ** P <0.01. Representative photographs of spheroids are shown below the diagrams in the mentioned five groups. ( f ) Cells were left untreated (CO) or were cultured in 1 μ M dexamethasone (DEX) for 2 to 24 h as indicated, followed by examination of SOX2 and Notch1 expression by western blot analysis. ( g ) Representative paraffin-embedded sections (HD 6065-1C, HD 5776-1A, HD 6144-1B, HD 5909-1E, HD 6055-1C, HD 5522-1A) derived from primary PDA tissue from patients with documented preoperative administration of glucocorticoids (GC, n =17) or not (CO, n =17) were evaluated by immunofluorescence staining to detect the expression of TGF β (red), SOX2 (red) and Notch1. The cell nuclei were counterstained with DAPI (blue). The sections were analyzed under × 400 magnification, and representative images are shown. For evaluation of the expression levels, a semiquantitative scoring system was used, as described in

Article Snippet: LY2109761 (10 mM, ≥99% pure, Selleckchem, Munich, Germany) and SP600125 (20 mM, ≥99% pure, Selleckchem) were prepared in DMSO.

Techniques: In Vitro, Expressing, Western Blot, Cell Culture, Enzyme-linked Immunosorbent Assay, MTT Assay, Control, Staining, Incubation, Derivative Assay, Immunofluorescence

Journal: Cell Death & Disease

Article Title: Dexamethasone mediates pancreatic cancer progression by glucocorticoid receptor, TGF β and JNK/AP-1

doi: 10.1038/cddis.2017.455

Figure Lengend Snippet: Dexamethasone mediates tumor progression by a complex signaling network. ( a ) AsPC-1 cells were left untreated (CO1) or were treated with vehicle alone (CO2: EtOH 1 : 25 000; CO3 EtOH 1 : 12 500; CO4 EtOH 1 : 12 500+DMSO 1 : 1000; CO5 EtOH 1 : 12 500+DMSO 1 : 2000) or with mifepristone/RU486 (RU, 1 μ M), LY2109761 (LY, 10 μ M) and SP600125 (SP, 10 μ M). After 30 min, 1 μ M dexamethasone (DEX) was added. After 48 h, 50 nM gemcitabine was added to the respective groups (GEM, D&G) for additional 96 h, followed by MTT assay. The percentage of viable cells in the control groups was set to 100%, and the significance was evaluated. ** P <0.01. ( b ) AsPC-1 cells were left untreated or were treated with mifepristone/RU486 (RU, 1 μ M), LY2109761 (LY, 10 μ M) and SP600125 (SP, 10 μ M) and the respective vehicle controls. After 30 min, 1 μ m dexamethasone (DEX) was added to the groups indicated and the expression of TGF β , P-Smad2, Notch1, SOX2, P-cJun and cJun was examined by western blot analysis. β -Actin served as loading control. ( c ) Column diagrams depicting the relative protein levels of the western blot bands above, as detected by Image Studio (Li-COR Biosciences GmbH, Bad Homburg vor der Höhe, Germany) after normalization to β -actin. The pixel density in the controls was set to 1, * P <0.05, ** P <0.01. ( d ) MIA-PaCa2 cells were treated as described in , followed by transplantation of 1 × 10 6 viable cells to fertilized chicken eggs ( n =15 per group) at day 9 of embryonic development. The tumor xenografts were analyzed as described in . Representative images of the tumor sizes are shown, * P <0.05, ** P <0.01. ( e ) Genomic DNA was isolated from CAM ( n =10) tissue directly adjacent to the tumor xenografts and the expression of human Alu sequences was evaluated as described in

Article Snippet: LY2109761 (10 mM, ≥99% pure, Selleckchem, Munich, Germany) and SP600125 (20 mM, ≥99% pure, Selleckchem) were prepared in DMSO.

Techniques: MTT Assay, Control, Expressing, Western Blot, Transplantation Assay, Isolation

Journal: Cell Death & Disease

Article Title: Dexamethasone mediates pancreatic cancer progression by glucocorticoid receptor, TGF β and JNK/AP-1

doi: 10.1038/cddis.2017.455

Figure Lengend Snippet: Model of dexamethasone mediated cross-signaling leading to tumor progression. Dexamethasone (DEX) enters the cytoplasm and binds to the glucocorticoid receptor (GR) to form glucocorticoid–glucocorticoid receptor homodimers that enter the nucleus, interact with other transcription factors such as cJun/AP-1 and bind to glucocorticoid receptor element-containing target genes, involving TGF β and cJun. This activates the expression of TGF β and cJun/AP-1. cJun/AP-1 binds to its own and to the TGF β promoter and reinforces the expression. Besides, cJun/AP-1 binds to other target genes and thereby activates, for example, the expression of Notch-1, SOX-2 and vimentin. Soluble TGF β ligand is secreted and binds to the surface receptor TGF β R-I/II kinase complex, leading to the intracellular recruitment and phosphorylation of Smad proteins. Activated Smad proteins associate with Smad4 and translocate to the nucleus, where they interact with transcription factors, coactivators, corepressors and chromatin remodeling factors that control the expression of numerous target genes. As a consequence, E-cadherin and vimentin are expressed. Soluble TGF β ligand can also activate other surface membrane receptors and thereby activates Ras/JNK signaling that contributes to the scenario by increasing cJun/AP-1 expression. All these amplifying mechanisms together may converge into proliferation, EMT and metastasis. The glucocorticoid receptor antagonist mifepristone (RU486), the TGF β R-I/II kinase inhibitor LY2109761 and the JNK inhibitor SP600125 interfere with this crosstalk, but none of the inhibitors on their own are able to completely block it

Article Snippet: LY2109761 (10 mM, ≥99% pure, Selleckchem, Munich, Germany) and SP600125 (20 mM, ≥99% pure, Selleckchem) were prepared in DMSO.

Techniques: Expressing, Phospho-proteomics, Control, Membrane, Blocking Assay