Journal: Scientific Reports

Article Title: Analysis of the relationship between the KRAS G12V oncogene and the Hippo effector YAP1 in embryonal rhabdomyosarcoma

doi: 10.1038/s41598-018-33852-7

Figure Lengend Snippet: Expression of oncogenic KRAS G12V in C2C12 myoblasts does not change Yap/Taz activity. ( A ) Representative Western blots showing pan-RAS, total Yap, Taz, and phosphorylated Yap protein levels in C2C12 myoblasts, with relevant β-actin or Gapdh as loading controls. ( B ) Effect of KRAS G12V expression on 8xGTIIC Yap/Taz Tead1-4 luciferase reporter normalised to Tk-renilla in C2C12 myoblasts. Data is presented as fold change compared to control (mean ± SD) from 3 independent experiments where an asterisk denotes significant difference (p < 0.05) between control and KRAS G12V using a Student’s t-test. ( C ) Confocal images of C2C12 myoblasts immunolabelled with Yap (green) and DAPI (blue). Scale bar equals 20 μm.

Article Snippet: The 8xGTIIC Yap/Taz Tead1-4 luciferase reporter (Addgene plasmid #34615) was co-transfected with TK-renilla plasmid.

Techniques: Expressing, Activity Assay, Western Blot, Luciferase, Control

Journal: Scientific Reports

Article Title: Analysis of the relationship between the KRAS G12V oncogene and the Hippo effector YAP1 in embryonal rhabdomyosarcoma

doi: 10.1038/s41598-018-33852-7

Figure Lengend Snippet: Expression of KRAS G12V does not compensate for Yap knockdown in C2C12 myoblasts. ( A ) Representative Western blots showing total Yap and Taz protein levels in C2C12 myoblasts, with β-actin as a loading control. ( B ) Effect of KRAS G12V expression on 8xGTIIC Yap/Taz Tead1-4 luciferase reporter in C2C12 myoblasts with low Yap activity (n = 3). Data is represented as (mean ± SD) from 3 independent experiments where an asterisk denotes significant difference (p < 0.05) between control, KRAS G12V or control and Yap knockdown and NS (non-significant) between KRAS G12V and Yap knockdown using a Student’s t-test.

Article Snippet: The 8xGTIIC Yap/Taz Tead1-4 luciferase reporter (Addgene plasmid #34615) was co-transfected with TK-renilla plasmid.

Techniques: Expressing, Knockdown, Western Blot, Control, Luciferase, Activity Assay

Journal: Scientific Reports

Article Title: Analysis of the relationship between the KRAS G12V oncogene and the Hippo effector YAP1 in embryonal rhabdomyosarcoma

doi: 10.1038/s41598-018-33852-7

Figure Lengend Snippet: Expression of KRAS G12V alone or in combination with p16 loss and small-T-antigen expression in U57810 cells does not change Yap/Taz transcriptional activity. ( A ) Representative Western blots showing total Yap, Taz, and phosphorylated Yap levels in U57810 cells, with Gapdh as loading control. ( B ) Effect of KRAS G12V expression on 8xGTIIC Yap/Taz Tead1-4 luciferase reporter normalised to Tk-renilla in U57810 cells. Data is presented as fold change compared to control (mean ± SD) from independent experiments. ( C ) Representative Western blots showing total Yap, Taz, and phosphorylated Yap S112/Taz S89 levels in U57810 cells, with Gapdh as a loading control. ( D ) Effect of KRAS G12V expression combined with p16 loss and small-T-antigen expression on 8xGTIIC Yap/Taz Tead1-4 luciferase reporter normalised to Tk-renilla in U57810 cells. Data is presented as fold change compared to control (mean ± SD) from 3 independent experiments.

Article Snippet: The 8xGTIIC Yap/Taz Tead1-4 luciferase reporter (Addgene plasmid #34615) was co-transfected with TK-renilla plasmid.

Techniques: Expressing, Activity Assay, Western Blot, Control, Luciferase

Journal: Cells

Article Title: IGF-1/IGF-1R/FAK/YAP Transduction Signaling Prompts Growth Effects in Triple-Negative Breast Cancer (TNBC) Cells

doi: 10.3390/cells9041010

Figure Lengend Snippet: FAK mediates the IGF-1-induced regulation of the YAP target genes CTGF and Cyr61. ( A ) YAP/TAZ luciferase reporter assay in MDA-MB 231 and SUM159 cells treated with IGF-1 (50 ng/mL) alone or in combination with the IGF-1R inhibitor OSI-906 (1 µM) or the FAK inhibitor VS-4718 (100 nM). CTGF ( B ) and Cyr61 ( C ) luciferase reporter assays in MDA-MB 231 and SUM159 cells treated with IGF-1 (50 ng/mL) alone or in combination with the YAP/TEAD complex suppressor Verteporfin (100 nM). CTGF ( D ) and Cyr61 ( E ) luciferase reporter assays in MDA-MB 231 and SUM159 cells treated with IGF-1 (50 ng/mL) alone and in combination with the IGF-1R inhibitor OSI-906 (1 µM) or the FAK inhibitor VS-4718 (100 nM). mRNA expression of CTGF ( F ) and Cyr61 ( G ) in MDA-MB 231 and SUM159 cells treated with IGF-1 (50 ng/mL) alone and in combination with the IGF-1R inhibitor OSI-906 (1 µM), the FAK inhibitor VS-4718 (100 nM) or the YAP/TEAD complex suppressor Verteporfin (100 nM). ( H ) Immunoblots showing the CTGF and Cyr61 protein levels in MDA-MB 231 and SUM159 cells treated with IGF-1 (50 ng/mL) alone and in combination with the IGF-1R inhibitor OSI-906 (1 µM), the FAK inhibitor VS-4718 (100 nM) or the YAP/TEAD complex suppressor Verteporfin (100 nM). Side panels show densitometric analysis of the immunoblots normalized to the β-actin, which was used as loading control. Data shown are representative of three independent experiments performed in triplicate. Error bars represent mean ± SD. * indicates p < 0.05 for cells treated with vehicle versus treatments.

Article Snippet: The YAP/TEAD complex suppressor Verteporfin was purchased from Med Chem Express (DBA, Milan, Italy).

Techniques: Luciferase, Reporter Assay, Expressing, Western Blot, Control

Journal: Cells

Article Title: IGF-1/IGF-1R/FAK/YAP Transduction Signaling Prompts Growth Effects in Triple-Negative Breast Cancer (TNBC) Cells

doi: 10.3390/cells9041010

Figure Lengend Snippet: The IGF-1/FAK/YAP signaling pathway triggers the growth of TNBC cells. Proliferation assay of MDA-MB 231 ( A ) and SUM159 ( B ) cells exposed for 4 days to IGF1 (50 ng/mL) alone and in combination with the IGF-1R inhibitor OSI-906 (1 µM), the FAK inhibitor VS-4718 (100 nM) or the YAP/TEAD inhibitor, verteporfin (100 nM). ( C ) Colony formation assay in MDA-MB 231 and SUM159 cells exposed to IGF1 (50 ng/mL) alone and in combination with the IGF-1R inhibitor OSI-906 (1 µM), the FAK inhibitor VS-4718 (100 nM) or the YAP/TEAD inhibitor verteporfin (100 nM). After 10 days of treatment, the plates were stained with crystal violet and colonies were counted, as indicated. ( D ) Colony formation assay in SUM159 cells exposed to IGF-1 (50 ng/mL) and transfected with shRNA or shCTGF. After 10 days of treatment the plates were stained with crystal violet and colonies were counted, as indicated. ( E ) Immunoblot showing the CTGF protein levels in SUM159 cells transfected with control shRNA or shCTGF. Side panels show densitometric analysis of the immunoblot normalized to the β-actin, which was used as loading control. ( F , G ) Spheroid formation assay in MDA-MB 231 and SUM159 cells exposed for 18 days to IGF-1 (50 ng/mL) alone and in combination with the IGF-1R inhibitor OSI-906 (1 µM), the FAK inhibitor VS-4718 (100 nM) or the YAP/TEAD complex suppressor verteporfin (100 nM). Scale bars, 100 µm. ( H ) Cartoon depicting the IGF-1/FAK/YAP signaling pathway promoting gene expression changes and the growth of TNBC cells. Data shown are representative of three independent experiments performed in triplicate. Error bars represent mean ± SD. (*) indicates p < 0.05 for cells treated with vehicle versus treatments.

Article Snippet: The YAP/TEAD complex suppressor Verteporfin was purchased from Med Chem Express (DBA, Milan, Italy).

Techniques: Proliferation Assay, Colony Assay, Staining, Transfection, shRNA, Western Blot, Control, Tube Formation Assay, Gene Expression