Journal: The EMBO Journal

Article Title: Wnt and Src signals converge on YAP‐TEAD to drive intestinal regeneration

doi: 10.15252/embj.2020105770

Figure Lengend Snippet: Src family kinase inhibition via Dasatinib treatment in control organoids enforces YAP cytoplasmic localisation after 4 h of treatment in organoids cultured for 3 days. n > 10 organoids in each experiment. Quantification of the percentage of nuclear YAP cells per control crypt bud as shown in panel (A), error bars show 1 SD (**** P < 0.0001). n > 10 organoids per condition (top). Quantification of the nuclear/cytoplasmic ratio of YAP per cell in Apc5 mutant organoids as shown in panel (C). n > 10 organoids per condition (bottom). Statistical analysis was performed with an unpaired Student's t ‐test. Src family kinase inhibition via Dasatinib or eCF506 treatment in Apc5 organoids causes relocalisation of YAP to the cytoplasm. n > 10 organoids in each experiment. qPCR analysis of the YAP‐TEAD target gene Ctgf reveals a strong inhibition of mRNA expression upon treatment with eCF506 (relative expression, error bars = 1 SD). Statistical analysis was performed with an unpaired Student's t ‐test. Src family kinase inhibition via Dasatinib or eCF506 treatment in Villin‐Cre ERt Lats1 / 2 dKO organoids (induced in vitro with 4‐OHT) fails to cause relocalisation of YAP to the cytoplasm after 4 h treatment in culture. n > 10 organoids in each experiment. Source data are available online for this figure.

Article Snippet: The Porcupine inhibitor LGK974 (Selleck chemicals, S7143) was used at 5 μM for 24 h. For Src inhibition experiments, organoids were treated with 100 nM of Dasatinib (Selleck chemicals, S1021) or 100 nM eCF506 for a period of 4 h. Organoid microscopy was performed with either a Leica SP5 or a Leica SP8 laser‐scanning confocal microscope.

Techniques: Inhibition, Control, Cell Culture, Mutagenesis, Expressing, In Vitro

Journal: eLife

Article Title: Cas phosphorylation regulates focal adhesion assembly

doi: 10.7554/eLife.90234

Figure Lengend Snippet: ( A ) Crk WT and Crk mGL genomic organization and Crk mGL mRNA structure. An artificial exon encoding the Crk C-terminus, linker, mGreenLantern (mGL), and polyA signal was inserted in intron 2. ( B ) Immunoblot showing expression of Crk mGL protein in Cas mSc Crk mGL HeLa cells. ( C ) Crk mGL clusters form shortly after Cas mSc clusters. Total internal reflection (TIRF) micrographs of Cas mSc Crk mGL HeLa cells. Upper panels: individual time frames. Arrowheads indicate a Cas mSc cluster (magenta) that is rapidly joined by Crk mGL (green). Lower panels: kymographs. ( D ) Median Δ t 1/2 (Crk-Cas) of multiple regions of interest (ROIs) from 10 to 21 spreading Cas mSc Crk mGL HeLa control and eCF506-treated cells. Error bars show mean and standard error of the mean (SEM). ***,p < 0.001 by Mann–Whitney U -test. ( E ) Upper panels: raw images. Lower panels: masks showing tracked ROIs, color coded by time of onset.

Article Snippet: eCF506 , Cayman Chemical, Cat: 19959 , 100 nM.

Techniques: Western Blot, Expressing, Control, MANN-WHITNEY

Journal: eLife

Article Title: Cas phosphorylation regulates focal adhesion assembly

doi: 10.7554/eLife.90234

Figure Lengend Snippet: ( A ) Representative images (ventral section) of Cas mSc MCF10A cells treated with control, Cas, or vinculin siRNA and fixed and stained with vinculin antibodies after 30 min of spreading on COLI. ( B ) Quantification of mean cell area and the number and mean intensities of Cas and/or vinculin clusters. Error bars show mean and standard error of the mean (SEM) for n = 10–50 cells from three biological repeats. ( C ) Median Δ t 1/2 (VCL-Cas) of multiple regions of interest (ROIs) from 8 to 20 spreading Cas mSc YFP-VCL MCF10A cells treated with Ctrl, Crk, CrkL, and Crk + CrkL siRNA. Error bars show mean and SEM. ( D ) Median Δ t 1/2 (VCL-Cas) from 13 to 16 time-lapse dual-color total internal reflection (TIRF) micrographs of spreading Cas mSc YFP-VCL MCF10A cells treated with DMSO or SFK inhibitor eCF506. ( E ) Mean cluster intensity of pY410Cas, Cas mSc and YFP-VCL in Cas mSc YFP-VCL MCF10A cells treated with control, Src, Fyn, or Yes1 siRNA and fixed after 30 min of spreading. Error bars show mean and SEM from n = 7–10 cells from three biological repeats. ( F ) Cas requirement for outside-in signaling. YFP-VCL MCF10A cells were treated with control or Cas siRNA and allowed to attach in the absence or presence of Mn 2+ for 30 min. Graphs show the mean cell spread area and mean intensity of YFP-VCL clusters. Error bars show mean and SEM for n = 6–20 cells from two biological repeats. ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 by Kruskal–Wallis followed by Dunn’s multiple comparison test ( A–E ) or pairwise Mann–Whitney U -tests ( F ).

Article Snippet: eCF506 , Cayman Chemical, Cat: 19959 , 100 nM.

Techniques: Control, Staining, Comparison, MANN-WHITNEY

Journal: eLife

Article Title: Cas phosphorylation regulates focal adhesion assembly

doi: 10.7554/eLife.90234

Figure Lengend Snippet:

Article Snippet: eCF506 , Cayman Chemical, Cat: 19959 , 100 nM.

Techniques: Concentration Assay