Journal: Nature communications

Article Title: Senescent cells perturb intestinal stem cell differentiation through Ptk7 induced noncanonical Wnt and YAP signaling.

doi: 10.1038/s41467-022-35487-9

Figure Lengend Snippet: Fig. 4 | Ptk7 acts through noncanonical Wnt signaling. a Schematic repre- sentation of canonical and noncanonical Wnt signaling pathways. While canonical and noncanonical Wnt signaling pathways share components such as FZD and Dvl, other components such as LRP5/6 and β-Catenin are specific for canonical Wnt signaling. The figure was created with BioRender.com. b Representative images of organoids cultured in corresponding conditioned media with DMSO or inhibitors. Scale bar, 200 μm. c Quantification of data from (b). The percentage of cystic organoids in organoids cultured in conditioned media is shown. d Representative images of organoids treated with DMSO (carrier), Wnt3a or Wnt5a. Scale bar, 200 μm. e Quantification of data from (d). The percentage of cystic organoids in organoids cultured in conditioned media is shown. f Representative images of organoids cultured in Wnt5a with DMSO or inhibitors. Scale bar, 200 μm. g Quantification of data from (f). mean ± s.d. percentage of cystic organoids in organoids cultured in conditioned media. h Representative images of organoids

Article Snippet: Proteins used in ELISAs were obtained from R&D Systems: PTK7-Fc (R&D Systems, #9799-TK), biotinylated Wnt3a (R&D Systems, #BT1324), biotinylated Wnt5a (R&D Systems, #BT645).

Techniques: Protein-Protein interactions, Cell Culture

Journal: Methods in molecular biology (Clifton, N.J.)

Article Title: In Vitro Analysis of Hedgehog Acyltransferase and Porcupine Fatty Acyltransferase Activities

doi: 10.1007/978-1-4939-9532-5_19

Figure Lengend Snippet: Sequences of the Shh (top) and Wnt3a (bottom) biotinylated peptides

Article Snippet: We used AnaSpec (Fremont, CA) to synthesize C-terminal biotinylated (Biotin–PEG NovaTag TM ), disulfide-bonded Wnt3a WT (MHL KC(S-)KC(S-)HG LSG SC(S-)E VKT C(S-)WW) and C-terminal biotinylated (Biotin–PEG NovaTagTM) disulfide-bonded Wnt3a S209A (MHL KC(S-)K C(S-)HG LAG SC(S-)E VKT C(S-)WW) and then analyzed by HPLC to assess purity to be greater than 95%.

Techniques:

Journal: PLoS ONE

Article Title: Differential Palmit(e)oylation of Wnt1 on C93 and S224 Residues Has Overlapping and Distinct Consequences

doi: 10.1371/journal.pone.0026636

Figure Lengend Snippet: Panel A: HEK293T cells were transfected with 8XSuperTop/FopFlash , renilla luciferase, and indicated pcDNA expression constructs. DNA concentrations were held constant within each series of experiments. Values shown reflect the experimental data (Firefly/Renilla) minus FopFlash (Firefly/Renilla). The relative luciferase activity of wild-type Wnt1/Wnt3a was set at 1. Single and double Wnt1/Wnt3a mutants show significantly less activity than wild-type Wnts (p<1×10 −10 ). Differences between C93S and C93A mutants are not significant nor are those between S224A and C93A/S224A. Error bars indicate standard error for at least 15 data points from 3 independent replicates. Panel B: HEK293T cells were transfected as in Panel A, lysed, subjected to SDS-PAGE, and blotted onto PVDF. Blots were probed with anti-Wnt1 or anti-ß-tubulin. Panel C: The Western blots were scanned and analyzed using NIH ImageJ. This experiment was performed 3 times. Error bars represent +/− standard error. Panel D: LS/L cells were then transiently transfected with constructs encoding GFP or wild-type/mutant Wnt1. As before, the relative luciferase activity of wild-type Wnt1 was set at 1. Wnt1 and Wnt1C93A show statistically significant increases in reporter activity as compared to GFP (p<1×10 −7 ) while Wnt1S224A and Wnt1C93AS224A do not. Error bars represent standard error from at least 3 independent replicates with a total of 12 data points.

Article Snippet: Other materials include: TX-114 (Fisher); Lipofectamine 2000, Phalloidin-Alexa Fluor 633 (Invitrogen); Fugene HD (Promega); Mouse anti-GFP JL-8 (Clontech); FITC or Cy3 conjugated goat anti-rabbit IgG (H+L), Cy3 conjugated goat anti-mouse IgG IgM (H+L) and goat anti-mouse IgG (H+L)-AP, anti-rabbit DyL 649 (Jackson Laboratories); Dual Luciferase Reporter (Promega); anti-ß-tubulin, anti-PCNA (Santa Cruz Biotechnology); Dual Light System (Applied Biosystems); HEK293T cells (Developmental Studies Hybridoma Bank); anti-HA (Upstate); anti-Flotillin (BD Biosciences); anti-PDI (Calbiochem); anti-Giantin (Abcam); Optiprep (Accurate Chemicals); Tran 35 S label (MP Biomedicals); Protein A/G agarose beads, Sulfo-NHS-LC-biotin, NeutraAvidin Agarose (Pierce); En 3 Hance (Perkin Elmer); purified mouse Wnt3a (R&D Systems).

Techniques: Transfection, Luciferase, Expressing, Construct, Activity Assay, SDS Page, Western Blot, Mutagenesis

Journal: PLoS ONE

Article Title: Differential Palmit(e)oylation of Wnt1 on C93 and S224 Residues Has Overlapping and Distinct Consequences

doi: 10.1371/journal.pone.0026636

Figure Lengend Snippet: Panel A: Mock-transfected L cells were treated with control-conditioned media (Control-CM) or Wnt1/3a-conditioned media for 24 hrs. Note that Wnt1-CM was produced by standard L cells while Wnt3a was produced by E-cadherin transfected L cells (EL cells). Cells were then fixed, immunostained for PDI, and imaged with a confocal microscope. The length of the long axis of cells was measured using Adobe Photoshop (version 9.0.2). Error bars represent standard error. This experiment was repeated twice. At least 100 cells in 5 fields were measured. A Student's t-test shows that the difference in cell length between cells treated with control-CM and Wnt1- or Wnt3a-CM is statistically significant (p<0.005). Panel B: LS/L cells were treated with control-CM from mock-transfected L cells, Wnt1 conditioned medium, 33 mM NaCl, or 33 mM LiCl for 20–30 hrs and then assayed for activation of the SuperTopFlash reporter. Panel C: L cells were treated with 33 mM NaCl or LiCl for 24 hrs and then analyzed for cell elongation as in Panel A. Error bars represent the standard error from over 150 cells in two independent experiments. Neither NaCl nor LiCl has any significant effect on the length of the cells. Panel D: Purified mWnt3a was added to LS/L cells 24 hrs prior to conducting a dual luciferase assay to measure the activation of the SuperTopFlash construct. Error bars represent the standard error from 2 independent replicates with a total of 8 data points. Panel E: Mock-transfected L cells were incubated with purified mWnt3a for 24 hrs. Cells were then fixed, immunostained with anti-PDI, and imaged via confocal microscopy. The length of the long axis of individual cells was measured in Adobe Photoshop. Error bars represent the standard error from 2 independent replicates with a minimum of 107 cells measured for each data point.

Article Snippet: Other materials include: TX-114 (Fisher); Lipofectamine 2000, Phalloidin-Alexa Fluor 633 (Invitrogen); Fugene HD (Promega); Mouse anti-GFP JL-8 (Clontech); FITC or Cy3 conjugated goat anti-rabbit IgG (H+L), Cy3 conjugated goat anti-mouse IgG IgM (H+L) and goat anti-mouse IgG (H+L)-AP, anti-rabbit DyL 649 (Jackson Laboratories); Dual Luciferase Reporter (Promega); anti-ß-tubulin, anti-PCNA (Santa Cruz Biotechnology); Dual Light System (Applied Biosystems); HEK293T cells (Developmental Studies Hybridoma Bank); anti-HA (Upstate); anti-Flotillin (BD Biosciences); anti-PDI (Calbiochem); anti-Giantin (Abcam); Optiprep (Accurate Chemicals); Tran 35 S label (MP Biomedicals); Protein A/G agarose beads, Sulfo-NHS-LC-biotin, NeutraAvidin Agarose (Pierce); En 3 Hance (Perkin Elmer); purified mouse Wnt3a (R&D Systems).

Techniques: Transfection, Control, Produced, Microscopy, Activation Assay, Purification, Luciferase, Construct, Incubation, Confocal Microscopy