Illustrator 6

Primary myocytes and H19C2 cells were fixed with 4% PFA, permeabilized with 0.5% Triton, and blocked with 10% normal serum before incubation with antibodies. Paraffin sections were deparaffinized and rehydrated according to standard protocols. Antigen retrieval was applied using citrate buffer (Abcam, 1b93679, Cambridge, MA) for 20 min and then maintained at a sub-boiling temperature for 10 min. Sections were treated with serum-free blocking solution (DAKO, X0909, Santa Clara, CA) and all antibodies (Supplementary file 3) diluted in antibody diluent solution (DAKO, S0809). Secondary staining was performed for 30 min at RT. Samples were imaged with a Carl Zeiss 880 laser scanning microscope using a 40× oil immersion objective. Images were composed and edited in ZEN&LSM image software provided by Carl Zeiss or Illustrator 6.0 (Adobe).

For fluorescence microscopy, a Zeiss AxioZoom microscope was used to obtain wide-field images with standard epifluorescence filter sets for DAPI (365 excitation, 395 beamsplitter, 445/50 emission), eGFP/AF488 (470/40 excitation, 495 beam splitter, 525/50 emission), tdTomato/AF546 (550/25 excitation, 570 beamsplitter, 605/70 emission) and Cy5/AF647 (640/30 excitation, 660 beamsplitter, 690/50 emission). Confocal imaging on sections and whole mount tissue was performed with a Zeiss LSM710 with diode lasers (473, 559, 653 nm) for excitation. Images were collected with a 10× 0.4 NA objective and a 60× oil 1.3 NA objective lens. Optical sectioning was optimized according to the microscope software. Images were processed and analyzed with Fiji software (Schindelin et al., 2012 (link); RRID: SCR_002285) or, for 3D reconstruction and movies, Imaris 9.5 software (Oxford Instruments; RRID: SCR_007370). Figures were prepared with Photoshop 22.4.2 and Illustrator 6.0 (Adobe).

For each seed sequence, an initial alignment was performed in Uniprot using the sequences in the 50% sequence identity cluster (Clusterref_50) with the “Align” option. The raw multiple sequence alignment from Uniprot was loaded into Jalview (2.8.1) (Waterhouse et al., 2009 (link)) for editing, visualization and analysis. Masking was performed to remove ambiguous regions of the alignment, and the initial tree was generated with the Neighbor-joining method using the BLOSUM 62 substitution matrix. Additional sequences were added to the alignment using a combination of Blast searches (Altschul et al., 1990 (link)) of Uniprot, and joining of additional 50% sequence identity (ID) clusters to provide further evolutionary depth when necessary. The generated tree in Newick format was loaded into the evolutionary tree builder MEGA6 (Tamura et al., 2013 (link)) and the root chosen using an outgroup from the accepted species tree (Murphy and Eizirik, 2009 ). The figure for the final tree was created in MEGA6 and further manipulated in Illustrator 6 (Adobe Systems). The number of Cys residues in the CSD motif was mapped onto the phylograms manually.

Cells were fixed and immunostained as described [58 (link)]. Images were acquired on a Zeiss LSM700 confocal microscopes in TIFF format. Images were arranged using Photoshop CS6 and Illustrator 6 (Adobe). Where indicated, images “masks” were created with Fiji software using maximum intensity projections of each image. For each projection, actin signals were thresholded for brightness and then converted to binary masks. Immunohistochemical staining of lungs to visualize SUM159T metastases was performed as described [7 (link)]. Briefly, formalin-fixed, paraffin-embedded tissue sections were deparaffinized in xylene and rehydrated in a series of alcohol washes. Antigen retrieval was performed by boiling slides in sodium citrate pH 6.0. Slides were blocked in 20% Aquablock (Abcam, Cambridge, MA) in TBS before being incubated overnight with the indicated antibodies, followed by species-specific secondary antibodies conjugated to AlexaFluor488, AlexaFluor546, or AlexaFluor647, and counterstained with Hoechst.

MCFDCIS cells were reverse transfected with 50 nM of siRNA in 8 well chamberslides (BD Biosciences) coated with poly-l-lysine at a density of 2500 cells per well. After 48 h, cells were fixed in formalin and immunostained as described [91 (link)]. Images were acquired on a Zeiss LSM700 confocal microscope in TIFF format. Quantification of E-cadherin signal intensity was performed using the ‘Measure…’ function of Image J to determine the pixel intensity in a region of interest at the cell-cell boundaries. The results were then normalized to the mean E-cadherin signal intensity for all cell-cell boundaries analyzed in the control cells for each experiment. Images were arranged using Illustrator 6 (Adobe).