Illustrator software

For the purposes of our analysis, we grouped all LOF variants together and compared cases to NFE gnomAD controls. A similar approach was used for evaluating VUS. In addition, we directly examined individual variants if observed in ≥3 cases.
We compared the grouped LOF variants or VUS and individual variants (in ≥3 cases) in our cohort and NFE gnomAD data using Fisher’s exact tests. We also conducted analyses restricting the case sample to those considered to be genetically enriched: probands from melanoma-prone families and MPM cases. For VUS only, we repeated the analysis including variants depending on whether they were located in one of the three ATM functional domains, i.e., FRAP–ATM–TRRAP (FAT), phosphatidylinositol 3-kinase/phosphatidylinositol 4-kinase (PI3K/PI4K), and FAT carboxy-terminal (FATC), which encompass residues 1940–2566, 2712–2962, and 3024–3056, respectively.
In addition, we also compared grouped LOF variants or VUS in cases and controls from the two centers that provided controls.
All analyses were two-sided and a 0.05 cutoff was used for statistical significance. Statistical analyses were performed within the R computational environment [14 ].
The lollipop plot was generated using cBioportal Mutation Mapper [15 (link), 16 (link)] and Adobe Illustrator® software.

To better elaborate the holistic mechanism of SZRP, three subnetworks were compiled by following the procedures: 1) All targets of SZRP were submitted to an online tool KEGG Search Pathway (https://www.genome.jp/kegg/tool/map_pathway1.html). 2) All result maps of KEGG were downloaded and integrated. 3) Maps related to “nervous,” “immune,” or “endocrine” were reserved. 4) For each label of “nervous,” “immune,” and “endocrine,” multiple pathways were integrated and overlapped according to cross-talk targets in these maps. Detailed information such as targets names and integrated pathways are shown in Table S3. 5) Subnetworks were drawn with Adobe Illustrator software, where intermediate genes were hidden for better display.

Immunohistochemistry was performed on PBS buffered formaline-fixed follicles and CL that were generated as described above, as well as on bovine tissues obtained from slaughterhouse. Paraffin-embedded tissues were cut at 4 μm thickness, mounted on SuperfrostPlus slides (Fischer Scientific, QC), deparaffined and rehydrated. Tissue sections were heat-treated as previously described [28 (link)], and were incubated for 14 h at 4°C with our anti-LAPTM4B antibody at a dilution of 1:500 in Tris-buffered saline (TBS; 150 mM NaCl, 0.1 M Tris pH 7.5) containing 1% bovine serum albumin, and 1% fat-free skim milk. Control tissue sections were incubated similarly with preimmune serum. The primary antibody and LAPTM4B antigen complexes were detected by incubation with a monoclonal anti-rabbit IgG conjugated with alkaline phosphatase (Sigma Chemicals) at a dilution of 1:200 for 2 h at room temperature, followed by several washes in TBS, and incubation with the NBT/BCIP alkaline phosphatase substrate (Roche Diagnostics). Sections were mounted in 5% gelatin, 27% glycerol, and 0.1% sodium azide. Photographs were taken under bright field illumination using a Nikon Eclipse E800 microscope equipped with a digital camera (Nikon DXM 1200). Digital images were processed by the Photoshop software (Adobe Systems Inc., San Jose, CA) and assembled by the Illustrator software (Adobe Systems Inc.).

MDCK cell lines were fixed at room temperature with 3% paraformaldehyde for 20–25 min, permeabilized (0.2% Triton X-100) for 45 min and incubated for 1 hr each with primary and then secondary antibodies as described previously (Ghosh et al., 2008 (link)). Dilutions of antibodies and reagents were as follows: anti-GIV (1:300); anti-phospho-Ser245-GIV (pS245-GIV; 1/250); anti-Occludin (1/250); anti-ZO-1 (1/250); anti-β-catenin (1/250); anti-E-cadherin (1/250); Phalloidin (1:1000); DAPI (1:2000); goat anti-mouse (488 and 594) Alexa-conjugated antibodies (1:500). Images were acquired using a Leica CTR4000 Confocal Microscope with a 63X objective. Z-stack images were obtained by imaging approximately 4-μm thick sections of cells in all channels. Cross-section images were obtained by automatic layering of individual slices from each Z-stack. Red-Green-Blue (RGB) graphic profiles were created by analyzing the distribution and intensity of pixels of these colors along a chosen line using ImageJ software. All individual images were processed using Image J software and assembled for presentation using Photoshop and Illustrator software (Adobe).

The Dof proteins of C. reinhardtii, P. patens, A. thaliana, O. sativa and eggplant were selected for phylogenetic analysis in planta. The ClustalX2 program was used to align Dof protein sequences with the Gonnet protein weight matrix (Larkin et al., 2007 (link)). The MEGA program (v6.06) was used to construct a neighbour-joining phylogenetic tree using the Jones-Taylor-Thornton (JTT) model with 500 bootstrap replicates (Tamura et al., 2013 (link)). The uniform rates and homogeneous lineages were adopted, whereas the partial deletion with a site coverage cutoff of 70% was used for gaps/missing data treatment. To clearly distinguish the genes in this phylogenetic tree, the term ‘Nta—’ was added as a prefix to indicate the genes were from tobacco. The MEGA program was also used to construct a neighbour-joining phylogenetic tree of Dof proteins in plants of Solanaceae, following previous methods. The frequency of each divergent branch was displayed when the value was higher than 50%. Adobe Illustrator software was used to clearly show the Dof branches after classification of all proteins based on the known background information.

Data are represented as mean and standard error of mean (Mean + SEM). Statistical analyses were performed using GraphPad Prism 7.0 software (GraphPad Prism Software Inc., San Diego, CA) and R. Normal distribution was confirmed using Shapiro–Wilk normality test before performing statistical analyses. For normally distributed data, comparison between two means was assessed by unpaired two-tailed Student’s t test and that between three or more groups were evaluated using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. In the case of Student’s t test, F test was performed to check whether the variance in the groups compared was significantly different. For data where significantly different variances were observed, a t test with Welch correction was performed. For data that did not follow a normal distribution, Mann–Whitney test was performed for comparison between two groups and Kruskal–Wallis test followed by Dunn’s multiple comparisons test was performed for comparing more than two groups. A p value of < 0.05 was considered statistically significant. Figures were generated using Adobe Illustrator software (San Jose, CA, USA).
Other methods are described in Additional file 1: Supporting materials and methods.

MAFFT version 7 (https://mafft.cbrc.jp/alignment/server/ (accessed on 8 December 2021)) was used to perform multi-sequence alignment on the TKS WRKY protein sequence domain with G-INS-1 (progressive method with an accurate guide tree) settings. The phylogenetic tree was constructed by MEGA software (version 7.0, Mega Limited, Auckland, New Zealand) with the neighbor-joining (NJ) method based on the comparison between TKS and Arabidopsis proteins; then we used Illustrator software (version 2019, Adobe, San Jose, CA, USA) to beautify and add color. The sequences of the WRKY protein from Arabidopsis, maize [50 (link)], rice [61 (link),67 (link)], grape [47 (link)], Brachypodium distachyon [68 (link)], pineapple [64 (link)], peach [69 ], and poplar [36 (link)] were obtained according to the corresponding literature and instructions downloaded from the Phytozome databases (https://phytozome.jgi.doe.gov/ (accessed on 27 December 2021)).