BUSCADOR RECOLECTA

Strategic Action in Health of the Institute of Health Carlos III Department of Regional Health of the Castilla y Léon Government Consejería de Educación, Junta de Castilla y León, Peer reviewed

Strategic Action in Health of the Institute of Health Carlos III Department of Regional Health of the Castilla y Léon Government Consejería de Educación, Junta de Castilla y León, Peer reviewed

Additional file 1: Supplementary Material 1. Minor Revision. Supplementary Material 2. DSMB. Supplementary Material 3. Full protocol., Peer reviewed

Figure S1: GenBank codes for the partial COI sequences of the Aedes albopictus lines used in this study; Figure S2: Mean female fecundity (left) and mean egg fertility (right) in Ae. albopictus ARwPL, ARwPBA, ARwPBN, and BN., Peer reviewed

Additional file 1: Supplementary Fig. 1. A) Box plot showing SLC3A2 gene expression levels in paired normal colon tissue and tumoral CRC tissue, using TNMplot online tool. FC = fold change. P value obtained using Mann Whitney U test. B) Box plot showing SLC3A2 gene expression levels in normal colon tissue and tumoral CRC tissue samples. Data were obtained from the GEPIA2. P value obtained using one-way ANOVA test. C) Box plot showing SLC3A2 gene expression levels in normal colon tissue and tumoral CRC tissue normal, using TNMplot online tool. P value obtained using Mann Whitney U test. D) Box plot showing the CD98hc score analyzed by immunohistochemistry in primary tumor, lymph node and liver metastases. P values were obtained using Student t test (two-sided). E) Immunohistochemical staining of CD98hc in representative samples from the study shown in D, assessed using with anti-CD98hcV509 antibody. Magnification: 40X. F) Box plot showing SLC3A2 gene expression levels in tumor and metastatic tissue of CRC patients. T = tumor, N = normal and M = metastatic. P value obtained using Kruskal–Wallis test. G) Heatmap (top) and boxplot (bottom) representative of the expression of CD98hc in different normal and tumoral tissues, obtained from the TNMplot database. Tissues written in red represent significant differences by the Mann–Whitney test. H) Expression of CD98hc in different normal and tumoral tissues. Data obtained from the GENT2 database., Instituto de Salud Carlos III Consejo Superior de Investigaciones Científicas Junta de Castilla y León ALMOM ACMUMA UCCTA CRIS Cancer Foundation Agencia Estatal de Investigación Consejo Superior de Investigaciones Cientificas (CSIC), Peer reviewed

Additional file 2: Supplementary Fig. 2. A) Internalization of the anti-CD98hcECTO antibody in SW480 cells, analyzed by immunofluorescence. Scale bar = 25 μm. The cells were seeded on coverslips and treated with 10 nM of anti-CD98hcECTO for the times indicated. The images at the right correspond to magnifications of a cell present in the images obtained at 24 h. The colocalization of CD98hc and LAPM1 is show in the merged images. B) Preparation of the antibody–drug conjugate targeting CD98hc. The coupling of DM1 to the anti-CD98hcECTO antibody was evaluated by Western blot using an anti-DM1 antibody. Twenty nanograms of anti-CD98hcECTO-DM1, the nude anti-CD98hcECTO, trastuzumab or T-DM1 were used to detect DM1 (upper panel) and the total amount of protein was evaluated by stain-free blot (lower image). Trastuzumab and T-DM1 were used as a negative and positive controls. C-E) FACS analyses in HT29 (C), cells dissociated from patient PDX BT6224 (D) and human tumoral organoid #48 (E) using anti-CD98hc-DM1 as primary antibody. The red histogram correspond to signals from cells incubated with the secondary antibody alone, whereas the blue histograms represent the fluorescence due to the expression of CD98hc., Instituto de Salud Carlos III Consejo Superior de Investigaciones Científicas Junta de Castilla y León ALMOM ACMUMA UCCTA CRIS Cancer Foundation Agencia Estatal de Investigación Consejo Superior de Investigaciones Cientificas (CSIC), Peer reviewed

Supplementary Fig. 1, Discussion and Tables 1–2., Peer reviewed

Additional file 3: Supplementary Fig. 3. A) Dose–response analyses of the effect of anti-CD98hc-DM1 on the proliferation of parental and CD98hc CRISPR #5 and #11 HT29 cells. Cells were treated with anti-CD98hc-DM1 at the indicated doses for four days. Results are shown as the mean ± SD of quadruplicates of an experiment repeated three times. B) Expression of CD98hc in normal human fibroblasts (NHF) and immortalized keratinocytes (HaCaT), compared to CRC cell lines. Cell extracts (20 µg) were used to identify CD98hc by Western blot with the anti-CD98hcV509 antibody. Calnexin was used as a loading control. C) Dose–response analyses of the anti-CD98hc-DM1 ADC on NHF and HaCaT, compared to HT29 cells. Cells were treated with the ADC for four days at the indicated doses. The data are plotted as the percentage of MTT metabolization with respect to control. Results are shown as the mean ± SD of quadruplicates of an experiment repeated two times. D) Evaluation of the effect of anti-CD98hc-DM1 (10 nM, 48 h) on the distribution of the different cell cycle phases in HT29 and HCT116 cell lines. E) Immunofluorecescence analyses of the action of anti-CD98hc-DM1 on spindle assembly and organization on HCT116 cells treated with CD98hc-DM1 (10 nM) for 48 h. β-Tubulin (green), DAPI (blue). Scale bars = 7.5 µm. F). Detection of giant multinucleated cells or altered nuclear structures after anti-CD98hc-DM1 treatment. HCT116 and HT29 cells were treated with 10 nM anti-CD98hcECTO-DM1 for 72 h, fixed and stained for nucleoporin p62 (red) and DNA (blue). Scale bar = 10 μm. The arrows indicate giant multinucleated cells., Instituto de Salud Carlos III Consejo Superior de Investigaciones Científicas Junta de Castilla y León ALMOM ACMUMA UCCTA CRIS Cancer Foundation Agencia Estatal de Investigación Consejo Superior de Investigaciones Cientificas (CSIC), Peer reviewed

Source Data Fig. 1: Raw data for resistivity measurements. Source Data Fig. 3: Data for scaled magneto-anisotropy. Source Data Fig. 4: (a) S1 to S4, data for the temperature dependence of resistivity anisotropy. (b) Theory, data for theoretically predicted anisotropy versus scaled temperature., Peer reviewed

[Description of methods used for collection/generation of data] 12870_2023_4431_MOESM1_ESM.xlsx. Orthologue/homoeologue sequences obtained from EnsemblPlants after BLASTn. 12870_2023_4431_MOESM2_ESM.xlsx. Genomic sequences of genes TraesCS7D02G094000, TraesCS4A02G397900, TRITD4Av1G231840 and TRITD4Av1G231510 were retrieved from Ensembl Plants and aligned using the multiple sequence alignment tool ClustalW. Primers were designed in the conserved 5’ and 3’ regions by using the NCBI Primer-Blast tool. SNP markers were designed following a Tetra-Primer ARMS (amplification refractory mutation system) strategy for SNPs detection. Primer1 web service (http://primer1.soton.ac.uk/primer1.html) was used for primer design. 12870_2023_4431_MOESM4_ESM.xlsx. Sequence editing, alignment and assembly were performed with SeqMan Pro Lasergene Software v17 (DNAStar, WI, US). The identity of the clones as GDSL esterase-lipase- like sequences was confirmed by BLASTn at NCBI. The coding sequences were predicted based on the exon-intron structure of TraesCS7D02G094000, and open reading frames were searched by using ORFfinder at NCBI (https://www.ncbi.nlm.nih.gov/orffinder). Signal peptide and cellular location of the expected proteins were predicted by SignalP 6.0 software 12870_2023_4431_MOESM5_ESM.xlsx. The genetic map was constructed using Joinmap(R) 5.0 [Kyazma(R), The Netherlands]. 12870_2023_4431_MOESM6_ESM.xlsx. Carotenoid content and profile was determined according to Rodríguez-Suárez C, Requena-Ramírez MD, Hornero-Méndez D, Atienza SG (2022). Chapter 4. The breeder’s tool-box for enhancing the content of esterified carotenoids in wheat: From extraction and profiling of carotenoids to marker-assisted selection of candidate genes. En: Carotenoids: Carotenoid and apocarotenoid biosynthesis, metabolic engineering and synthetic biology. pp. 99-125. Editor. Eleanore T. Wurtzel. Book series: Methods in Enzymology. Editorial: Academic Press. Elsevier. Hardcover ISBN: 9780323913539., This research was financed by project PID2021-122152NB-I00 funded by MCIN/AEI/10.13039/501100011033/ and by ERDF “ERDF A way of making Europe”. M.D.R.-R. was supported by PRE2018-084037 funded by MCIN/AEI/10.13039/501100011033 and ESF “ESF investing in your future”. DH-M is member of the Spanish Carotenoid Network (CaRed), grant RED2022-134577-T. SA, CR-S and MDR-R are members of CeReS Network, grant RED2022-134922-T. Both networks are funded by MCIN/AEI/10.13039/501100011033., 12870_2023_4431_MOESM1_ESM.xlsx 12870_2023_4431_MOESM2_ESM.xlsx 12870_2023_4431_MOESM3_ESM.xlsx 12870_2023_4431_MOESM4_ESM.xlsx 12870_2023_4431_MOESM5_ESM.xlsx 12870_2023_4431_MOESM6_ESM.xlsx 12870_2023_4431_MOESM1_ESM.xlsx contains the results of the Search for XAT candidate genes in durum wheat genomes using XAT-7D (TraesCS7D02G094000) from common wheat as gene model. 12870_2023_4431_MOESM2_ESM.xlsx contains the list of primers designed in this work. 12870_2023_4431_MOESM3_ESM.xlsx contains the list of landraces showing positive amplification for XAT candidate gene. 12870_2023_4431_MOESM4_ESM.pdf contains the alignment of XAT-7A1 and XAT-7D proteins. The predicted active sites are shown with black triangles. Signal peptide in positions 1–22 is highlighted. 12870_2023_4431_MOESM5_ESM.xlsx contains the genetic map for the F2 population derived from the cross BGE047535 × 'Athoris' using DArTSeq markers. 12870_2023_4431_MOESM6_ESM.xlsx contains the carotenoid content and profile of the F2 population derived from the cross BGE047535 x Athoris., Peer reviewed