2026-10-14T12:54:38Z https://api.figshare.com/v2/oai

oai:figshare.com:article/34017695 2026-09-28T17:52:59Z category_1 category_4 category_915 category_7 category_12 category_13 category_14 category_16 category_873 category_734 category_61 category_128 category_135 portal_5 item_type_1 month_year_09_2026

<p>Integrated workflow for cell cycle synchronization and nuclear proteome profiling in <i>T. cruzi.</i></p> Ana Paula Menezes (25132835) Camila Gachet-Castro (11579152) Suzanne McGill (11916067) Richard Burchmore (178746) Ariel Silber (25132838) Maria Carolina Elias (9906069) Júlia P. C. da Cunha (25132841) Biophysics Biochemistry Physical Sciences not elsewhere classified Medicine Cell Biology Genetics Molecular Biology Physiology Chemical Sciences not elsewhere classified Biological Sciences not elsewhere classified Developmental Biology Hematology Computational Biology unique cellular features transition using hydroxyurea regulating fundamental processes pyruvate kinase activities maintaining genomic integrity hu ), followed cd ), represents specific expression patterns previously underappreciated role gene expression regulation canonical regulatory mechanisms >, potentially influencing trypanosoma cruzi </ purified nuclear fraction metabolic enzymes including gene expression metabolic enzymes cruzi </ pivotal role variant histones valuable model temporal compartmentalization specialized organelles revealing distinct results suggest provide insights proteins involved proteins identified protein synthesis nucleoside metabolisms nuclear region nuclear proteome nuclear division microtubule dynamics increased levels immunofluorescence supported identified 2 eukaryotic models etiological agent dna replication diverging eukaryotes citrate synthase chromosome condensation <p>Workflow to cell cycle synchronization and nuclear proteome. Exponential epimastigotes cultures (CL Brener) were synchronized in G1/S, S and G2/M using 20mM of hydroxyurea for 24h(HU). The cell synchronization was confirmed by propidium iodide (PI) staining and flow cytometry. The nuclei extraction was realized followed Marques Porto, 2002. Briefly, parasites were lysed parasites with sucrose and NP-40 solutions. After sequentially washing the detergent residues, the pellet was put on percoll gradient. Through ultracentrifugation (65000 x g – 120min), the nucleus was obtained and lysed with an 8M urea solution to release the nuclear proteins. Nuclear protein extracts were cleaned and digested with FASP Protein Digestion Kit (Expedeon-Abcam-USA) and labelled with TMT (Thermo-TMT 16plex Mass Tag Labeling kit cat. 90110) following the manufacturer’s recommendation. Mass spectrometry was performed using Orbitrap Fusion Tribrid. The TMT data was processed in Thermo Scientific Proteome Discoverer, software version 2.5, with SEQUEST searc. Created in BioRender. Cunha, J. (2026) <a href="https://BioRender.com/n9hj1f2" target="_blank">https://BioRender.com/n9hj1f2</a>.</p> <p>(TIF)</p> 2026-09-28T17:59:59Z Image Figure 10.1371/journal.pntd.0013299.s001 https://figshare.com/articles/figure/_p_Integrated_workflow_for_cell_cycle_synchronization_and_nuclear_proteome_profiling_in_i_T_cruzi_i_p_/34017695 CC BY 4.0