Uncropped full-length images of westernblotting membranes

Uncropped full-length images of westernblotting membranes. 12885_2023_10975_MOESM9_ESM.pdf (1.0M) GUID:?0A760526-A52B-47B3-9E09-11CFB66FE51E Additional file 10: Supplementary Fig. Additional file 9: Supplementary Fig. 9. Uncropped full-length images of westernblotting membranes. 12885_2023_10975_MOESM9_ESM.pdf (1.0M) GUID:?0A760526-A52B-47B3-9E09-11CFB66FE51E Additional file 10: Supplementary Fig. 10. Uncropped full-length images of westernblotting membranes. 12885_2023_10975_MOESM10_ESM.pdf (798K) GUID:?2D98F31C-CF11-41DC-A488-9F4EC2F3C5E6 Data Availability StatementAll data generated or analyzed during this study SH3RF1 are included in this published article and its supplementary information files. Abstract Background Thrombocytopenia is a common complication in cancer patients undergoing chemotherapy. Chemotherapy-induced thrombocytopenia (CIT) leads to dose reduction and treatment delays, lowering chemotherapy efficacy and survival rate. Thus, rapid recovery and continuous maintenance of platelet count during chemotherapy cycles are crucial in patients with CIT. Thrombopoietin (TPO) and its receptor, myeloid proliferative leukemia (MPL) protein, play a major role in platelet production. Although several MPL agonists have been developed to regulate thrombopoiesis, none have been approved for the management of CIT due to concerns regarding efficacy or safety. Therefore, the development of effective MPL agonists for treating CIT needs to be further expanded. Methods Anti-MPL antibodies were selected from the human combinatorial antibody phage libraries using phage display. We identified 2R13 as the most active clone among the binding antibodies via cell proliferation assay using BaF3/MPL cells. The effect of 2R13 on megakaryocyte differentiation was evaluated in peripheral blood CD34+ cells by analyzing megakaryocyte-specific differentiation markers (CD41a+ and CD42b+) and DNA ploidy using flow cytometry. The 2R13-induced platelet production was examined in 8- to 10-week-old wild-type BALB/c female mice and a thrombocytopenia mouse model established by intraperitoneal injection of 5-fluorouracil (150?mg/kg). The platelet counts were monitored twice a week over 14?days post-initiation of treatment with a single injection of 2R13, or recombinant human TPO (rhTPO) for seven consecutive days. Results We found that 2R13 specifically interacted with MPL and activated its signaling pathways. 2R13 stimulated megakaryocyte differentiation, evidenced by increasing the proportion of high-ploidy (?8N) megakaryocytes in peripheral blood-CD34+ cells. The platelet count was increased by a single injection of 2R13 for up to 14?days. Injection of 5-fluorouracil considerably reduced the platelet count by day ML241 4, which was recovered by 2R13. The platelets produced by 2R13 sustained a higher count than that achieved using seven consecutive injections of rhTPO. Conclusions Our findings suggest that 2R13 is a promising therapeutic agent for CIT treatment. Supplementary Information The online version contains supplementary material available at 10.1186/s12885-023-10975-3. Keywords: Chemotherapy-induced thrombocytopenia, Thrombopoietin receptor, Agonist antibody, Megakaryopoiesis, Platelet production Background Thrombocytopenia frequently occurs in patients with cancer because of chemotherapy, ML241 the malignancy itself, or infection [1]. Chemotherapy-induced thrombocytopenia (CIT, platelet count??100,000/L after chemotherapy), occurs in approximately 16%C30% of patients receiving platinum-, taxane-, or gemcitabine-based regimens [2, 3]. However, no CIT management agents have received approval from the United States Food and Drug Administration (FDA). CIT is associated with hematologic toxicity caused by hematopoiesis suppression, a significant adverse effect of chemotherapy. Since bleeding events are fatal in patients with CIT, surgical procedures, radio-, and chemotherapy are cautiously administered [4]. Therefore, the current standard management for CIT involves the postponement of chemotherapy cycles and decreasing doses to restore the platelet count to ML241 the desired level for subsequent treatments. Unfortunately, such treatment approaches lead to reduced relative dose intensity (RDI), which in turn substantially lowers the efficacy of chemotherapy and the survival rate of patients [5]. Thrombopoiesis is regulated at multiple levels by various cytokines, the most important of which is thrombopoietin (TPO). TPO and its receptor, myeloid proliferative leukemia (MPL) protein, govern the megakaryocytic lineage from hematopoietic stem cells and stimulate megakaryocyte (MK) maturation in the.