Nr4a3

Cells were lysed in RIPA buffer (Beyotime, Shanghai, China) containing protease inhibitors (Sigma‑Aldrich), and the BCA Protein Assay Kit (Thermo Fisher Scientific) was used to quantify the concentration of proteins. Cell extracts were separated by 10% SDS-PAGE, transferred to NC membranes (Sigma‑Aldrich) and blocked in QuickBlock Blocking Buffer (Beyotime) for 10 min at room temperature. The membranes were incubated with primary antibodies against NR4A3 (Abcam, Cambridge, UK), PDK1 (Cell Signaling Technology, Danvers, USA), cyclinD1 (Cell Signaling Technology), and PCNA (Abcam) or GAPDH (Cell Signaling Technology) at 4°C overnight. After three washes with PBS, the membranes were incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. Finally, the immunoblots were detected with BeyoECL Plus (Beyotime).

Cells were lysed in a buffer containing 100 mM Tris (pH 7.6), 1% Triton X-100, 150 mM NaCl, 0.1 mg aprotinin, 35 mg/ml PMSF 10 mM Na3VO4, 100 mM NaF, 10 mM Na4P2O7, and 4 mM EDTA (approximately 5 × 10⁶ cells). Samples were centrifuged at 4°C for 20 min to remove cell debris. Protein concentration was measured using the Bradford Assay (Bio-Rad). Laemmli buffer containing 100 mmol/L of dithiothreitol was added to the protein extracts and heated at 100°C for 5 min. Samples were run on a 10% SDS-PAGE. After the run, the proteins were transferred to nitrocellulose membranes (Millipore). Membranes were immunoblotted with NR4A3 (Abcam, ab41918), HnRNPK (Abcam, ab32969), SF3B2 (ProteinTech, 10919-1-AP), HnRNPA1 (ProteinTech, 11176-1-AP), PARP1 (Santa Cruz, sc-56197), Lamin B1 (Santa Cruz, sc-6127), and GAPDH (Santa Cruz, sc-32233) antibodies. K-562 protein samples were obtained from three independent experiments, all bands are shown in the Supplementary Material; however, only one patient sample of HSC CD34+ cells (which is a rare population of hematopoietic progenitors) was available for protein extraction. Band intensity was quantified using UVITEC alliance software.