Fig 2: Topo IIα localizes with PICH in PICH bodies and on UFBs.(a–c) Cells were arrested in prometaphase using nocodazole, released and imaged by time-lapse or immunofluorescent microscopy. Scale bars, 5 μm. (a) Representative images of PICH bodies in PICH−/− +Topo IIα-GFP (green), +hPICH-mCherry (red) cells in mitosis, stained with Hoechst 33342 (blue). (a–b) The zoomed images denoted by white boxes highlight PICH bodies. (b) Representative microscopy images of PICH−/− +Topo IIα-GFP (green) +hPICH cells. UBF (red) was immunofluorescently stained and DNA (blue) was stained with DAPI. Arrows and zoomed images highlight UBF-Topo IIα foci. (c) Representative microscopy images of UFBs in PICH−/− +Topo IIα-GFP (green) +hPICH- or +hPICH-K128A cells. PICH (red) was immunofluorescently stained and DNA (blue) was stained with DAPI. The zoomed images denoted by white boxes highlight UFBs.

Fig 3: PICH is overexpressed in Burkitt Lymphoma.A PICH mRNA expression (TPM, Transcripts per million) in the different cancer types from The Cancer Genome Atlas PanCancer Study and paired normal tissue (Adapted from GEPIA). Fold-change in PICH expression in tumors compared to normal tissue is shown in the right y-axis. Studies are ranked by fold-change. The study acronyms are LUAD: Lung adenocarcinoma, DLBC Lymphoid Neoplasm Diffuse Large B-cell Lymphoma, THYM Thymoma, CHOL Cholangiocarcinoma, GBM Glioblastoma multiforme, PAAD Pancreatic adenocarcinoma, READ Rectum adenocarcinoma, LUSC Lung squamous cell carcinoma, COAD Colon adenocarcinoma, BRCA Breast invasive carcinoma, STAD Stomach adenocarcinoma, SARC Sarcoma, BLCA Bladder Urothelial Carcinoma, SKCM Skin Cutaneous Melanoma, LIHC Liver hepatocellular carcinoma, CESC Cervical squamous cell carcinoma and endocervical adenocarcinoma, TGCT Testicular Germ Cell Tumors, UCEC Uterine Corpus Endometrial Carcinoma, OV Ovarian serous cystadenocarcinoma, UCS Uterine Carcinosarcoma, LGG Brain Lower Grade Glioma, KIRP Kidney renal papillary cell carcinoma, ACC Adrenocortical carcinoma, PCPG Pheochromocytoma and Paraganglioma, ESCA Esophageal carcinoma, HNSC Head and Neck squamous cell carcinoma, KICH Kidney Chromophobe, PRAD Prostate adenocarcinoma, THCA Thyroid carcinoma, LAML Acute Myeloid Leukemia. B Box plot of PICH mRNA expression in different lymphoma subtypes in the Mature B-cell malignancies study from MD Anderson Cancer Center. Boxplot shows 10–90 percentile. n = 60 (Burkitt Lymphoma), n = 95 (Diffuse Large B-cell Lymphoma), n = 65 (Follicular Lymphoma), n = 43 (Mantle Cell Lymphoma), and n = 23 (Marginal Zone Lymphoma). Mean and SEMs are indicated. The levels of PICH expression are significantly higher in BL than in the other four lymphoma subtypes (Diffuse Large B-Cell L., Follicular L., Mantle Cell L., and Marginal Zone L.). Significance was assessed by Tukey’s multiple comparisons test. ****p ≤ 0.0001. The levels are also significantly higher in Diffuse Large B-Cell L. than in Follicular L., Mantle Cell L., and Marginal Zone L. with a P-value ****p ≤ 0.0001 with Tukey’s multiple comparisons test. C PICH expression in human cancer cell lines from the Cancer Cell Line Encyclopedia, adapted from DepMap. Cell lines are grouped by lineage and subtypes are shown for the lymphoid lineage. Boxplot shows 10–90 percentile. D Representative immunoblots of PICH protein levels in BL cell lines and the primary non-tumor cell line RPE-1. β-Actin was used as a control for protein load. E Representative pictures (left) and quantification (right) of PICH expression in BL and normal lymph node tissue array assessed by IHC staining. Scale bars are indicated, 100 μm. Quantification of the percentage of PICH-positive cells is shown on the right. Data shown correspond to six fields per tissue, counting at least 1000 cells per field. Four normal lymph nodes and three BL tumors were assessed. Means and SEMs are indicated. Significance was assessed by unpaired t-test ****p ≤ 0.0001. F Representative images of spleens collected from Myc wild-type (Myc+/+) and Emu-Myc transgenic (Myc+/tg) mice (left) and immunohistochemical staining against Pich on paraffin sections of spleens from mice with the indicated genotypes (right). Scale bar, 50 μm.

Fig 4: PICH is required for the maintenance of HSC function. A) Cell cycle analysis of the indicated LT‐HSCs through staining with DAPI and Ki67, n = 4 for each. ∗∗∗p ≤ 0.001. B) Percentage of large, intermediate, and small colonies from a single colony forming assay of sorted LT‐HSCs. C) Cell death analysis of LSK cells in FL of WT and Pich‐KO mice using flow cytometry, n = 3 for each. ∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001. D) The survival rate of lethally irradiated WT recipients with adopted WT or Pich KO fetal liver cells, or PBS as a control. E) Representative FACS plots and percentage of donor‐derived bone marrow cells at 12 weeks after competitive fetal liver cell transplantation, n for each. ∗∗∗p ≤ 0.001.

Fig 5: IFNAR1 deletion alleviates the HSC dysfunction in Pich‐KO mice. A) Flow cytometry analysis of FL HSCs in WT, Pich‐KO, Ifnar1‐KO, and DKO mice. Sca‐1+c‐Kit+ cells in the Lin− cells were gated out for LT‐HSC analysis. B) Percentages of the indicated cells in WT, Pich‐KO, Ifnar1‐KO, and DKO mice, n = 3 for each. ∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001. C) Cell numbers of the indicated cells in WT, Pich‐KO, Ifnar1‐KO, and DKO mice. D) Cell cycle analysis of LT‐HSCs through staining with DAPI and Ki67, n = 3 for each. ∗p ≤ 0.05. E) Cell death analysis of LSK cells in FL of WT and Pich‐KO mice using flow cytometry, n = 3 for each. ∗∗p ≤ 0.01. F) Quantification of total cell number in FL of WT, Pich‐KO, Ifnar1‐KO, and DKO mice, n = 3 for each. ∗p ≤ 0.05. G) E14.5 embryos were isolated and visualized under a dissecting microscope. Scale bar = 1mm. A blow‐up of the fetal liver in each case is shown.