Additional file 3 of EP300-ZNF384 transactivates IL3RA to promote the progression of B-cell acute lymphoblastic leukemia
Creators: name: Hou, Zhijie
name: Ren, Yifei
name: Zhang, Xuehong
name: Huang, Dan
name: Yan, Fanzhi
name: Sun, Wentao
name: Zhang, Wenjuan
name: Zhang, Qingqing
name: Fu, Xihui
name: Lang, Zhenghui
name: Chu, Chenyang
name: Zou, Boyang
name: Gao, Beibei
name: Jin, Bilian
name: Kang, Zhijie
name: Liu, Quentin
name: Yan, Jinsong
Publisher: name: figshare
Year: 2024
Subjects: subject: Biochemistry
subject: Space Science
subject: Medicine
subject: Microbiology
subject: FOS: Biological sciences
subject: Cell Biology
subject: Genetics
subject: Molecular Biology
subject: Pharmacology
subject: Biotechnology
subject: Immunology
subject: FOS: Clinical medicine
subject: Biological Sciences not elsewhere classified
subject: Developmental Biology
subject: Cancer
subject: Hematology
subject: Infectious Diseases
subject: FOS: Health sciences
subject: Virology

description: Supplementary Material 3: Fig. 1 EP300-ZNF384 promotes the expression of IL3RA in both B and myeloid leukemia cells. PLVX eukaryotic expression vectors encoding EP300-ZNF384 and wild-type ZNF384 were transfected into the acute B lymphoblastic leukemia (B-ALL) cell line BALL-1 (A), the Burkitt’s lymphoma cell line Daudi (B), and the acute myeloid leukemia cell line KG-1α (C) by lentivirus-mediated gene transfer. Total RNA was isolated from the transfectants, IL3RA expression was quantified by reverse-transcription quantitative PCR (RT-qPCR) in relative to ACTB expression. *P &lt; 0.05, **P &lt; 0.01, ***P &lt; 0.001, ns, no significance. (D) Surface expression of CD123 was evaluated by flow cytometry on B-ALL cells with the empty vector, EP300-ZNF384 fusion gene, and ZNF384. The proportion of CD123-positive cells and the median fluorescence intensity of CD123 was quantified. **P &lt; 0.01, ***P &lt; 0.001. Fig. 2 IL3RA plays an important role in EP300-ZNF384 induced cell proliferation and STAT5 activation with or without IL-3. (A) Immunoblot analysis of pSTAT5 and STAT5 in empty vector, EP300-ZNF384-, or ZNF384-expressing NALM-6 cells with or without IL-3. (B) shIL3RA vectors (shIL3RA-1, shIL3RA-2, shIL3RA-3, shIL3RA-3 and empty vector) were transfected into NALM-6 cells. The knockdown efficiency was evaluated by reverse transcription quantitative PCR (RT-qPCR). **P &lt; 0.01, ***P &lt; 0.001, ns, no significance. (C) ShIL3RA expressing NALM-6 cells were subjected to a colony-forming cell assay in the presence of IL-3 (10 ng/mL) or doxycycline (Dox, 2 µg/mL) or the combination of IL-3 with Dox. Quantitative analysis of colony size (diameter) and colony number was presented. *P &lt; 0.05, ***P &lt; 0.001, ns, no significance. (D) ShIL3RA-4-expressing NALM-6 cells were transfected with EP300-ZNF384 expressing vector. Cells were collected and RT-PCR was performed to assess the expression of EP300-ZNF384 and IL3RA. (E) Cells derived from D were subjected to immune blot analysis to determine the expression of EP300-ZNF384 and the phosphorylation of STAT5 in response to IL-3 treatment (10 ng/mL). Fig. 3 EP300-ZNF384 promotes the expression of CD123 on the cell membrane and the secretion of IL-3 in B precursor cells. Statistical analysis of surface CD123 expression on B precursor cells derived from 5 healthy volunteers with the empty vector, EP300-ZNF384 fusion gene, and ZNF384. (A) The proportion of CD123-positive cells and (B) the median fluorescence intensity of CD123 were quantified. Data represent the mean ± SD. ns, no significance. (C) The expression pattern of IL3 in hematopoiesis was presented. (D) CD19-positive cells were enriched with magnetic beads from PBMCs of healthy volunteers. Empty vector, EP300-ZNF384-, and ZNF384-expressing vectors were transfected into CD19-positive cells by lentivirus-mediated gene transfer. After 48 h, cells were collected for reverse transcription quantitative PCR assay to assess the transcription of IL3. Data represents mean ± SD. *P &lt; 0.05, **P &lt; 0.01, ***P &lt; 0.001, ns, no significance. (E) Empty vector-, EP300-ZNF384-, and ZNF384-expressing vectors were respectively transfected into healthy volunteer derived CD19-positive cells in serum-free media by lentivirus-mediated gene transfer. Culture media was collected and IL-3 secretion was determined by ELISA. (n = 7) *P &lt; 0.05, ns, no significance. Fig. 4 Doxorubicin specifically sensitized EP300-ZNF384-expressing B-ALL cells. The empty vector-, EP300-ZNF384- and ZNF384-expressing NALM-6 cells were treated with increasing doses of doxorubicin (A), dexamethasone (B), pegaspargase (C), and vincristine (D) for 48 h. Cell viability was evaluated using the CCK-8 assay. Fig. 5 Doxorubicin inhibited the infiltration of EP300-ZNF384-expressing B-ALL cells in bone marrow. Flow cytometry dot plots illustrating the infiltration of empty vector or EP300-ZNF384 or ZNF384-expressing NALM-6 cells in the bone marrow of mice in response to doxorubicin treatment (n = 7).