Additional file 1 of Deletion of CD36 exhibits limited impact on normal hematopoiesis and the leukemia microenvironment
Creators: name: Meng, Yiting
name: Pospiech, Mateusz
name: Ali, Atham
name: Chandwani, Ritu
name: Vergel, Mary
name: Onyemaechi, Sandra
name: Yaghmour, George
name: Lu, Rong
name: Alachkar, Houda
Publisher: name: figshare
Year: 2023
Subjects: subject: Biochemistry
subject: Medicine
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Physiology
subject: Chemical Sciences not elsewhere classified
subject: Immunology
subject: FOS: Clinical medicine
subject: Developmental Biology
subject: Cancer
subject: Hematology

description: Additional file 1: Table S1. Cd36 Gene Expression in Mouse Normal Hematopoiesis. Table S2. Gene Expression for Cd36 in Mouse Normal Hematopoiesis. Table S3. Gene Expression for CD36 in Human Normal Hematopoiesis. Table S4. Cd36 expression in mouse normal hematopoietic system. Table S5. CD36 expression in normal human Hematopoiesis. Figure S1. Cd36 differential gene expression patterns in normal hematopoiesis between mouse and human. (A-B) Log2 transformed gene expression level for Cd36 in mouse normal hematopoietic system obtained from GSE14833 and GSE6506 datasets and CD36 expression in normal human hematopoiesis obtained from datasets GSE17054, GSE19599, GSE11864, and E-MEXP-1242, which all datasets were downloaded from BloodSpot database. Data are presented as the mean of gene expression among each cell population and each colored dot represents the expression value of a single sample. Unpaired t-test analysis was used (***, P &lt; 0.001; **, P &lt; 0.01; *, P &lt; 0.05). Figure S2. Confirmation of Cd36 reduced expression in Cd36-KO mice compared with WT mice. (A) Genotype confirmation of wildtype and Cd36 knockout mice by gel electrophoresis of DNA fragments generated by standard PCR with recommended primers. Lane 1 and 2: WT mice liver tissues with WT primers; Lane 3 and 4: WT liver mice tissues with Cd36-KO primers; Lane 5 and 6: Cd36-KO liver mice tissues with WT primers; Lane 7 and 8: Cd36-KO mice tissues with Cd36-KO primers. (B-D) qPCR quantification of Cd36 knockout efficiency in healthy mouse bone marrow cells, spleen cells, and liver cells (n = 6 mice per group). The bar graph represents the mean of Cd36 mRNA level in per group with standard error of the mean. Welch's t-test was used to analyze the significant difference (**, P &lt; 0.01). (E-H) Cd36 knockout was confirmed using flow cytometry by comparing the cell surface Cd36+ population in bone marrow, spleen, liver, and blood tissues (n = 6 mice per group). (I) The spleens and livers were collected from Cd36-KO and WT mice. (J-K) The weight of spleen and liver organs were compared. The bar graph represents the mean of spleen and liver mass in Cd36-KO and WT mice with standard error of the mean. Each colored dot represents the mass value of every single mouse tissue (n = 6 mice per group). The differences between Cd36-KO and WT mice tissues were analyzed by unpaired t-test (**, P &lt; 0.01; Abbreviation: ns, not significant). (L-O) Cd36 knockout efficiency was confirmed using flow cytometry analysis by comparing the cell surface Cd36+ population and Cd36 MFI in BM, spleen, liver, and blood cells. Data are presented as the mean of Cd36+ surface expression or CD36 MFI and each colored dot represents the value of a single sample. (n = 6 mice per group). The differences between Cd36-KO and WT mice tissues were analyzed by Welch's t-test (*, P &lt; 0.05; ***, P &lt; 0.001; ****, P &lt; 0.0001). Figure S3. Hematological analysis reveals similar blood counts between Cd36-KO and WT mice. (A-H) Data are presented as the mean of blood count between Cd36-KO mice and WT mice for white blood cell, red blood cell, hemoglobin, hematocrit, neutrophil, lymphocyte, monocyte, and platelet. Each colored single dot represents the count for every single mouse (n = 6 mice per group). The differences between Cd36-KO and WT group were analyzed by unpaired t-test (*, P &lt; 0.05; Abbreviation: ns, not significant). Figure S4. Cd36-KO mice exhibit similar T cell phenotypes compared with WT mice. (A-B) Representative flow cytometry of the lymphocytes population from mouse spleen cells stained with CD3+, CD4+, CD8+, and CD25+ flow antibody to evaluate the different T cell population in WT mice and Cd36-KO mice (n = 6 mice per group). Figure S5. Chracterization of hematopoietic stem and progenitors in WT and Cd36-KO mice. (A) Quantification results of the positive cell population percentages in freshly unriched BM cells were represented by the bar graph, in which each bar represents the mean with standard error of population percentage for Cd36-KO and WT mice (n = 4 female mice per group). Shown here are an early form of murine hematopoietic stem cell (KLS), common lymphoid progenitor (CLP), common myeloid progenitor (CMP), megakaryocyte-erythroid progenitor (MEP), and granulocyte-macrophage progenitor (GMP). The differences between groups were analyzed using unpaired t-test (Abbreviation: ns, not significant). (B) Quantification results of the positive cell population percentages in frozen unriched BM cells were represented by the bar graph, in which each bar represents the mean with standard error of population percentage for Cd36-KO and WT mice (n = 2 male mice per group). Shown here are an early form of murine hematopoietic stem cell (KLS), common lymphoid progenitor (CLP), common myeloid progenitor (CMP), megakaryocyte-erythroid progenitor (MEP), and granulocyte-macrophage progenitor (GMP). The differences between groups were analyzed using unpaired t-test (Abbreviation: ns, not significant). Figure S6. The homing rate was similar between WT and Cd36-KO mice. Representative flow cytometry result showing CD45.2+ WT or Cd36-KO HSPCs homing in CD45.1+ recipient BM niches (n = 7 in WT including 5 female and 2 male in WT group and n = 9 in Cd36-KO including 5 female and 4 male). Figure S7. Cd36 is dispensable for normal HSPCs homing and engraftment in the bone marrow. (A) Spleens and livers were collected from CD45.1 allele bearing mice injected with either 2.4 x 107 CD45.2+ BM cells (WT; n = 5 mice) or 2.4 x 107 CD36-KO BM cells (KO; n = 5 mice), one CD45.1 allele bearing blank mice, and one CD45.2 allele bearing blank mice. (B-C) The weights of spleens and livers were measured, and data are presented as the mean of tissue mass from WT and KO group and each colored dot represents the mass of a single mouse tissue (n = 5 mice per group). The differences between Cd36-KO and WT groups were analyzed by Mann-Whitney test (Abbreviation: ns, not significant). (D-F) Representative flow cytometry result showing BM transplant of healthy CD45.2+ WT or CD36-KO BM cells in the BM, spleen, and blood cells of CD45.1 allele bearing mice (n = 5 mice per group). Figure S8. Cd36-KO BM cells are less engrafted than WT BM cells in the competitive repopulation assay. (A-C) Representative flow cytometry result showing BM transplant and quantification of engraftment of mixed CD45.1+ WT and CD45.2+ Cd36-KO BM cells in the BM and spleen cells of CD45.1+ CD45.2+ recipient mice (n = 1 male and 1 female). The differences between groups were analyzed using ratio paired t-test (Abbreviation: *, P &lt; 0.05). Figure S9. Cd36-KO mice exhibit similar AML engraftment with WT mice. (A) Spleens and livers were collected from Cd36-KO and WT mice engrafted with 5 x 106 FLT3-ITD/MLL-PTD mouse leukemic cells (n = 5 mice in KO; n = 6 mice in WT), one CD45.1 allele bearing blank mice, and one CD45.2 allele bearing blank mice. (B-C) The weights of spleens and livers were measured, and data are presented as the mean of tissue mass from WT and KO group and each colored dot represents the mass of a single mouse tissue (n = 6 mice in WT group; n = 5 mice in Cd36-KO group). The differences between Cd36-KO and WT groups were analyzed by Mann-Whitney test (Abbreviation: ns, not significant). (D-G) Representative flow cytometry result showing AML engraftment of FLT3-ITD/MLL-PTD mouse leukemic cells in the BM, spleen, liver, and blood cells of WT and Cd36-KO mice (n = 6 mice in WT group; n = 5 mice in Cd36-KO group). Figure S10. Cd36-KO mice exhibit similar AML engraftment with WT mice. (A) Spleens and livers were collected from Cd36-KO and WT mice engrafted with 1 x 106 FLT3-ITD/MLL-PTD mouse leukemic cells (n = 4 mice per group), one CD45.1 allele bearing blank mice, and one CD45.2 allele bearing blank mice. (B-C) The weights of spleens and livers were measured, and data were presented as the mean of tissue mass from WT and KO group and each colored dot represents the mass of a single mouse tissue (n = 4 mice per group). The differences of tissue weight between Cd36-KO and WT groups were analyzed by Mann-Whitney test (Abbreviation: ns, not significant). (D-G) Representative flow cytometry result showing AML engraftment and quantification of engraftment of FLT3-ITD/MLL-PTD mouse leukemic cells in the BM, spleen, liver, and blood cells of WT and Cd36-KO mice (n = 4 mice). The difference between groups were analyzed using unpaired t-test (Abbreviation: ns, not significant).