Fig 2: NOX2 Is Expressed in HSPCs, and Its Deficiency Compromises Steady-State and Regenerative Hematopoiesis(A) Schematics showing the subunits making up the canonical NADPH oxidase 2 complex.(B) mRNA expression of all 4 murine paralogs of NADPH oxidase genes in purified LSK cells.(C) mRNA expression levels of NOX2 in primitive hematopoietic cells. Data are mined from previously reported RNA-seq results (Cabezas-Wallscheid et al., 2014)(D) mRNA expression of all 4 murine paralogs of NADPH oxidase genes in MPP3 cells.(E) Frequency of LT-HSCs, MPP1, and MPP4 cells in total live BM from age- and sex-matched WT and NOX2 KO mice. n = 8, two independent experiments.(F) Frequency of MPP2 and MPP3 cells in total live BM from age- and sex-matched WT and NOX2 KO mice. n = 8, two independent experiments.(G) Differential cell count on the peripheral blood (PB) of age- and sex-matched WT and NOX2 KO mice. n = 5, representative of 3 independent experiments.(H) Representative flow cytometric plot showing the composition of PB (left) and the frequency of Gr1/CD11b+ myeloid cells in total CD45+ live BM from age- and sex-matched WT and NOX2 KO mice (right). n = 5.(I) Frequency of Gr1/CD11b+ myeloid cells in total CD45+ live BM from age- and sex-matched WT and NOX2 KO mice. n = 5.(J) Competitive transplantation of 500,000 whole BM cells from WT and NOX2 KO mice into irradiated WT recipients. The contribution of donor and competitor cells in PB is shown.Bars represent mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant, unpaired Student’s t test.

Fig 3: NOX2 and FOXC1 Are Important for the Growth of Primary Human Myeloid Leukemia Cells(A) The Cancer Genome Atlas (TCGA) database for AML was used to analyze the expression levels of several NADPH-dependent oxidases (NOX1–NOX5), accessory subunits (NCF1, NCF2, NCF4, NOXA1, NOXO1, p22Phox, RAC1, and RAC2) as well as related oxido-reductase enzymes (dual-oxidases 1,2, A2). The reads per kilobase of transcript per million mapped reads (RPKM) values for each gene in a total of 188 AMLs are shown.(B) RNA-seq analysis was performed on functionally validated leukemic stem cells isolated from human primary AMLs. The RPKM value for each gene is shown. Unpublished data are used with permission. Additional supporting data are shown in Figure S7A.(C) Equal numbers of control or shNOX2-transduced primary AML cells were cultured in vitro in the presence of 10 ng/mL of IL-3, SCF, and FL3, and the relative percent expansion is reported for each specimen. n = 3, mean ± SD. *p < 0.05; **p < 0.01.(D) Control and shNOX2 primary AML cells were purified and cultured in vitro for 12 days. Annexin V, DAPI staining was performed to evaluate the degree of apoptotic cell death. A representative flow plot (left) and quantitation of 3 technical triplicates (right) are shown. Additional data are shown in Figure S7B.(E) The relative expression level of NOX2, CEBPe, Elane, and CTSG is shown in AML specimens in which NOX2 was knocked down using shRNAs. Additional supporting data are provided in Figure S7D.(F) Histogram showing the relative expression of the myeloid surface antigen CD11b in control and NOX2 KD primary human AML cells cultured in vitro.(G) Images showing the morphology of control and NOX2 KD primary AML cells analyzed via May-Grünwald-Giemsa staining.(H) Relative level of expression of NOX2 is shown in control and shNOX2 primary AML cells used in xenograft analysis.(I) Representative flow cytometric plot showing the level of GFP+ (human leukemic cells bearing control or shNOX2) leukemic cells explanted from the BM of recipient mice (left), and quantitation of the relative normalized engraftment, defined as(% CD45+GFP+ BM at harvest/% CD45+ GFP+ BM pre-transplant) × 100 (right). Data are shown as whiskers minimum-to-maximum plot; the line inside the box represents the mean, and the top and bottom lines represent the 75% and 25% percentiles. The lines above and below represent SD. n = 9. ****p < 0.0001, unpaired Student’s t test.(J) Relative engraftment levels of a bc-CML specimen in which NOX2 is knocked down.(K) Relative engraftment levels of an AML specimen in which NOX2 is knocked down.(L) The relative level of expression of FOXC1 is shown in control and shFOXC1 primary bc-CML specimen used in xenograft analysis.(M) Similar analysis as in (I) but performed for FOXC1 knockdown xenografts of the bc-CML specimen.(N) Relative engraftment levels of control and FOXC1 KD groups in an AML specimen expressing high levels of FOXC1.(O) Relative engraftment levels of control and FOXC1 KD groups in an additional AML specimen expressing high levels of FOXC1.

Fig 4: Genetic deletion of Eif4e reduces eIF4E protein expression in p190 cells(A) Established p190 WT and eIF4E+/- leukemia cells have similar immunophenotype characterized by B220 and CD43 expression. Representative flow cytometry plots of p190 cells.(B) Protein analyses of eIF4E, 4E-BP-1, 4E-BP-2 and actin in WT and Het p190 cells. Relative protein quantification performed by densitometric analysis using ImageJ64 software. Data are expressed as mean ± SEM. Fold change was calculated using actin as loading control and normalized to WT sample for each independent experiment. Significance was calculated using unpaired t test (*p < 0.05; ****p < 0.001, n = 7).(C) p190s were generated from WT or Het mice and we used a bicistronic dual Renilla-Firefly luciferase reporter construct to measure cap-dependent translation (Renilla luciferase) relative to cap-independent, Coxsackie virus IRES mediated translation (firefly luciferase) as an internal control. MLN0128 (100 nM) treated cells were used as a control. Data are represented as mean ± SEM. Fold change was calculated using WT vehicle condition. Significance was calculated using a paired one tailed student's t test. (*p < 0.05; **p < 0.01; n = 3 or 4 per group).(D) Western blots of eIF4E, 4E-BP-1, 4E-BP-2, and total ERK (tERK) in eIF4E+/+, eIF4E fl/+ and eIF4E fl/fl p190 after 72hr of 4OHT (1 µM) treatment.(E) Relative protein quantification performed by densitometric analysis using ImageJ64 software. Fold change calculated using tERK as loading control. Data are expressed as mean ± SEM. Significance was calculated using paired t test (*p < 0.05; **p < 0.01; ****p < 0.001, n = 4–6 per group).

Fig 5: Treg-LTaß and LEC-LTßR interaction regulates graft survival and Treg migration and stabilityBALB/c islet-allograft survival in streptozotocin-induced diabetic C57BL/6 background recipients.(A) WTfl and KOfl diabetic recipients treated with tamoxifen received 1 × 106 tTregs from WT Foxp3GFP mice mixed with islets or islets alone (no Tregs). Graft survival compared by log rank (Mantel-Cox) test; *p < 0.05, MST (days).(B) 1 × 106 tTregs from WT or LTa-/- Foxp3GFP mixed with BALB/c islets and transplanted to diabetic CD45.1+ C57BL/6. Grafts and dLNs were harvested after 4 days and analyzed for Treg content by flow cytometry.(C) 1 × 106 tTregs from CD45.1+ WT Foxp3GFP mixed with islets and transplanted to diabetic, tamoxifen-treated CD45.2+ WTfl or KOfl. Recipient mice were treated with tamoxifen and rested for 10 days before streptozotocin administration. Islet transplantation performed at least 1 week after streptozotocin treatment. Percentage and numbers of adoptively transferred Tregs in each population are measured.(D) Cells from groups treated as in (B) analyzed for changes in Tregs. tTregs from WT or LTa-/- Foxp3GFP plus islets transplanted to diabetic CD45.1+ C57BL/6. Grafts and dLNs were harvested 4 days after transplantation. Transferred cells were analyzed for CD25 and Foxp3 expression. Average cell percentages for CD25hiFoxp3hi tTregs and CD25loFoxp3lo exTregs are shown.Data were pooled from 3 independent experiments with 3 recipient mice in each group. Errors bars are SD. Unpaired t test with Welch’s correction for comparison of cell numbers and percentages.