Figure 7(A, Tumor progression in Il7+/+ (grey), Il7+/- (dashed) and Il7-/- (black) mice transplanted with Pax5Jak2-Luc/+ tumor cells, as determined by bioluminescence measurements at the indicated days after cell transfer. Images of three transplanted mice for each Il7 genotype are shown at the indicated days after cell transfer B) Tumor progression in Il7+/+ (grey), Il7+/- (dashed) and Il7-/- (black) mice transplanted with Pax5Jak2-Luc/+ tumor cells, as determined by bioluminescence measurements at the indicated days after cell transfer. The bioluminescence measurements of the transplanted mice are shown as mean radiance values with SEM (B) and were analyzed by two-way ANOVA with Tukey's multiple comparison test: ns P > 0.05, ****P < 0.0001. n, number of mice analyzed. (C) Kaplan-Meier survival analysis of Il7+/+, Il7+/- and Il7-/- mice transplanted with Pax5Jak2-Luc/+ tumors cells. Statistical analysis of the survival curves was performed with the log-rank (Mantel-Cox) test; ns P > 0.05, *P < 0.05, **P < 0.01. (D) Phosphorylation (p) of the STAT5 protein in B220low B-ALL cells (black) and control non-leukemic B220+ B cells (grey) from the lymph node of a 11-week-old Pax5Jak2/+ mouse. The p-STAT5 level was determined by intracellular staining with an anti-STAT5 (pY694) antibody. (E) Quantification of the median fluorescence intensity (MFI) values of the p-STAT5 levels determined by intracellular staining of splenic FO B cells from Pax5+/+ (grey), Pax5Jak2/+ (black) and Pax5Jak2-KD/+ (white) mice at the age of 3-10 weeks Statistical data are presented as mean values with SEM and were analyzed by the two-tailed unpaired Student's t-test: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Each dot corresponds to one mouse. (F) Flow cytometric analysis of p-STAT5 levels in pro-B cells (B220+CD19+Kit+IgM-) and leukemic B220low B cells of Pax5Jak2/+ mice (black) as well as in control pro-B cells of Pax5+/+ (WT) mice (grey) at the age of 4-5 weeks. A representative flow cytometric analysis of the bone marrow of a Pax5Jak2/+ mouse (left) and representative intracellular p-STAT5 stainings (middle) are shown. The p-STAT5 levels in pro-B and leukemic B220low B cells (right) are quantified as MFI values relative to that of Pax5+/+ pro-B cells. Statistical data are presented as mean values with SEM and were analyzed by the two-tailed unpaired Student's t-test: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Each dot corresponds to one mouse. (G) STAT5 binding at regulated and non-regulated genes in Pax5Jak2/+ B-ALL cells. The regulated genes corresponded to the 254 up- and 144 down-regulated genes (> 3-fold) identified in Pax5Jak2/+ B-ALL cells relative to control Pax5+/- Cdkn2ab+/- B-ALL cells The 4,264 non-regulated genes were defined by an expression difference between > -1.25 and < 1.25-fold. The percentages indicate the overlap of the regulated and non-regulated genes with the 1,606 STAT5-bound genes, which were determined by ChIP-seq analysis in wild-type pro-B cells. The P value was calculated with the Pearson's chi-squared test with Yates' continuity correction. (H) GSEA analysis of the 19 activated STAT5 target genes identified in pro-B cells as compared with the ranked log2-fold gene expression changes in Pax5Jak2/+ (PJ) B-ALLs versus control Pax5+/- Cdkn2ab+/- B-ALLs. NES, normalized enrichment score. (I) Upregulation of the genes Cxcr5, Syndig1l and Sema4a in Pax5Jak2/+ (PJ) B-ALL cells. Mean TPM values with SEM are shown for the following RNA-seq experiments: 2 (Ctrl B-ALL), 4 (PJ B-ALL) (J, Identification of Cxcr5, Syndig1l and Sema4a as activated STAT5 target genes. Expression of the three genes in pro-B cells of Vav-Bcl2 Rag1-Cre Stat5fl/fl (KO) and control (Ctrl) Vav-Bcl2 Rag1-Cre Stat5fl/+ mice (J) was determined by RNA-seq Mean TPM values with SEM are shown for the following RNA-seq experiments: 2 (Ctrl pro-B) and 2 (KO pro-B), K) Identification of Cxcr5, Syndig1l and Sema4a as activated STAT5 target genes. ChIP-seq analysis identified STAT5-binding regions at all three loci (K). Horizontal bars below the ChIP-seq track indicate STAT5-binding regions identified by MACS peak calling. RPM, reads per million.