Additional file 2 of Overexpression of wild type RRAS2, without oncogenic mutations, drives chronic lymphocytic leukemia

Additional file 2: Figure S2. a, Flow cytometry analysis of GFP populations in 23 wk-old Rosa26-RRAS2fl/flxSox2-Cre mouse spleen. Representative two-color contour plots of GFPhigh and GFPlow populations in total B cells (CD19+), CD5+ leukemic and CD23+ follicular B cells. Bottom, representation of GFP populations in T lymphocytes (CD3+). b, Percentage of GFPhigh cells in the indicated populations determined by flow cytometry. Data show means ± SEM from n = 8 mice (23 wk-old mice). ****p < 0.0001 (one-way ANOVA test). c, Western blot analysis of R-RAS2 expression of sorted GFPlow and GFPhigh leukemic cells from the spleen of a 25 wk-old Rosa26-RRAS2fl/flxSox2-Cre mouse (β-actin as loading control). d, Dot plot representation of GFPlow CD5+ leukemic B cell evolution in mb1-Cre mice over time, showing each mouse individually (n = 14). Data points were adjusted to a linear fit. These data were retrieved from the same mice as in Fig. 2i. e, Percentage of CD5+ cells in the indicated populations comparing GFPhigh and GFPlow distribution. Data show means ± SEM from n = 4 30 wk-old mice. Two-way ANOVA test. f, Heatmap of RNAseq expression data showing the genes differentially regulated in wild-type, follicular B cells (n = 6, 12wk-old), leukemic CD19 + CD5+ B cells (n = 6, 54wk-old), CD19+ GFPhigh (n = 2, 54wk-old) and CD19+ GFPlow (n = 2, 54wk-old) populations. Only genes significantly different between GFPhigh GFPlow populations (p < 0.05) and with a difference of 2-fold or more were used. Gene expression is shown in normalized log2 fold change.