Additional file 6 of Overexpression of wild type RRAS2, without oncogenic mutations, drives chronic lymphocytic leukemia

Additional file 6: Figure S6. a, Mutations found in human cancer involving the RRAS2 gene. Data obtained from cBioPortal (97,250 patients/100669 samples). Refseq: NM_012250. Ensembl: ENST00000256196. CCDS: CCDS7814. Uniprot: RRAS2_HUMAN. Missense mutations (green dots): 36. Truncating mutations (black dots): 6. Splice mutations (orange dots): 5. b, Quantification by RT-qPCR or total mouse (Rras2) and human (RRAS2) mRNA expression in purified splenic CD19+ B cells from Rras2(Q72L)fl/fl xmb1-Cre (Q72L) mice compared to purified B CD19+ B cells from control WT C57BL/6 mice and to CD19 + CD5+ leukemic B cells from Rosa26-RRAS2fl/flxmb1-Cre mice. Results show data obtained in triplicate normalized to the C57BL/6 control for n = 3 mice per group. All mice were 14 month-old. Data show means ± SEM for three mice per group. *p < 0.05; ns. Not significant (one-way ANOVA test). c, Left, quantification by flow cytometry of total B-cell number in spleens of 14 month-old control and Rras2(Q72L)fl/fl xmb1-Cre mice. Right, two-parameter flow cytometry plot showing frequency of IgM + CD5+ cells within CD19+ splenic B cells of control and Rras2(Q72L)fl/fl xmb1-Cre mice. d, Left, concentration of B-cells per microliter in blood of control and Rras2(Q72L)fl/fl xmb1-Cre mice. Right, two-parameter flow cytometry plot showing frequency of CD19 + CD5+ cells within blood B cells of control and Rras2(Q72L)fl/fl xmb1-Cre mice. e, Frequency of marginal zone (MZ) phenotype (CD21high, CD23low), and follicular (CD21low, CD23high) B cells within CD19+ splenic B cells of control and Rras2(Q72L)fl/fl xmb1-Cre mice. f, Phosflow cytometry analysis of different elements from PI3K-Akt-mTOR, Raf-Erk and proximal BCR signaling pathways. Wild-type CD19+ follicular B cells, CD19 + CD5+ leukemic cells from spleens of Rosa26-RRAS2fl/flxmb1-Cre mice and CD19+ non-leukemic B cells from Rras2(Q72L)fl/fl xmb1-Cre are shown. In grey, background fluorescence of the secondary antibodies. All mice were 23 wk-old. Data show means ± SEM from three mice per group. *p < 0.05; **p < 0.01; ****p < 0.0001 (one-way ANOVA test). g, Phosflow cytometry analysis of different elements from PI3K-Akt-mTOR, Raf-Erk and proximal BCR signaling pathways. CD19 + CD5+ leukemic cells from 30 wk-old Rosa26-RRAS2fl/flxmb1-Cre mice are compared with WT Control follicular (CD23highCD21−), marginal zone (MZ, CD23−CD21high), B1a (CD11b + CD5+) and B1b (CD11b + CD5−) spleen B cell populations. Data show means ± SEM from n = 3 mice per group. **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant (one-way ANOVA test).