RasGRP1 KO and RasGRP 1/3 DKO show inefficient thymocyte development beyond DN3.

<p><b>A.</b> CD44 by CD25 profiles of DN thymocytes (CD4⁻CD8^–Thy1.2⁺CD3^lo) from B6 (n = 9), 1KO (n = 6), 3KO (n = 9) and DKO (n = 13) thymi. <b>B.</b> left, numbers of DN3 (CD4⁻CD8⁻CD3^loCD44⁻CD25⁺) thymocytes and DN4 (CD4⁻CD8⁻CD3^loCD44⁻CD25⁻) thymocytes; right, ratio of frequencies of DN3/DN4.</p

RasGRP1 KO and RasGRP1/3 DKO mice display inefficient generation of DP thymocytes.

<p><b>A.</b> Numbers of thymocytes from B6 (n = 9), 1KO (n = 6), 3KO (n = 9) and DKO (n = 13) thymi. <b>B.</b> Numbers and frequencies of DN (CD4⁻CD8⁻Thy1.2⁺CD3^lo) from B6 (n = 9), 1KO (n = 6), 3KO (n = 9) and DKO (n = 13) thymi. <b>C.</b> left, numbers of DP (CD4⁺CD8⁺Thy1.2⁺); right, ratio of frequencies of DP/DN. *p<0.05, **p<0.01 and ***p<0.001.</p

RasGRP1 KO and RasGRP1/3 DKO DN4 thymocytes show an increased frequency of γδ T cells, despite normal numbers and frequencies of mature thymic γδ T cells. A.

<p>CD3 by γδTCR profiles of bulk thymocytes from B6 (n = 7), 1KO (n = 3), 3KO (n = 6) and DKO (n = 9) thymi. <b>B.</b> Frequencies and numbers of mature CD3⁺γδTCR⁺ γδ T cells. <b>C.</b> left, frequencies of surface TCRβ⁺ DN4 (CD4⁻CD8⁻Thy1.2⁺CD44⁻CD25⁻) cells from B6 (n = 8), 1KO (n = 6), 3KO (n = 9) and DKO (n = 12) thymi; right, frequencies of surface γδTCR⁺ DN4 cells. ***p<0.001.</p

RasGRP1 KO and RasGRP1/3 DKO thymocytes display impaired proliferation of DN3 and inefficient transition from DN3E to DN3L. A.

<p>Intracellular TCRβ (TCRβ_i.c.) by forward scatter (FSC) profiles of DN3 (CD4⁻CD8⁻Thy1.2⁺CD44⁻CD25⁺) from B6 (n = 8), 1KO (n = 6), 3KO (n = 9) and DKO (n = 12) thymi. <b>B.</b> Frequencies of DN3 (CD4⁻CD8⁻Thy1.2⁺CD44⁻CD25⁺) and DN4 (CD4⁻CD8⁻Thy1.2⁺CD44⁻CD25⁻) expressing intracellular TCRβ (TCRβ_i.c.). <b>C.</b> Ratio of frequencies of TCRβ_i.c.⁺ DN3E/DN3L ((DN3E/DN3L)TCRβ_i.c.⁺). <b>D.</b> CD98 by CD25 profiles of Thy1.2⁺CD44⁻ cells from 1 and 2 day DN3E-OP9-DL1 co-cultures; data are representative of 3 independent experiments. <b>E.</b> Frequencies of BrdU⁺ DN3 (CD4⁻CD8⁻Thy1.2⁺CD44⁻CD25⁺) and DN4 (CD4⁻CD8⁻Thy1.2⁺CD44^–CD25⁻) from B6 (n = 5), 1KO (n = 5), 3KO (n = 6) and DKO (n = 7) mice injected with BrdU i.p. 2h prior to euthanasia. *p<0.05, **p<0.01 and ***p<0.001.</p

RasGRP1 KO, RasGRP3 KO and RasGRP1/3 DKO thymocytes show intact survival.

<p>Percentages of DN3 (CD4⁻CD8⁻Thy1.2⁺CD44⁻CD25⁺), DN4 (CD4⁻CD8⁻Thy1.2⁺CD44⁻CD25⁻) and DP (CD4⁺CD8⁺Thy1.2⁺) showing active caspase 3.</p

DN3 require RasGRP1 for ERK phosphorylation in response to CXCR4 activation.

<p><b>A, B.</b> Bulk thymocytes were pre-treated with (right panels) or without (left panels) 20 µM of the PI3K inhibitor LY294002 and were stimulated with 10 nM SDF1α for 0, 1, 2, 3 or 5 minutes. <b>A.</b> Induction of p-ERK (pT202/pY204) was measured in DN3 (CD4⁻CD8⁻Thy1.2⁺CD3^lo Thy1.2⁺CD44⁻CD25⁺) from B6 and DKO thymi. <b>B.</b> Induction of p-AKT (pS473) was measured in DN3 (CD4⁻CD8⁻Thy1.2⁺CD3^lo Thy1.2⁺CD44⁻CD25⁺) from B6 and DKO thymi. <b>C.</b> Bulk thymocytes were stimulated with 10 nM SDF1α for 0, 1, 2, 3 or 5 minutes and the induction of p-ERK (pT202/pY204) was measured in DN3 (CD4⁻CD8⁻Thy1.2⁺CD3^lo Thy1.2⁺CD44⁻CD25⁺) from B6, 1KO, 3KO and DKO thymi. Data are representative of between 3 and 7 independent experiments. * represents a significant statistical comparison between B6 and DKO. # represents a significant statistical comparison between B6 and 1KO. */# p<0.05.</p