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A PI3K p110β–Rac signalling loop mediates Pten-loss-induced perturbation of haematopoiesis and leukaemogenesis

Yuzugullu, Haluk, Baitsch, Lukas, Von, Thanh, Steiner, Allison, Tong, Haoxuan, Ni, Jing, Clayton, Linda K., Bronson, Roderick, Roberts, Thomas M., Gritsman, Kira, Zhao, Jean J.·2015·Nature Communications
DOI 10.1038/ncomms9501PMC PMC4598950
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Abstract

The tumour suppressor PTEN, which antagonizes PI3K signalling, is frequently inactivated in haematologic malignancies. In mice, deletion of PTEN in haematopoietic stem cells (HSCs) causes perturbed haematopoiesis, myeloproliferative neoplasia (MPN) and leukaemia. Although the roles of the PI3K isoforms have been studied in PTEN-deficient tumours, their individual roles in PTEN-deficient HSCs are unknown. Here we show that when we delete PTEN in HSCs using the Mx1–Cre system, p110β ablation prevents MPN, improves HSC function and suppresses leukaemia initiation. Pharmacologic inhibition of p110β in PTEN-deficient mice recapitulates these genetic findings, but suggests involvement of both Akt-dependent and -independent pathways. Further investigation reveals that a p110β–Rac signalling loop plays a critical role in PTEN-deficient HSCs. Together, these data suggest that myeloid neoplasia driven by PTEN loss is dependent on p110β via p110β–Rac-positive-feedback loop, and that disruption of this loop may offer a new and effective therapeutic strategy for PTEN-deficient leukaemia.

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Figure 1 Ablation of p110β prolonged survival of animals on Pten loss. (a) Survival of PtenΔ/Δ (blue; n=9; median survival=26 days), PtenΔ/Δ;p110αΔ/Δ (green; n=9; median survival=31 days), PtenΔ/Δ;p110βΔ/Δ (red; n=9; median survival=55 days), PtenΔ/Δ;p110δ−/− (orange; n=7; median survival=37 days) and control animals (black) represented as DPI. The log-rank test was used to compare survival between groups. (b) Survival time and the development of MPN and T-ALL in each group of mice as indicated. Survival of PtenΔ/Δ (n=9; median survival=26 days), PtenΔ/Δ;p110αΔ/Δ (n=9; median survival=31 days), PtenΔ/Δ;p110βΔ/Δ (n=9; median survival=55 days), PtenΔ/Δ;p110δ−/− (n=7; median survival=37 days) and control animals (black) represented as DPI. Black dots represent MPN disease cases and red dots represent T-ALL cases in each group. The log-rank test was used to compare survival between groups. (c) Histopathology of the spleen (× 1 and × 40) and liver (× 100) taken at 26 DPI. Increased splenomegaly and disruption of spleen architecture as well as increased myeloid cell infiltration after Pten deletion. Scale bar, 5 μm. (d) Total cellularity of the spleen is represented as mean±s.d. of n=5 except PtenΔ/Δ;p110δ−/− (n=10). Two-way ANOVA test was applied to compare the spleen cellularity. (e) Representative flow cytometry plots of Mac1+Gr1+ cells in the spleen. Samples were analysed at 26 DPI. Bar graphs represent the mean±s.d. of n=9 (PtenΔ/Δ;p110δ−/−), 10 (PtenΔ/Δ;p110αΔ/Δ), 11 (ctrl) or 13 (PtenΔ/Δ and PtenΔ/Δ;p110βΔ/Δ). Two-way ANOVA test was applied to compare the Mac1-Gr1 cells in the spleen. *P<0.05, **P<0.01, ***P<0.001
Figure 1 Ablation of p110β prolonged survival of animals on Pten loss. (a) Survival of PtenΔ/Δ (blue; n=9; median survival=26 days), PtenΔ/Δ;p110αΔ/Δ (green; n=9; median survival=31 days), PtenΔ/Δ;p110βΔ/Δ (red; n=9; median survival=55 days), PtenΔ/Δ;p110δ−/− (orange; n=7; median survival=37 days) and control animals (black) represented as DPI. The log-rank test was used to compare survival between groups. (b) Survival time and the development of MPN and T-ALL in each group of mice as indicated. Survival of PtenΔ/Δ (n=9; median survival=26 days), PtenΔ/Δ;p110αΔ/Δ (n=9; median survival=31 days), PtenΔ/Δ;p110βΔ/Δ (n=9; median survival=55 days), PtenΔ/Δ;p110δ−/− (n=7; median survival=37 days) and control animals (black) represented as DPI. Black dots represent MPN disease cases and red dots represent T-ALL cases in each group. The log-rank test was used to compare survival between groups. (c) Histopathology of the spleen (× 1 and × 40) and liver (× 100) taken at 26 DPI. Increased splenomegaly and disruption of spleen architecture as well as increased myeloid cell infiltration after Pten deletion. Scale bar, 5 μm. (d) Total cellularity of the spleen is represented as mean±s.d. of n=5 except PtenΔ/Δ;p110δ−/− (n=10). Two-way ANOVA test was applied to compare the spleen cellularity. (e) Representative flow cytometry plots of Mac1+Gr1+ cells in the spleen. Samples were analysed at 26 DPI. Bar graphs represent the mean±s.d. of n=9 (PtenΔ/Δ;p110δ−/−), 10 (PtenΔ/Δ;p110αΔ/Δ), 11 (ctrl) or 13 (PtenΔ/Δ and PtenΔ/Δ;p110βΔ/Δ). Two-way ANOVA test was applied to compare the Mac1-Gr1 cells in the spleen. *P<0.05, **P<0.01, ***P<0.001CC BY · PubMed Central
Figure 2 Ablation of p110β blocked MPN development in a cell-autonomous manner. (a) Colony-forming assays were performed with 1 × 104 (BM) or 5 × 104 (spleen) cells obtained from corresponding animals. Cells were cultured in M3434 medium. Colonies were counted 7 days after incubation. Data are represented as mean±s.e.m. (n=3, in duplicates). *P<0.05, **P<0.01, ***P<0.001. (b) BM cells were isolated from CD45.2+ WT, PtenΔ/Δ and PtenΔ/Δ;p110βΔ/Δ animals and transplanted into CD45.1+ mice as shown in the figure (n=10 for each group). (c) The myeloid cell reconstitution potential of the CD45.2+ cells was measured at regular intervals for 16 weeks. WT and PtenΔ/Δ;p110βΔ/Δ-derived myeloid cells have a stable amount of CD45.2+ myeloid cells; however, PtenΔ/Δ-derived myeloid cells expanded significantly and reached to 60% at week 16 (n=10 for all groups, **P<0.001). Two-way ANOVA test was applied to compare the myeloid cell potential.
Figure 2 Ablation of p110β blocked MPN development in a cell-autonomous manner. (a) Colony-forming assays were performed with 1 × 104 (BM) or 5 × 104 (spleen) cells obtained from corresponding animals. Cells were cultured in M3434 medium. Colonies were counted 7 days after incubation. Data are represented as mean±s.e.m. (n=3, in duplicates). *P<0.05, **P<0.01, ***P<0.001. (b) BM cells were isolated from CD45.2+ WT, PtenΔ/Δ and PtenΔ/Δ;p110βΔ/Δ animals and transplanted into CD45.1+ mice as shown in the figure (n=10 for each group). (c) The myeloid cell reconstitution potential of the CD45.2+ cells was measured at regular intervals for 16 weeks. WT and PtenΔ/Δ;p110βΔ/Δ-derived myeloid cells have a stable amount of CD45.2+ myeloid cells; however, PtenΔ/Δ-derived myeloid cells expanded significantly and reached to 60% at week 16 (n=10 for all groups, **P<0.001). Two-way ANOVA test was applied to compare the myeloid cell potential.CC BY · PubMed Central
Figure 3 p110β deletion restores HSCs to both the bone marrow (BM) and the spleen. (a) Representative flow cytometric plots of ctrl, PtenΔ/Δ and PtenΔ/Δ;p110βΔ/Δ BM showing LSK cells (Lin–Sca-1+c-kit+), ST-HSC (CD150–CD48−Lin−Sca-1+c-kit+) and LT-HSC (CD150+CD48−Lin−Sca-1+c-kit+) cells (left panels) gated on total living CD45.2+Lin– cells. Total counts LSK (top) and LT-HSC (bottom right) of lineage-negative cells in the BM measured at 26 DPI in ctrl (n=13), PtenΔ/Δ (n=12) and PtenΔ/Δ;p110βΔ/Δ (n=18) animals. Two-way ANOVA test was applied to compare the stem cell numbers. (b) Representative flow cytometry plots of control, PtenΔ/Δ and PtenΔ/Δ;p110βΔ/Δ spleen cells showing LSK cells (Lin–Sca-1+c-kit+), ST-HSC (CD150–CD48−Lin−Sca-1+c-kit+) and LT-HSC (CD150+CD48−Lin−Sca-1+c-kit+) cells (left panels). Total LSK (top) and ST-HSC and LT-HSC (bottom right) cells in the spleen measured at 26 DPI. Numbers represent total cell counts per spleen (n=7 (ctrl, PtenΔ/Δ) and 10 (PtenΔ/Δ;p110βΔ/Δ)). Two-way ANOVA test was applied to compare the stem cell numbers. *P<0.05, **P<0.01, ***P<0.001
Figure 3 p110β deletion restores HSCs to both the bone marrow (BM) and the spleen. (a) Representative flow cytometric plots of ctrl, PtenΔ/Δ and PtenΔ/Δ;p110βΔ/Δ BM showing LSK cells (Lin–Sca-1+c-kit+), ST-HSC (CD150–CD48−Lin−Sca-1+c-kit+) and LT-HSC (CD150+CD48−Lin−Sca-1+c-kit+) cells (left panels) gated on total living CD45.2+Lin– cells. Total counts LSK (top) and LT-HSC (bottom right) of lineage-negative cells in the BM measured at 26 DPI in ctrl (n=13), PtenΔ/Δ (n=12) and PtenΔ/Δ;p110βΔ/Δ (n=18) animals. Two-way ANOVA test was applied to compare the stem cell numbers. (b) Representative flow cytometry plots of control, PtenΔ/Δ and PtenΔ/Δ;p110βΔ/Δ spleen cells showing LSK cells (Lin–Sca-1+c-kit+), ST-HSC (CD150–CD48−Lin−Sca-1+c-kit+) and LT-HSC (CD150+CD48−Lin−Sca-1+c-kit+) cells (left panels). Total LSK (top) and ST-HSC and LT-HSC (bottom right) cells in the spleen measured at 26 DPI. Numbers represent total cell counts per spleen (n=7 (ctrl, PtenΔ/Δ) and 10 (PtenΔ/Δ;p110βΔ/Δ)). Two-way ANOVA test was applied to compare the stem cell numbers. *P<0.05, **P<0.01, ***P<0.001CC BY · PubMed Central
Figure 4 p110β deletion rescued long-term HSC (LT-HSC) reconstitution and suppressed leukaemia initiation and homing defects after Pten deletion. (a) Competitive repopulation experiment: bone marrow (BM) cells were isolated at 7 days post pIpC (DPI) from CD45.2+ ctrl, PtenΔ/Δ, PtenΔ/Δ;p110αΔ/Δ and PtenΔ/Δ;p110βΔ/Δ animals, and transplanted into lethally irradiated recipient animals along with CD45.1+ BM cells. The donor chimaerism in the peripheral blood was measured at regular intervals for 20 weeks, represented as the percentage of CD45.2+ cells among all leukocytes in the peripheral blood (n=9 for all groups). Two-way ANOVA test was applied to compare the donor derived cell numbers. (b) Disease-free survival of animals in the competitive repopulation experiment. Statistical analysis was performed at week 20 post transplantation using Fisher’s exact test. All samples were compared with the control group. Fischer exact test was applied to test overall survival. (c) After 20 weeks, the competitive repopulation experiment was terminated, and the remaining numbers of LSK, ST-HSC and LT-HSCs in the BM were measured by flow cytometry (n=4 for each group except n=5 for PtenΔ/Δ;p110δ−/−). Two-way ANOVA test was applied to compare the donor derived stem cell numbers. (d) Homing potential of CD45.2-positive carboxyfluorescein diacetate succinimidyl ester (CFSE)-labelled control, PtenΔ/Δ and PtenΔ/Δ;p110βΔ/Δ cells in 24 h in irradiated recipients (n=5) for each group. Two-way ANOVA test was applied to compare the donor derived stem cell numbers. *P<0.05, **P< 0.01, ***P<0.001
Figure 4 p110β deletion rescued long-term HSC (LT-HSC) reconstitution and suppressed leukaemia initiation and homing defects after Pten deletion. (a) Competitive repopulation experiment: bone marrow (BM) cells were isolated at 7 days post pIpC (DPI) from CD45.2+ ctrl, PtenΔ/Δ, PtenΔ/Δ;p110αΔ/Δ and PtenΔ/Δ;p110βΔ/Δ animals, and transplanted into lethally irradiated recipient animals along with CD45.1+ BM cells. The donor chimaerism in the peripheral blood was measured at regular intervals for 20 weeks, represented as the percentage of CD45.2+ cells among all leukocytes in the peripheral blood (n=9 for all groups). Two-way ANOVA test was applied to compare the donor derived cell numbers. (b) Disease-free survival of animals in the competitive repopulation experiment. Statistical analysis was performed at week 20 post transplantation using Fisher’s exact test. All samples were compared with the control group. Fischer exact test was applied to test overall survival. (c) After 20 weeks, the competitive repopulation experiment was terminated, and the remaining numbers of LSK, ST-HSC and LT-HSCs in the BM were measured by flow cytometry (n=4 for each group except n=5 for PtenΔ/Δ;p110δ−/−). Two-way ANOVA test was applied to compare the donor derived stem cell numbers. (d) Homing potential of CD45.2-positive carboxyfluorescein diacetate succinimidyl ester (CFSE)-labelled control, PtenΔ/Δ and PtenΔ/Δ;p110βΔ/Δ cells in 24 h in irradiated recipients (n=5) for each group. Two-way ANOVA test was applied to compare the donor derived stem cell numbers. *P<0.05, **P< 0.01, ***P<0.001CC BY · PubMed Central
Figure 5 Pharmacological inhibition of p110β suppresses leukaemia in vitro and in Pten-deficient mice. (a) Colony-forming assays were performed with 1 × 104 (BM) or 5 × 104 (spleen) cells obtained from 7 DPI PtenΔ/Δ animals. Cells were cultured with added DMSO, BYL719, TGX221,GS1101, NVSPI35, GDC0941 or RAD001 with the following concentrations based on published data: 1, 0.5, 0.1 μM (BYL719, TGX221 and GDC0941); 5, 1, 0.5 μM (GS1101); 0.1, 0.05, 0.01 μM (NVSPI35 and RAD001). Colonies were counted 7 days after incubation. Data are represented as mean±s.e.m. (n=3, measured in duplicates) and all data are compared with the DMSO control for statistical analysis. *P<0.05; **P<0.001. Two-way ANOVA test was applied to compare the colony formation. (b) Schematic representation of drug treatment. (c) Survival of PtenΔ/Δ animals treated with vehicle (blue; n=7; median survival=23 days), BYL719 (green; n=8; median survival=32 days), KIN193 (red; n=7; median survival=50 days), IC87114 (orange; n=7; median survival=28 days) and AS605240 (brown; n=6; median survival=23 days). *P<0.05; **P<0.001; ***P<0.0001. The log-rank test was applied to compare survival. (d) Spleen weight of vehicle-treated (n=4) and KIN193-treated animals (n=3) at the moribund stage. *P<0.05. Student’s t-test was applied. (e) Western blot analysis of Akt signalling in whole BM cells at 7 DPI. Freshly isolated BM cells were treated with DMSO, BYL719, KIN193, GS1101 or NVSPI35 at the 1-μM dose for 2 h (n=6) for each treatment. (f) Phospho-flow analysis of p-Akt on isoform-selective inhibitor-treated animals. Freshly isolated lineage-negative BM cells were treated with inhibitors as in (e) and subjected to flow cytometry for LSK staining and intracellular P-Akt staining (n=3) for each, and median fluorescence intensities were normalized to control.
Figure 5 Pharmacological inhibition of p110β suppresses leukaemia in vitro and in Pten-deficient mice. (a) Colony-forming assays were performed with 1 × 104 (BM) or 5 × 104 (spleen) cells obtained from 7 DPI PtenΔ/Δ animals. Cells were cultured with added DMSO, BYL719, TGX221,GS1101, NVSPI35, GDC0941 or RAD001 with the following concentrations based on published data: 1, 0.5, 0.1 μM (BYL719, TGX221 and GDC0941); 5, 1, 0.5 μM (GS1101); 0.1, 0.05, 0.01 μM (NVSPI35 and RAD001). Colonies were counted 7 days after incubation. Data are represented as mean±s.e.m. (n=3, measured in duplicates) and all data are compared with the DMSO control for statistical analysis. *P<0.05; **P<0.001. Two-way ANOVA test was applied to compare the colony formation. (b) Schematic representation of drug treatment. (c) Survival of PtenΔ/Δ animals treated with vehicle (blue; n=7; median survival=23 days), BYL719 (green; n=8; median survival=32 days), KIN193 (red; n=7; median survival=50 days), IC87114 (orange; n=7; median survival=28 days) and AS605240 (brown; n=6; median survival=23 days). *P<0.05; **P<0.001; ***P<0.0001. The log-rank test was applied to compare survival. (d) Spleen weight of vehicle-treated (n=4) and KIN193-treated animals (n=3) at the moribund stage. *P<0.05. Student’s t-test was applied. (e) Western blot analysis of Akt signalling in whole BM cells at 7 DPI. Freshly isolated BM cells were treated with DMSO, BYL719, KIN193, GS1101 or NVSPI35 at the 1-μM dose for 2 h (n=6) for each treatment. (f) Phospho-flow analysis of p-Akt on isoform-selective inhibitor-treated animals. Freshly isolated lineage-negative BM cells were treated with inhibitors as in (e) and subjected to flow cytometry for LSK staining and intracellular P-Akt staining (n=3) for each, and median fluorescence intensities were normalized to control.CC BY · PubMed Central
Figure 6 Pten-deficient haematopoietic cells depend on the p110β–Rac1 signalling axis. (a) Rac–GTP activity assay was performed on BM after 7 DPI in the corresponding genotypes. Rac–GTP and total Rac were measured and normalized (n=3; mean with s.e.m.). Two-way ANOVA test was applied to compare the RAC-GTPase/RAC levels. (b) PtenΔ/Δ;p110βΔ/Δ BM cells were transduced with retroviruses expressing HA-tagged wild-type P110β or RBD-mutant P110β, and briefly selected in the presence of puromycin. HA immunoblotting was performed to show expression of RBD-mutant and wild-type P110β (n=3 for each group). (c) Cells from WT control, PtenΔ/Δ or PtenΔ/Δ;p110βΔ/Δ animals were cultured in methylcellulose with myeloid growth factors in the presence of puromycin for 7 days, and colonies were counted at the end of day 7 (n=3 for each group). Two-way ANOVA test was applied to compare the colony numbers. (d) Lineage-negative cells were isolated from WT control, PtenΔ/Δ or PtenΔ/Δ;p110βΔ/Δ mice, and the Rac–GTP activity assay was performed as in (a). Rac–GTP and total Rac were measured and normalized to WT (n=6; mean with s.e.m.). Two-way ANOVA test was applied to compare the RAC-GTPase/RAC levels. **P< 0.01, ***P<0.001
Figure 6 Pten-deficient haematopoietic cells depend on the p110β–Rac1 signalling axis. (a) Rac–GTP activity assay was performed on BM after 7 DPI in the corresponding genotypes. Rac–GTP and total Rac were measured and normalized (n=3; mean with s.e.m.). Two-way ANOVA test was applied to compare the RAC-GTPase/RAC levels. (b) PtenΔ/Δ;p110βΔ/Δ BM cells were transduced with retroviruses expressing HA-tagged wild-type P110β or RBD-mutant P110β, and briefly selected in the presence of puromycin. HA immunoblotting was performed to show expression of RBD-mutant and wild-type P110β (n=3 for each group). (c) Cells from WT control, PtenΔ/Δ or PtenΔ/Δ;p110βΔ/Δ animals were cultured in methylcellulose with myeloid growth factors in the presence of puromycin for 7 days, and colonies were counted at the end of day 7 (n=3 for each group). Two-way ANOVA test was applied to compare the colony numbers. (d) Lineage-negative cells were isolated from WT control, PtenΔ/Δ or PtenΔ/Δ;p110βΔ/Δ mice, and the Rac–GTP activity assay was performed as in (a). Rac–GTP and total Rac were measured and normalized to WT (n=6; mean with s.e.m.). Two-way ANOVA test was applied to compare the RAC-GTPase/RAC levels. **P< 0.01, ***P<0.001CC BY · PubMed Central
Figure 7 Pten-deficient haematopoietic cells depend on the p110β–Rac1 signalling axis. (a) NSC23766 was given at 2.5 μg g−1 daily intraperitoneally (IP). Spleen size and cellularity of vehicle-treated (n=3) and NSC23766-treated PtenΔ/Δ animals (n=3) at 17 DPI. *P<0.05; **P<0.001; ***P<0.0001. Two-way ANOVA test was applied to compare spleen size. (b) Quantification of the numbers of LSK (Lin–Sca-1+c-kit+), ST-HSC (CD150–CD48−Lin−Sca-1+c-kit+) or LT-HSC (CD150+CD48−Lin−Sca-1+c-kit+) cells in the spleens of control, PtenΔ/Δ animals treated with vehicle, or PtenΔ/Δ animals treated with NSC23766 at 17 DPI, determined by flow cytometry. The numbers represent total cell counts per spleen (n=3; ctrl, PtenΔ/Δ vehicle and NSC23766 treated). (c) Survival of PtenΔ/Δ animals treated with vehicle (blue; n=7; median survival=22 days) or NSC23766 (red; n=7; median survival=42 days). **P<0.005. The log-rank test was applied to compare survival. (d) Lineage-negative BM cells were isolated from control, PtenΔ/Δ or PtenΔ/Δ:P110βΔ/Δ PtenΔ/Δ:P110αΔ/Δ on 7 DPI, and lineage-negative cells were pretreated for 3 h with KIN193, NSC23766 (50 μM) or pertussis toxin (PTX; 100 ng ml−1). Migration towards the lower chamber (supplemented with CXCL12, 50 nM) was measured as the fraction of migrated cells relative to untreated Pten-null bone marrow-derived cells. Numbers of replicates are n=4 (basal, PtenΔ/Δ no Cxcl12; PtenΔ/Δ+PTX), 10 (PtenΔ/Δ), 6 (PtenΔ/Δ:P110βΔ/Δ) and 6 (PtenΔ/Δ+KIN193). Statistical comparisons were all performed relative to the PtenΔ/Δ group. (e) PtenΔ/Δ and PtenΔ/Δ;p110βΔ/Δ BM cells were transfected with two different siRNAs targeting scrambled, mouse Rac1, mouse Rac2 alone, or both Rac1 and Rac2 and subjected to immunoblotting 48 h later. (f) PtenΔ/Δ and PtenΔ/Δ;p110βΔ/Δ BM cells from knockdown were transfected with two different siRNAs targeting scrambled, mouse Rac1, mouse Rac2 alone, or both Rac1 and Rac2, or treated with NSC23766 (50 μM) and then colony assays were performed in methylcellulose with myeloid growth factors. Colonies were counted after 7 days (n=3) for each group. Two-way ANOVA test was applied to calculate colony formation. (g) A working model of signalling cascades downstream of P110β and the signalling loop of P110β and Rac1.
Figure 7 Pten-deficient haematopoietic cells depend on the p110β–Rac1 signalling axis. (a) NSC23766 was given at 2.5 μg g−1 daily intraperitoneally (IP). Spleen size and cellularity of vehicle-treated (n=3) and NSC23766-treated PtenΔ/Δ animals (n=3) at 17 DPI. *P<0.05; **P<0.001; ***P<0.0001. Two-way ANOVA test was applied to compare spleen size. (b) Quantification of the numbers of LSK (Lin–Sca-1+c-kit+), ST-HSC (CD150–CD48−Lin−Sca-1+c-kit+) or LT-HSC (CD150+CD48−Lin−Sca-1+c-kit+) cells in the spleens of control, PtenΔ/Δ animals treated with vehicle, or PtenΔ/Δ animals treated with NSC23766 at 17 DPI, determined by flow cytometry. The numbers represent total cell counts per spleen (n=3; ctrl, PtenΔ/Δ vehicle and NSC23766 treated). (c) Survival of PtenΔ/Δ animals treated with vehicle (blue; n=7; median survival=22 days) or NSC23766 (red; n=7; median survival=42 days). **P<0.005. The log-rank test was applied to compare survival. (d) Lineage-negative BM cells were isolated from control, PtenΔ/Δ or PtenΔ/Δ:P110βΔ/Δ PtenΔ/Δ:P110αΔ/Δ on 7 DPI, and lineage-negative cells were pretreated for 3 h with KIN193, NSC23766 (50 μM) or pertussis toxin (PTX; 100 ng ml−1). Migration towards the lower chamber (supplemented with CXCL12, 50 nM) was measured as the fraction of migrated cells relative to untreated Pten-null bone marrow-derived cells. Numbers of replicates are n=4 (basal, PtenΔ/Δ no Cxcl12; PtenΔ/Δ+PTX), 10 (PtenΔ/Δ), 6 (PtenΔ/Δ:P110βΔ/Δ) and 6 (PtenΔ/Δ+KIN193). Statistical comparisons were all performed relative to the PtenΔ/Δ group. (e) PtenΔ/Δ and PtenΔ/Δ;p110βΔ/Δ BM cells were transfected with two different siRNAs targeting scrambled, mouse Rac1, mouse Rac2 alone, or both Rac1 and Rac2 and subjected to immunoblotting 48 h later. (f) PtenΔ/Δ and PtenΔ/Δ;p110βΔ/Δ BM cells from knockdown were transfected with two different siRNAs targeting scrambled, mouse Rac1, mouse Rac2 alone, or both Rac1 and Rac2, or treated with NSC23766 (50 μM) and then colony assays were performed in methylcellulose with myeloid growth factors. Colonies were counted after 7 days (n=3) for each group. Two-way ANOVA test was applied to calculate colony formation. (g) A working model of signalling cascades downstream of P110β and the signalling loop of P110β and Rac1.CC BY · PubMed Central

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Cited by

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Metadata

Authors
Yuzugullu, H., Baitsch, L., Von, T., Steiner, A., Tong, H., Ni, J., Clayton, L. K., Bronson, R., Roberts, T. M., Gritsman, K., & Zhao, J. J.
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2015-10-07
Volume
6
Issue
1
ISSN
2041-1723
DOI
10.1038/ncomms9501
PMC
PMC4598950
Cited by
47
References
6
Language
en
Licence

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