Figure 2.
Cell-intrinsic requirements for Tlr2 expression in AML. (A) Western blot analysis of Tlr2 pathway (NF-κB, p42/44, p38) activation in response to Pam3CSK4 or PBS treatment in Dnmt3a+/−Flt3ITD AML cells. Treatments were performed in vitro for the indicated times. β-actin served as loading control. (B) Average ± SEM relative expression of Infg by RT-qPCR in response to Pam3CSK4 or PBS treatment in Dnmt3a+/−Flt3ITD AML cells. Treatments were performed in vitro for the indicated times (n = 3/group). (C) Kaplan-Meier survival curves of de novo Dnmt3a+/−Flt3ITD AML and Tlr2−/−Dnmt3a+/−Flt3ITD AML mice (n = 10/group). (D) Histopathology of BM, spleen, and liver sections from moribund de novo Dnmt3a+/−Flt3ITD AML and Tlr2−/−Dnmt3a+/−Flt3ITD AML or age-matched WT mice. (E) Average ± SEM total BM counts (left) and average ± SEM percent Lin−cKit+ (LK) and Lin−cKit+Sca1+ (LSK) (right) populations in BM from age-matched WT (n = 5) and moribund de novo Dnmt3a+/−Flt3ITD AML (n = 6) and Tlr2−/−Dnmt3a+/−Flt3ITD AML (n = 5) mice. (F) Average ± SEM (left) total SP counts and average ± SEM percent LK and LSK (right) populations in spleens from moribund de novo Dnmt3a+/−Flt3ITD AML (n = 6) and Tlr2−/−Dnmt3a+/−Flt3ITD AML (n = 5) mice. (G) Schematic (left) of CD45.2+Tlr2−/−Dnmt3a+/−Flt3ITD AML-transplanted mice into CD45.1+ WT recipient mice, treatment schematic in panel D. Kaplan-Meier survival curves (right) of Tlr2−/−Dnmt3a+/−Flt3ITD AML-transplanted mice (n = 6/group). Arrow indicates treatment on day 14 after transplant. (H) Average ± SEM total BM counts of Dnmt3a+/−Flt3ITD AML-transplanted WT recipients (AML:WT, n = 9/group) and Tlr2−/−Dnmt3a+/−Flt3ITD AML-transplanted WT recipients (Tlr2−/−AML:WT, n = 9/group) 24 hours after treatment with Pam3CSK4 or PBS control. (I) Average ± SEM total BM counts of Dnmt3a+/−Flt3ITD AML-transplanted WT recipients 24 hours after treatment with indicated TLR agonist or PBS control (n = 3/group). (J) Average ±SEM percent of cleaved caspase 3/7+ BM cells from Dnmt3a+/−Flt3ITD AML-transplanted WT recipients 24 hours after treatment with indicated TLR agonist or PBS control (n = 3/group). Statistical significance by ordinary 1-way analysis of variance (ANOVA) using Tukey multiple comparison test (B,E-F,H-I,J, right) or unpaired t test (J, left), or Kaplan-Meier log rank test (C,G). ∗∗∗∗P < .0001, ∗∗∗P < .001, ∗∗P < .01, ∗P < .05.

Cell-intrinsic requirements for Tlr2 expression in AML. (A) Western blot analysis of Tlr2 pathway (NF-κB, p42/44, p38) activation in response to Pam3CSK4 or PBS treatment in Dnmt3a+/−Flt3ITD AML cells. Treatments were performed in vitro for the indicated times. β-actin served as loading control. (B) Average ± SEM relative expression of Infg by RT-qPCR in response to Pam3CSK4 or PBS treatment in Dnmt3a+/−Flt3ITD AML cells. Treatments were performed in vitro for the indicated times (n = 3/group). (C) Kaplan-Meier survival curves of de novo Dnmt3a+/−Flt3ITD AML and Tlr2−/−Dnmt3a+/−Flt3ITD AML mice (n = 10/group). (D) Histopathology of BM, spleen, and liver sections from moribund de novo Dnmt3a+/−Flt3ITD AML and Tlr2−/−Dnmt3a+/−Flt3ITD AML or age-matched WT mice. (E) Average ± SEM total BM counts (left) and average ± SEM percent Lin−cKit+ (LK) and Lin−cKit+Sca1+ (LSK) (right) populations in BM from age-matched WT (n = 5) and moribund de novo Dnmt3a+/−Flt3ITD AML (n = 6) and Tlr2−/−Dnmt3a+/−Flt3ITD AML (n = 5) mice. (F) Average ± SEM (left) total SP counts and average ± SEM percent LK and LSK (right) populations in spleens from moribund de novo Dnmt3a+/−Flt3ITD AML (n = 6) and Tlr2−/−Dnmt3a+/−Flt3ITD AML (n = 5) mice. (G) Schematic (left) of CD45.2+Tlr2−/−Dnmt3a+/−Flt3ITD AML-transplanted mice into CD45.1+ WT recipient mice, treatment schematic in panel D. Kaplan-Meier survival curves (right) of Tlr2−/−Dnmt3a+/−Flt3ITD AML-transplanted mice (n = 6/group). Arrow indicates treatment on day 14 after transplant. (H) Average ± SEM total BM counts of Dnmt3a+/−Flt3ITD AML-transplanted WT recipients (AML:WT, n = 9/group) and Tlr2−/−Dnmt3a+/−Flt3ITD AML-transplanted WT recipients (Tlr2−/−AML:WT, n = 9/group) 24 hours after treatment with Pam3CSK4 or PBS control. (I) Average ± SEM total BM counts of Dnmt3a+/−Flt3ITD AML-transplanted WT recipients 24 hours after treatment with indicated TLR agonist or PBS control (n = 3/group). (J) Average ±SEM percent of cleaved caspase 3/7+ BM cells from Dnmt3a+/−Flt3ITD AML-transplanted WT recipients 24 hours after treatment with indicated TLR agonist or PBS control (n = 3/group). Statistical significance by ordinary 1-way analysis of variance (ANOVA) using Tukey multiple comparison test (B,E-F,H-I,J, right) or unpaired t test (J, left), or Kaplan-Meier log rank test (C,G). ∗∗∗∗P < .0001, ∗∗∗P < .001, ∗∗P < .01, ∗P < .05.

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