Figure 5.
TREM2 was identified as a functional receptor mediating the effects of IL-34 on myeloid differentiation and AML progression. (A) Western blots confirming the KO of TREM2 expression in primary AML cells (AML01). (B) Growth curves of AML01-shTREM2, and AML01-NC cells after treatment with IL-34 (100 ng/mL) at the indicated time points (n = 3). (C) Colony-forming ability of primary AML cells after short hairpin RNA (shRNA)-mediated KO of TREM2, with or without treatment with IL-34 (n = 5). (D) Flow cytometry analysis of CD11b expression in AML01-shTREM2, and AML01-NC cells after IL-34 treatment (n = 5). (E) Representative Giemsa staining images showing the morphology of AML01-shTREM2, and AML01-NC cells after IL-34 treatment. Arrows highlight distinct morphology indicative of differentiation. Scale bars, 10 μm. (F) Western blots showing the KO efficiency of shTREM2 in THP-1 cells. (G) Growth curves of THP-1-shTREM2, and THP-1-NC cells after treatment with IL-34 (100 ng/mL) at the indicated time points (n = 3). (H) Colony-forming ability of THP-1 cells after shRNA-mediated knockdown of TREM2 with or without treatment with IL-34 (n = 5). (I) Flow cytometry analysis of CD11b expression in THP-1-shTREM2, and THP-1-NC cells after IL-34 treatment (n = 5). (J) Representative Giemsa staining images showing the morphology of THP-1-shTREM2, and THP-1-NC cells after IL-34 treatment. Arrows highlight distinct morphology indicative of differentiation. Scale bars, 10 μm. (K) Kaplan-Meier survival curves of mice that received transplantation with AML01-shTREM2, and AML01-NC cells treated with IL-34. Statistical assessment using log-rank test (n = 8). (L) Kaplan-Meier survival curves of mice that received transplantation with with THP-1-shTREM2, and THP-1-NC cells treated with IL-34. Statistical assessment by log-rank test (n = 8). (M) Percentage of CD11b+Gr-1+ cells in the BM of mice with AML01-shTREM2 grafts or AML01-NC grafts (n = 5). (N) Percentage of CD11b+Gr-1+ cells in the BM of mice with THP-1-shTREM2 grafts or THP-1-NC grafts (n = 5). (O) Coimmunoprecipitation assay of endogenous TREM2 and IL-34 in BV-2 microglial cells. (P) Coimmunoprecipitation assay of endogenous TREM2 and IL-34 in WT bone marrow–derived macrophages (BMDMs). (Q) The interaction between endogenous TREM2 and IL-34 in TREM2-deficiency BMDMs. (R-S) Representative immunostaining for IBA-1 (red) in primary BMDMs from WT (R) or TREM2-KO mice (S), after 3 days treatment with 10 ng/mL M-CSF (vehicle) or 100 ng/mL IL-34. Scale bars, 10 μm. Statistical significance was calculated using one-way ANOVA with Tukey multiple comparison test. Western blot images were representative of at least 3 independent experiments.

TREM2 was identified as a functional receptor mediating the effects of IL-34 on myeloid differentiation and AML progression. (A) Western blots confirming the KO of TREM2 expression in primary AML cells (AML01). (B) Growth curves of AML01-shTREM2, and AML01-NC cells after treatment with IL-34 (100 ng/mL) at the indicated time points (n = 3). (C) Colony-forming ability of primary AML cells after short hairpin RNA (shRNA)-mediated KO of TREM2, with or without treatment with IL-34 (n = 5). (D) Flow cytometry analysis of CD11b expression in AML01-shTREM2, and AML01-NC cells after IL-34 treatment (n = 5). (E) Representative Giemsa staining images showing the morphology of AML01-shTREM2, and AML01-NC cells after IL-34 treatment. Arrows highlight distinct morphology indicative of differentiation. Scale bars, 10 μm. (F) Western blots showing the KO efficiency of shTREM2 in THP-1 cells. (G) Growth curves of THP-1-shTREM2, and THP-1-NC cells after treatment with IL-34 (100 ng/mL) at the indicated time points (n = 3). (H) Colony-forming ability of THP-1 cells after shRNA-mediated knockdown of TREM2 with or without treatment with IL-34 (n = 5). (I) Flow cytometry analysis of CD11b expression in THP-1-shTREM2, and THP-1-NC cells after IL-34 treatment (n = 5). (J) Representative Giemsa staining images showing the morphology of THP-1-shTREM2, and THP-1-NC cells after IL-34 treatment. Arrows highlight distinct morphology indicative of differentiation. Scale bars, 10 μm. (K) Kaplan-Meier survival curves of mice that received transplantation with AML01-shTREM2, and AML01-NC cells treated with IL-34. Statistical assessment using log-rank test (n = 8). (L) Kaplan-Meier survival curves of mice that received transplantation with with THP-1-shTREM2, and THP-1-NC cells treated with IL-34. Statistical assessment by log-rank test (n = 8). (M) Percentage of CD11b+Gr-1+ cells in the BM of mice with AML01-shTREM2 grafts or AML01-NC grafts (n = 5). (N) Percentage of CD11b+Gr-1+ cells in the BM of mice with THP-1-shTREM2 grafts or THP-1-NC grafts (n = 5). (O) Coimmunoprecipitation assay of endogenous TREM2 and IL-34 in BV-2 microglial cells. (P) Coimmunoprecipitation assay of endogenous TREM2 and IL-34 in WT bone marrow–derived macrophages (BMDMs). (Q) The interaction between endogenous TREM2 and IL-34 in TREM2-deficiency BMDMs. (R-S) Representative immunostaining for IBA-1 (red) in primary BMDMs from WT (R) or TREM2-KO mice (S), after 3 days treatment with 10 ng/mL M-CSF (vehicle) or 100 ng/mL IL-34. Scale bars, 10 μm. Statistical significance was calculated using one-way ANOVA with Tukey multiple comparison test. Western blot images were representative of at least 3 independent experiments.

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