Figure 3.
ANXA5 deficiency in the BMM results in acceleration of AML via establishment of a proinflammatory BMM and activation of NF-κB. (A) Representative images of bone sections from WT or ANXA5 KO recipient mice with MLL-AF9–induced AML (retroviral transduction/transplantation model) stained for reticulin (left) and its quantification (right). Nuclei were counterstained with nuclear fast red-aluminum sulfate solution (P = .0455; t test; n = 5). The scale bar represents 50 μm. (B) Representative immunoblot for pp65S536 (∼62 kDa), p65 (60 kDa), nuclear factor of kappa light polypeptide gene enhancer in B-cell inhibitor α (IκBα; 36 kDa), and vinculin (124 kDa) as housekeeping control performed on lysates from WT or ANXA5 KO MSCs treated with TNF-α (15 ng/mL) for the indicated times (n = 4-5). (C-E) Representative immunofluorescence images (top) and quantification (bottom) of primary WT (red) or ANXA5 KO (blue) MSCs (C), macrophages (Mϕ) (D) and fibroblasts (E) treated with TNF-α (15 ng/mL) or IL-1β (10 ng/mL) for 5 minutes, stained with an antibody to p65 (green) and DAPI (blue) (2-way ANOVA, Sidak test, n = 4). (F) Relative expression of prostaglandin-endoperoxide synthase 2 (Ptgs2; COX2) by quantitative real time polymerase chain reaction analysis in WT (red) vs ANXA5 KO (blue) MSCs treated with TNF-α (15 ng/mL) for the indicated times (P = .043; 2-way ANOVA; Sidak test; n = 4). (G-H) Binding of p65 to the promoter of Ptgs2 (COX2) in WT (red) or ANXA5 KO (blue) MSCs treated with TNF-α (15 ng/mL) vs vehicle for 30 minutes in a chromatin immunoprecipitation assay using 2 different primer pairs: set 1 (G) and set 2 (H) (2-way ANOVA; Sidak test; n = 4). (I) Levels of PGE2, measured by enzyme-linked immunosorbent assay (ELISA), in the conditioned medium of WT (red) or ANXA5 KO (blue) MSCs treated with TNF-α (15 ng/mL) for the indicated times (P = .011; 2-way ANOVA; Sidak test; n = 4). (J) Levels of PGE2, measured by ELISA, in the conditioned medium of WT (red) or ANXA5 KO (blue) MSCs treated with TNF-α (15 ng/mL) ± celecoxib (250 μM) for 4 hours (P = .004; P = .019; 2-way ANOVA; Tukey test; n = 4). The data are normalized over the control (vehicle). Ctrl, control.

ANXA5 deficiency in the BMM results in acceleration of AML via establishment of a proinflammatory BMM and activation of NF-κB. (A) Representative images of bone sections from WT or ANXA5 KO recipient mice with MLL-AF9–induced AML (retroviral transduction/transplantation model) stained for reticulin (left) and its quantification (right). Nuclei were counterstained with nuclear fast red-aluminum sulfate solution (P = .0455; t test; n = 5). The scale bar represents 50 μm. (B) Representative immunoblot for pp65S536 (∼62 kDa), p65 (60 kDa), nuclear factor of kappa light polypeptide gene enhancer in B-cell inhibitor α (IκBα; 36 kDa), and vinculin (124 kDa) as housekeeping control performed on lysates from WT or ANXA5 KO MSCs treated with TNF-α (15 ng/mL) for the indicated times (n = 4-5). (C-E) Representative immunofluorescence images (top) and quantification (bottom) of primary WT (red) or ANXA5 KO (blue) MSCs (C), macrophages (Mϕ) (D) and fibroblasts (E) treated with TNF-α (15 ng/mL) or IL-1β (10 ng/mL) for 5 minutes, stained with an antibody to p65 (green) and DAPI (blue) (2-way ANOVA, Sidak test, n = 4). (F) Relative expression of prostaglandin-endoperoxide synthase 2 (Ptgs2; COX2) by quantitative real time polymerase chain reaction analysis in WT (red) vs ANXA5 KO (blue) MSCs treated with TNF-α (15 ng/mL) for the indicated times (P = .043; 2-way ANOVA; Sidak test; n = 4). (G-H) Binding of p65 to the promoter of Ptgs2 (COX2) in WT (red) or ANXA5 KO (blue) MSCs treated with TNF-α (15 ng/mL) vs vehicle for 30 minutes in a chromatin immunoprecipitation assay using 2 different primer pairs: set 1 (G) and set 2 (H) (2-way ANOVA; Sidak test; n = 4). (I) Levels of PGE2, measured by enzyme-linked immunosorbent assay (ELISA), in the conditioned medium of WT (red) or ANXA5 KO (blue) MSCs treated with TNF-α (15 ng/mL) for the indicated times (P = .011; 2-way ANOVA; Sidak test; n = 4). (J) Levels of PGE2, measured by ELISA, in the conditioned medium of WT (red) or ANXA5 KO (blue) MSCs treated with TNF-α (15 ng/mL) ± celecoxib (250 μM) for 4 hours (P = .004; P = .019; 2-way ANOVA; Tukey test; n = 4). The data are normalized over the control (vehicle). Ctrl, control.

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