Fig 1: Ca2+/CaM-Src kinase model validation. a Cartoon schematic of Src kinase activation of the Ca2+/CaM signaling cascade and the pro-apoptotic protein—Bax—following hypoxic neonatal brain injury, as well as pharmacological inhibition of this pathway using PP2. b Simulation of our model yields activated Src dynamics c activated CaMKK2 (blue) and CaMKIV (cyan), d cytoplasmic (maroon) and nuclear Ca2+ (grey), ATP (purple), and e Bax (black) over simulation time course
Fig 2: Phenotype of WT and Camkk2-/- BMDM generated in the presence or absence of tumor-conditioned medium. WT and Camkk2-/- BMDM were generated in the presence of RM or TCM. a Left: CD206 and MHC II expression on CD11b+ F4/80+ gated BMDM. Right: percentages of CD206+/MHC II- and CD206-/MHC II+ subsets (mean ± SEM; N = 3 biological replicates). b Expression of MHC II on CD11b + F4/80 + gated BMDM (mean of MFI ± SEM; N = 3 biological replicates). c Cxcl9 expression (mean ± SEM; N = 3 biological replicates). These experiments were replicated at least three times. d Tumors of comparable size (500–700 mm3) were removed from WT and Camkk2-/- mice, and myeloid cells were then identified and sorted by flow cytometry. Bar graph reports mean ± SEM of CD206+/MHC II- and CD206-/MHC II+ TAM percentages gated on “Mac” subset in the Supplementary Fig. 2 (N = 5 and 4 tumors removed from WT and Camkk2-/- mice, respectively). e Cxcl9 expression in TAM sorted from E0771 tumors removed from WT and Camkk2-/- mice (mean ± SEM; N = 3 and 3 tumors for each genotype)
Fig 3: AMPKa1 and CaMKK2 co-immunoprecipitate with IQGAP1.A, HEK-293 cells were transfected with GFP-AMPKa1. 48 h after transfection, cells were lysed and endogenous IQGAP1 was immunoprecipitated (IP) with anti-IQGAP1 antibody (IQ1). Nonimmune rabbit serum (NIRS) was used as control. Samples were analyzed by SDS-PAGE and Western blotting using anti-GFP and anti-IQGAP1 antibodies. 1% of the lysate used for IP was resolved in parallel (Input). B, HEK-293 cells were transfected with GFP or GFP-AMPKa1. 72 h after transfection, cells were lysed and GFP-tagged proteins were immunoprecipitated with GFP-Trap Agarose. Samples were analyzed by SDS-PAGE and Western blotting using anti-GFP and anti-IQGAP1 antibodies. 1% of the lysate used for IP was resolved in parallel (Input). C, HEK-293 cells were transfected with GFP-CaMKK2. Immunoprecipitation of endogenous IQGAP1 and Western blotting were performed as described for panel (A). D, HEK-293 cells were transfected with GFP or GFP-CaMKK2. Immunoprecipitation of GFP proteins and Western blotting were performed as in panel (B). The positions of migration of molecular weight markers are indicated on the left. All data are representative of at least three independent experiments.
Fig 4: AMPKa1 and CaMKK2 bind directly to IQGAP1 via its IQ domain.A, schematic representation of IQGAP1 constructs. The identified protein interaction motifs (CHD, calponin homology domain; WW, two tryptophan-containing domain; IQ, IQ domain; GRD, GAP-related domain; RGCT, RasGAP C_terminus) and amino acid residues of each construct are indicated. These constructs correspond to full-length IQGAP1 (F, amino acids 2–1657), the N-half (N, 2–863), the IQ domain (IQ, 717–916), and the C-half (C, 864–1657) of IQGAP1. IQGAP1?746-860 has amino acids 746 to 860 deleted. B, a Coomassie-stained gel of the GST proteins (GST-AMPKa1, GST-CaMKK2, or GST alone) used for binding assays. Data are representative of two independent experiments. C, fragments of IQGAP1 (F, N, IQ or C) were expressed and labeled with [35S]methionine using the TNT system. The IQGAP1 (IQ1) fragments were incubated with purified recombinant GST-AMPKa1, GST-CaMKK2, or GST alone. Complexes were pulled down (PD) with glutathione-Sepharose beads and analyzed by SDS-PAGE and autoradiography. 1% of the TNT products were analyzed in parallel (Input, right panel). The positions of migration of molecular weight markers are indicated on the left. Data are representative of at least two independent experiments. D, TNT products of full-length IQGAP1(F) or IQGAP1?746-860 (?746-860) were labeled with biotinylated-lysine and were incubated with purified GST alone, GST-AMPKa1, or GST-CaMKK2. Complexes were pulled down and analyzed by SDS-PAGE. The gel was cut at ~120 kDa. The upper portion of the gel (containing IQGAP1) was processed by Western blotting using IRDye-conjugated streptavidin (Strept.). The lower portion of the gel was stained with Coomassie blue. 1% of the TNT products were analyzed in parallel (Input). Data are representative of five independent experiments.
Fig 5: Pharmacological inhibition of CaMKK2 attenuates mammary tumor growth in immunocompetent mice. a E0771 (4 × 105) cells were orthotopically grafted into syngeneic WT mice. Starting at day 2 after grafting, mice were treated three times/week with vehicle or STO-609 (IP, 100 µmoles/kg body weight), and subsequently tumor volumes measured (mean ± SEM; N = 6 in each group); two-way ANOVA was used to calculate p-values. b, c T lymphocytes and myeloid cells within E0771 mammary tumors treated with STO-609 or vehicle. Tumors of comparable size (500–700 mm3) were removed, digested and single-cell suspensions were stained for myeloid and lymphoid markers, and analyzed using the gating strategy reported in Supplementary Fig. 3C and 11. Treatment with STO-609 resulted in the accumulation of CD8+ T cells and CD11b+ MHC II+ myeloid cells (b and c, respectively). Bar graph shows mean ± SEM; N = 5 and 4 tumors in Veh and STO-609 groups, respectively. A t test was used to calculate p-values. d STO-609 failed to affect mammary tumor growth in CD8+ T cell-depleted Camkk2-/- mice. Camkk2-/- mice were treated with anti-CD8 or control isotype antibodies. Subsequently, E0771 cells were orthotopically grafted, and mice treated with STO-609 or vehicle. Tumor volumes were measured (mean ± SEM; N = 3, 7, and 8 in isotype/Veh, CD8/Veh and CD8/STO groups, respectively). Two-way ANOVA test was used to calculate p-values. Asterisks refer to *p < 0.05
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