(A.) Bone marrow-derived macrophages were pre-treated for 1 hour with increasing concentrations (0.2–10µM) of Sorafenib or vehicle control (DMSO, 0µM), then stimulated with LPS (10ng/ml) with PGE₂ (10⁻⁸M). 16 hour culture supernatants were measured for IL-12p40 (black bars) or IL-10 (hatched bars) by ELISA. LPS stimulation alone is present as a control. (B.,C.) BMMΦ were pre-treated and stimulated as in (A). The presence of IL-12/23p40 (B.) and IL-10 (C.) mRNA was measured at 0’, 60’, 90’, 120’, 150’, and 180’ post-stimulation by real-time PCR after stimulation with LPS (circles), LPS+PGE (triangles), or LPS+PGE in the presence of Sorafenib (5µM, squares). mRNA levels are presented as relative to the expression at 0’. (D.) BMMΦ were pre-treated with vehicle or 1–5µM Sorafenib then stimulated with LPS alone. IL-12/23p40 protein was measured by ELISA. Culture supernatants were diluted 1:50 prior to ELISA. (E., F.) BMMΦ were pretreated and simulated as in (D.), and then IL-12/23p40 and IL-10 expression was measured by real-time PCR 2 and 3 hours after stimulation. (G.) Peritoneal macrophages were pretreated with vehicle or 5µM Sorafenib as in (A) and stimulated with LPS or LPS+ PGE₂. ELISAs were performed from 16h culture supernatants. Each bar represents the mean ± SD triplicate samples. *Indicates a p-value <0.05 and **indicates a p-value <0.01 as determined by student’s T-test comparing drug to vehicle control. Figures are representative of three independent experiments.

(A.) BMMΦ were stimulated for 1, 2, and 3 hours with LPS or LPS+PGE and assayed for STAT3 phosphorylation at Tyr705 and Ser727. Total STAT3 is present as a loading control. (B.) BMMΦ were pretreated with vehicle or Sorafenib (5µM) for one hour, then stimulated with LPS+ PGE₂. STAT3 phosphorylation was measured at Tyr705 at 1, 2, and 3h by western blot. Total STAT3 is present as a loading control. (C.) BMMΦ were stimulated with LPS, LPS+ PGE₂ (L/P), or LPS+PGE in the presence of Sorafenib for three hours. The presence of SOCS-3, STAT-3 phosphorylation at Tyr705, and actin were measured by western blotting. (D.) BMMΦ from wild type or IL-10^−/− BMMΦ were stimulated with LPS, LPS+PGE, and LPS+PGE in the presence of Sorafenib (5 and 10µM). Concentrations of IL-12/23p40 were measured by ELISA from 16h stimulated cultures. Each bar represents the mean ± SD of triplicate samples. *Indicates a p-value <0.0001. (E.) STAT3 phosphorylation at Tyr705 was assay by western blotting as in (A. and B.). Upper. Wild-type or IL-10^−/− BMMΦ were stimulated with LPS or LPS+PGE for 1, 2, or 3h. Lower. Wild-type or IL-10^−/− BMMΦ pretreated for 1 hour with vehicle or Sorafenib (5µM) were stimulated with LPS+PGE. Total STAT3 is present as a loading control. Figures are representative of at least two independent experiments.

(A.) BMMΦ pretreated with vehicle or Sorafenib (5µM) were stimulated with LPS+PGE for 15’, 30’, 45’, or 60’. Lysates were assayed for pAKT (Ser473), p-GSK3α/β (Ser21/9), and total AKT by western blotting. (B.) Image quantitation of AKT and GSK3β phosphorylation from (A.) (C.) BMMΦ were stimulated for LPS or LPS+PGE for 15’, 30’, 45’, 60’. Western blotting was performed as in (A.). Figures are representative of at least two independent experiments.

(A.) BMMΦ stimulated with LPS in the presence of cAMP analogs at 50–100µM. A general cAMP analog (8-Bromo cAMP), a PKA-activating analog (6-BNZ-cAMP), or an EPAC activating analog (8-CPT-2′-O-Me-cAMP) were used. Macrophages were either pretreated with vehicle (black bars) or 7µM Sorafenib (hatched bars). Concentrations of IL-12/23p40 were measured by ELISA. Stimulation with LPS alone and LPS+PGE are present as controls. (B.) Macrophages with stimulated with LPS+50µM 6-BNZ-cAMP in the presence of various doses of Sorafenib (2–7µM) or vehicle (0µM). Concentrations of IL-12/23p40 were measured by ELISA. LPS stimulation alone is present as a control. (C.) BMMΦ were stimulated with LPS or LPS+Cholera Toxin (1ng/ml) in the presence of increasing concentrations of Sorafenib (0–5µM). Concentrations of IL-12/23p40 and IL-10 are presented as in (A.). (D.–E.) 4T1 and NT2.5 mouse mammary tumor cells were cultured overnight at approximately 10⁶ cells/ml of macrophage culture media overnight as a source of tumor culture supernatants. Media was collected and centrifuged, then added to BMMΦ cultures at final concentrations of 20% (D.) and 40% (E.). (D.) BMMΦ were stimulated with LPS or LPS+20% 4T1 culture supernatants in the presence or absence of increasing concentrations of Sorafenib (0–5µM). Concentrations of IL-12/23p40 (black bars) or IL-10 (hatched bars) were measured by ELISA. (E.) BMMΦ were stimulated with LPS or LPS+40% NT2.5 culture supernatants in the presence or absence of 5µM Sorafenib. Each bar represents the mean ± SD of triplicate samples. *Indicates a p-value <0.01 in (B.) and (E.). **Indicates a p-value <0.001, comparing drug to vehicle control in A., B., C., and D., as well as comparing LPS alone to LPS+PGE, LPS+8-Bromo cAMP, and LPS+6-Bnz cAMP in (A.). Figures are representative of three independent experiments.

(A.) BMMΦ pretreated with Sorafenib were stimulated with LPS+PGE for 15’, 30’, or 45’. Lysates were assayed for MEK1/2 (Ser217/221), ERK1/2 (Thr202/Tyr204), and MSK-1 (Thr581) phosphorylation by western blotting. Total ERK is present as a loading control. (B) BMMΦ were pretreated and stimulated as in (A.). p-ERK1/2, p-p38 (Thr180/Tyr182), ERK1/2, and p38 were measured by western blotting. (C.) BMMΦ were stimulated with LPS or LPS+PGE for 15’, 30’, 45’, or 60’. Lysates were assayed for MSK1 phosphorylation or total MSK1 by western blotting. (D.) BMMΦ pretreated with vehicle or Sorafenib were stimulated with LPS alone. Phospho- or total ERK1/2, p38, and MSK1 were measured by western blotting. (E.) BMMΦ were pretreated with vehicle or Sorafenib, then stimulated with LPS+PGE for 30’ or 60’. Phospho-histone H3 Ser10 was determined by western blotting. Figures are representative of three independent experiments.

(A.) BMMΦ were pretreated with Sorafenib (5µM), the MEK1/2 inhibitor U0126 (5µM), the p38 inhibitor SB203580 (5µM), U0+SB (5µM each), AKTi inhibitor IV (1µM), or AKTi+SB (1µM and 5µM, respectively), then stimulated with LPS+ PGE₂. The concentration of IL-12/23p40 was determined by ELISA 16 hours after stimulation. Concentrations of inhibitors were titrated based on the highest IL-12/23p40 concentration after stimulation with LPS+ PGE₂. (B.) BMMΦ were pretreated with Sorafenib or MAPK inhibitors and stimulated with LPS+PGE as in (A.) for 45’. Lysates were blotted for phospho- and total MSK-1, p38, and ERK-1/2. (C.) BMMΦ were pretreated with Sorafenib or the AKT inhibitor and stimulated with LPS+PGE as in (A.) or left unstimulated for 45’. Lysates were blotted for p-GSK3α/β, p-AKT, and total AKT. Figures are representative of three independent experiments.