Fig 5.

SHH signaling forms a feedback loop to modulate the TLR2 signaling cohort. (A) SHH was ectopically expressed in macrophages by transfection with plasmid pcDNA3-SHH. Control cells were transfected with the pcDNA3 vector alone. Immunoblotting was performed in cells overexpressing SHH or vector alone with antibodies specific to phospho-p85 (pp85), phospho-4EBP1 (p4EBP1), phospho-PKCδ (pPKCδ), phospho-PKCα (pPKCα), phospho-ERK1/2 (pERK1/2), and phospho-p38 (pp38). Blots are representative of 3 independent experiments. (B) Macrophages were treated with betulinic acid for 1 h prior to purmorphamine treatment. Cell lysates obtained from M. bovis BCG-infected macrophages or uninfected macrophages were analyzed using anti-pp85, anti-p4EBP1, anti-pPKCδ, anti-pPKCα, anti-pERK1/2, and anti-pp38. Blots are representative of 3 separate experiments. (C) Betulinic acid-treated macrophages were stimulated with TNF-α, followed by infection with M. bovis BCG. Immunoblotting was performed on equal amounts of proteins, as described for panel B. (D) Expression of VEGF-A protein in the supernatants of macrophages treated with purmorphamine, TNF-α, or betulinic acid and infected with M. bovis BCG assayed by sandwich ELISA. Data are the means of three replicates. *, P < 0.05 versus M. bovis BCG-infected macrophages. (E) The levels of SOCS-3, MMP-9, and COX-2 expression were monitored in macrophages pretreated with betulinic acid and stimulated with TNF-α, followed by infection with M. bovis BCG. DMSO was used as the vehicle control. Blots were probed with anti-β-actin antibody to ensure equal loading of proteins. Results for a representative panel of at least 3 independent experiments are shown. (F) RAW 264.7 macrophages were transfected with control siRNA or Shh siRNA. After 72 h of transfection, cells were stimulated with TNF-α prior to M. bovis BCG infection. The levels of expression of TLR2 signaling-responsive genes were assayed as discussed for panel E. Blots are representative of 3 independent experiments.