Cytokine signaling in fibroblasts.

<p>(A) HGFs grown on fibronectin with 2% serum for 3 hours and then stimulated under serum free conditions with vehicle, IL-1β (40 ng/mL) or TNF-α (10 ng/mL) for 4 hours. Undiluted conditioned media were used to screen for human MMP/TIMP in antibody arrays. Array key is displayed on the left of the blots. (B) Immunohistochemical assessment of MMP-3 expression detected with Cy-2 labeled-streptavidin (green) and the nuclei with DAPI stain (blue) in gingival lamina propria (red box; corresponds to blue box in the inset of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070659#pone-0070659-g004" target="_blank">Figure 4A</a>) and in periodontal ligament (yellow box; corresponds to green box in the inset of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070659#pone-0070659-g004" target="_blank">Figure 4A</a>). Tissue designations: Tissue designations: AB: alveolar bone, D: dentin, GE: gingival epithelium, Lig: Ligature, LP: gingival lamina propria, PDL: periodontal ligament. (C) Whole cell lysates from HGFs that were cultured on fibronectin under low (2%) serum and treated in serum free medium with vehicle, IL-1β (40 ng/mL) or TNF-α (10 ng/mL) as in (A) for 15 minutes, were immunoblotted for phospho-ERK and total ERK. (D) Mouse embryonic fibroblasts that are WT (PTPα^WT) or null (PTPα^Null) for PTPα were cultured on FN or PLL-coated tissue culture plastic for 3 hours in presence of 2% serum and stimulated with or without IL-1β for 15 minutes. Equal amounts of whole cell lysates, as determined by BCA protein assay, were separated and blotted for phospho-ERK and total ERK. Alternatively, conditioned media collected from cells plated overnight on FN or PLL coated dishes in the presence or absence of IL-1β (40 ng/mL), were concentrated 20x and equal amounts of proteins were loaded onto polyacrylamide gels to detect MMP-3 by Western blot. (E) PTPα^Null cells transfected with HA tagged full length construct (PTPα^Rescue) or the construct lacking D2 domain (PTPα^ΔD2) or the construct lacing both D1 and D2 domains (PTPα^ΔD1/D2) as described in the Methods were plated on FN as above and lysates were immunoblotted for phospho-ERK, ERK, MMP-3 and HA tag (right panel inset).