Figure 4.

A: Cell density causes a dramatic increase in Cx43 levels in QU-DB cells. QU-DB (lanes 5-8) or A549 (lanes 1-4) or nontransformed E10 (lane 9) cells were grown to different densities as indicated and extracts probed for Cx43 or Hsp90 as a loading control. Note the absence of Cx43 in A549 cells and the increase in Cx43 with density in QUDB. B: Stat3 knockdown reduces Cx43 levels. QU-DB cells infected with the lentiviral vector carrying the Stat3-specific shRNA (lane 2) or not infected (lane 1) were grown to 2 days post-confluence and lysates probed for Cx43 or Hsp90 as a loading control. C: CPA7 or Stat3-knockdown with shRNA reduce Stat3-ptyr705 levels in A549 cells. A549 cells were grown to increasing densities and treated with the Stat3 inhibitor, CPA7 (lanes 6-8) or the DMSO carrier (lanes 1-5) for 15 hrs and cell extracts probed for Stat3-ptyr705 or tubulin as a loading control. Parallel cultures were infected with a vector expressing a Stat3-specific, shRNA [37], and cell extracts from stable lines produced were probed as above. D: CPA7 or Stat3-knockdown with shRNA reduce Stat3 transcriptional activity in A549 cells. A549 cells were transfected with a plasmid expressing a firefly luciferase gene under control of a Stat3-responsive promotor (▪) and a Stat3-independent promotor driving a Renilla luciferase gene (□) (see Methods). After transfection, cells were plated to different densities and treated with CPA7 or the DMSO carrier alone for 24 hrs, at which time firefly and Renilla luciferase activities were determined. Parallel cultures expressing the sh-Stat3 construct were transfected with the plasmids and firefly and Renilla luciferase activities determined.