FIGURE 4.

Increased interleukin-6 production triggers STAT3 signaling after Pard3 knockdown. A, medium was collected from cultures of NICD1-mMECs infected with lentivirus to express the shRNAs indicated and analyzed by ELISA for IL-6 levels (n = 3). B, mRNA was isolated from NICD1-mMECs infected with lentivurus expressing the shRNAs indicated and reverse transcribed into cDNA, and expression of Il6 mRNA was analyzed by qPCR. 18 S rRNA was used as a normalization control (n = 4). C, NICD1-mMECs were treated for 20 min with the indicated concentrations of recombinant IL-6 and then harvested, and equal amounts of lysate were immunoblotted for phospho-STAT3, total STAT3, and β-tubulin (loading control). D, mRNA was isolated from NICD1-mMEC/shPAR3 cells infected with lentivirus to express shRNAs against Il6 and reverse transcribed into cDNA, and expression of the genes indicated was analyzed by qPCR. GAPDH was used as a normalization control (n = 3). E, medium was collected from cultures of NICD1-mMEC/shPAR3 cells infected with the shIl6 constructs indicated and analyzed by ELISA for IL-6 levels (n = 3). Error bars, S.E. F, cells infected with lentivirus to express the shRNA constructs indicated were harvested, and equal amounts of lysate were immunoblotted for phospho-STAT3, total STAT3, and β-tubulin (loading control). G, quantification of immunoblot shown in E (n = 4). H, NICD1-mMECs expressing shRNAs indicated from lentiviral vectors were treated for 20 min with recombinant IL-6 as indicated, and lysates were immunoblotted for phospho-STAT3, total STAT3, and β-tubulin (loading control). I and J, NICD1-mMECs or NMuMGs expressing the shRNAs indicated from lentiviral vectors and treated with an IL-6-neutralizing antibody as indicated were harvested, and lysates were immunoblotted for phospho-STAT3, total STAT3, and GAPDH (loading control).