Fig. 6.

NMIIA deletion enhances the activity of NF-κB, Sox2, and Nanog in cells grown on a stiff substrate. (A) Two PTEN-deleted GBM cell lines were infected with nontargeting (NT) or NMIIA-targeting shRNA-encoding lentiviruses. They were transiently transfected with a luciferase gene under control of an NF-κB response element and a mammalian expression vector for β-galactosidase (pCMV-βgal). NF-κB–dependent luciferase activity is plotted as mean ± SEM (n = 12). (B) NF-κB luciferase reporter assays were performed MDA-MB-231 cells expressing nontargeting or either of two NMIIA-targeting shRNA as above. Data are plotted as mean ± SEM (n = 9). (C) NF-κB luciferase reporter assays were performed on nontransformed keratinocytes that were treated with either NT shRNA or two different NMIIA-targeting shRNAs. (n = 8). (D) The MDA-MB-231 cells depleted for NMIIA were stably transfected with an shRNA-resistant GFP-NMIIA heavy-chain fusion protein and sorted for GFP expression. While NF-κB luciferase activity was significantly increased in cells depleted of NMIIA, expression of the GFP-NMIIA heavy chain returned NF-κB activity to baseline. Data are plotted as mean ± SEM (n = 17). (E) GBM cell lines treated with NT or NMIIA-targeting shRNA were transiently transfected with the Sox2 response element luciferase reporter and normalized luciferase activity was measured (n = 6). (F) MDA-MB-231 cells were transiently transfected with luciferase reporter construct under the control of Nanog transcriptional response element along with a control vector (pCMV-Renilla). Promoter activity was measured by luciferase activity. Values were normalized to Renilla activity to correct for transfection efficiency (n = 9). Data were analyzed with two-tailed t test. Significant at ****P < 0.0001, *** P = 0.0001 to 0.001, and **P = 0.001 to 0.01. Data are presented as the mean ± SEM.