Further characterization of Act1 induced gene expression.

<p>(A) HBE1 cells were transfected with Act1-FLAG and harvested 24h post-transfection for RNA extraction and real-time PCR analysis of various IL-17 target genes. (B) A549 or NHBE cells were transfected with empty vector (EV) or Act1-FLAG and harvested 24h post-transfection for RNA extraction and real-time PCR analysis of <i>DEFB4</i> expression. (C) HBE1 cells were transfected with empty vector, Act1-FLAG or untagged Act1 and harvested 24h post-transfection for RNA extraction and real-time PCR analysis of <i>DEFB4</i> expression. C(t) values were normalized to <i>GAPDH</i> and calibrated to empty vector controls. Error bars represent SEM of 3 independent experiments. *p<0.05 compared to empty vector (EV) transfected cells.</p

Further characterization of Act1 induced gene expression.

<p>(A) HBE1 cells were transfected with Act1-FLAG and harvested 24h post-transfection for RNA extraction and real-time PCR analysis of various IL-17 target genes. (B) A549 or NHBE cells were transfected with empty vector (EV) or Act1-FLAG and harvested 24h post-transfection for RNA extraction and real-time PCR analysis of <i>DEFB4</i> expression. (C) HBE1 cells were transfected with empty vector, Act1-FLAG or untagged Act1 and harvested 24h post-transfection for RNA extraction and real-time PCR analysis of <i>DEFB4</i> expression. C(t) values were normalized to <i>GAPDH</i> and calibrated to empty vector controls. Error bars represent SEM of 3 independent experiments. *p<0.05 compared to empty vector (EV) transfected cells.</p

PCR Primers.

<p>PCR Primers.</p

Nuclear Act1 binds to the DEFB4 promoter regions.

<p>HBE1 cells were transfected with Act1-FLAG and fixed 24h post-transfection. Isolated nuclei were sonicated and chromatin immunoprecipitation (chIP) was performed using an anti-FLAG antibody or non-specific mouse IgG. PCR was used to detect <i>DEFB4</i> promoters in the immunoprecipitated material. The data was presented as % of the input as described in Materials and Methods.</p

Act1 drives target gene promoter activity.

<p>(A) HBE1 cells were transiently co-transfected with <i>DEFB4</i> promoter-luciferase constructs, pRL-TK and either empty vector or Act1-FLAG (n = 6) and lysed 24 hours post-transfection. *p<0.05 compared to empty vector (EV) transfected cells. (B) HBE1 cells were co-transfected with a <i>CCL20</i> promoter-luciferase construct (CCL20-LUC), pRL-TK and either empty vector or Act1-FLAG (n = 4) and lysed 24 hours post-transfection. *p<0.05 compared to empty vector (EV) transfected cells. (C) HBE1 cells were co-transfected with a <i>DEFB4</i> promoter-luciferase construct in which all 3 NF-κB binding sites are mutated (DEFB4-mut-LUC), pRL-TK and either empty vector or Act1-FLAG. 24 hours post-transfection, cells were left unstimulated (-IL-17A) or stimulated with 100 ng/mL IL-17A for 6 hours (n = 4). Lysate was measured for firefly and <i>Renilla</i> luciferase activity. Firefly luciferase activity was normalized to <i>Renilla</i> luciferase activity and calibrated to empty vector transfected controls. *p<0.05 compared to empty vector (EV) transfected cells. NS: not significant when IL-17A treated cells (+IL-17) were compared to non-treated cells (-IL-17). (D) Lysate was separated by SDS-PAGE and immunoblotted to confirm Act1-FLAG expression. β-actin was used as a loading control.</p

Nuclear localization of Act1.

<p>(A) HBE1 were left unstimulated or stimulated with 100 ng/mL IL-17A for 1h prior to nuclear protein extraction. 10 μg nuclear extract was separated by SDS-PAGE and analyzed for Act1 protein expression by Western blot analysis. Nucleolin was used as a protein loading control. (B) HBE1 cells plated on glass coverslips were left unstimulated or stimulated with 100 ng/mL IL-17A for 1h. Endogenous Act1 was immunostained with a rabbit anti-Act1 antibody, followed by an Alexa-488 conjugated secondary antibody (green) and counterstained with DAPI to visualize nuclei (blue). (C) HBE1 cells were transfected with a myc-tagged Act1 expression construct and/or a FLAG-tagged Act1 expression construct. 24 hours post-transfection, cells were fixed and co-stained with rabbit anti myc antibody and mouse anti FLAG antibody, followed by an Alexa-488 conjugated anti-rabbit antibody (green) or Alexa-562 conjugated anti-mouse antibody (red) and counterstained with DAPI to visualize nuclei (blue). Co-localization is indicated in yellow. All confocal images were taken using a 63X objective lens. Bar = 20 μm.</p

Regions of Act1 necessary for gene expression.

<p>(A) Schematic representation of various FLAG-tagged Act1 derivatives. Numbers represent amino acid residue location. (B) HBE1 cells were transiently transfected with empty vector or various Act1 derivatives. 24h post-transfection, cells were lysed for RNA extraction and real-time PCR analysis of <i>DEFB4</i> gene expression. C(t) values were normalized to <i>GAPDH</i> and calibrated to the empty vector control. Error bars represent SEM of 3 independent experiments. * p< 0.05 compared to Act1-FL (C) 30 μg whole cell lysate was immunoblotted with anti-FLAG antibody to confirm expression of FLAG-tagged Act1 derivative. β-actin was used as a loading control.</p

Act1 drives gene expression independent of IL-17 stimulation.

<p>(A) HBE1 cells were transfected with Act1 siRNA or a non-targeting (NT) siRNA control. 48h post transfection, cells were treated with media only (-IL-17A) or 100 ng/mL IL-17A (+IL-17A) for an additional 17h, then harvested for RNA extraction and real-time PCR analysis of <i>DEFB4</i> expression. C(t) values were normalized to a housekeeping gene, <i>GAPDH</i>, and calibrated to NT siRNA/-IL-17A. Error bars represent SEM of 3 independent experiments. *p<0.05 compared to NT siRNA/-IL-17A samples, **p<0.05 compared to NT siRNA/+IL-17A samples. (B) HBE1 cells were transfected with increasing amounts of Act1-FLAG plasmid. Total transfected DNA was normalized using p3XFLAG-CMV-14. 24h post-transfection, cells were treated with media only or 100 ng/mL IL-17A for an additional 17h, then harvested for RNA extraction and real-time PCR analysis of <i>DEFB4</i> expression. *p<0.05 compared to 0 ng transfected (mock transfection) (C) To confirm increasing Act1-FLAG protein expression, 30 μg whole cell extracts from transfected cells were analyzed by Western blot for Act1-FLAG. β-actin was used as a loading control. (D) DEFB4 protein was quantitatively measured in cell culture supernatant by ELISA (n = 3). *p<0.05 compared to empty vector (EV) transfected cells.</p