Knockdown of optineurin by shRNA enhances TNFα-induced NF-κB activity.

<p>(A) HeLa cells were infected with Ad-shOptn1, Ad-shOptn2 or control viruses and after 72 hours cell lysates were subjected to western blotting using antibodies for optineurin, IκBα and p65 NF-κB. Control virus (AdC sh) expresses shRNA of unrelated sequence of same length. (B) HeLa cells were infected with Ad-shOptn1, Ad-shOptn2 or control viruses. After 48 h of infection these cells were transfected with NF-κB reporter plasmid (25 ng) along with β-galactosidase expression plasmid. After 22 hours of transfection the cells were treated with TNFα (10 ng/ml) for 4 hours. Cell lysates were then made for reporter assays. The data represent luciferase activities relative to untreated control taken as 1.0 (n = 3). (C) HeLa cells were infected with indicated adenoviruses and after 72 hours treated with TNFα for 6 min or left untreated. Cell lysates were subjected to western blot analysis using antibodies for IκB, optineurin and tubulin (loading control). AdC, control virus not expressing any shRNA.</p

Induction of optineurin gene expression and promoter activation by TNFα.

<p>(A), A549 cells were treated with TNFα (10 ng/ml) for indicated time (3–24 hours). Total RNA was then isolated and the level of optineurin mRNA was determined by real time RT-PCR. GAPDH was used as a control. (B) Effect of TNFα on optineurin protein level. A549 cells were treated with TNFα (10 ng/ml) for indicated time. The cell lysates were then prepared for immunoblotting which was performed using antibodies against optineurin, IRF-1 and tubulin (loading control). The numbers at the top indicate relative amount of protein. Activation of optineurin promoter by TNFα in A549 cells (C) or HeLa cells (D). Cells grown in 24 well plates were transfected with 100 ng of optineurin promoter-reporter plasmid (full length construct pGL-FP or deletion construct pGL-DP) along with pCMV.SPORT β-gal plasmid. After 6 hours of transfection TNFα was added (10 ng/ml). After another 18 hours cell lysates were prepared for reporter assays. Luciferase activities relative to untreated control (taken as 1.0) are shown (n = 3) after normalizing with β-galactosidase enzyme activities.</p

NF-κB mediates optineurin gene expression.

<p>(A) NF-κB p65 activates optineurin promoter through NF-κB site. Optineurin minimal promoter constructs pGL-DP or pGL-mDP (100 ng) were transfected without or with NF-κB p65 expression plasmid (100 ng) in A549 cells. After 24 hours of transfection cell lysates were prepared for reporter assays. Luciferase activities relative to control pGL-DP (taken as 1.0) are shown (n = 3). (B) IκBα inhibits TNFα-induced NF-κB activity. pGL-DP was transfected without or with IκBα super repressor expression plasmid (100 ng). TNFα was added 6 hours after transfection. Luciferase activities relative to control are shown (n = 3). (C) Blocking of NF-κB activation inhibits TNFα-induced optineurin gene expression. A549 cells were preincubated with 25 µM MG132 or solvent DMSO (0.1%) for 30 minutes prior to treatment with TNFα. After 6 hours of treatment with TNFα, RNA was isolated and the level of optineurin mRNA was determined by real time RT-PCR analysis. GAPDH was used as a control. (D) A549 cells were treated with MG132 as in panel C and after 6 hours of treatment with TNFα cell lysates were subjected to Western blotting. (E) A549 cells were treated with 100 µg/ml of SN-50 peptide for 30 minutes prior to treatment with TNFα for 6 hours. The picture shows RT-PCR analysis for optineurin gene expression.</p

Optineurin and E50K mutant inhibit TNFα-induced NF-κB activity.

<p>(A) NF-κB-Luc reporter plasmid (25 ng) was transfected without or with optineurin expression plasmid (100 ng) in HeLa cells. After 22 hours of transfection cells were treated with TNFα for 4 hours. Luciferase activities relative to untreated control are shown (n = 3). (B) Western blot showing expression of optineurin and its mutants using HA tag antibody. Cdk2 was used as control. (C) Optineurin and E50K mutant inhibit TNFα-induced nuclear translocation of NF-κB p65. HeLa cells grown on coverslips were transfected with optineurin expression plasmid. After 24 hours of transfection cells were treated with TNFα for 30 min. The cells were then fixed and stained for optineurin (HA tag, FITC green) and p65 (Cy3, red) and visualized using a fluorescence microscope. (D) HeLa cells were infected with adenoviruses for expressing optineurin (Optn-AdV), its E50K mutant (E50K-AdV) or control virus (AdC). After 36 hours of infection, the cells were treated with TNFα for 6 min or 12 min or left untreated. Cell lysates were then prepared for western blotting with antibodies for IκBα, HA tag and Cdk2.</p