Fig 1: IRF3 regulates GLI3 by directly binding to IRF3 binding sites.(A) Schematic diagram of ~ 3000 bp upstream of ATG located in exon 2 of GLI3. Several candidate IRF3 binding sites (BS) were identified. (B) Untreated MM6 cells (10 × 106) were lysed and a chromatin immunoprecipitation (ChIP) assay was performed followed by qPCR using three primer sets for the areas containing IRF3 binding sites upstream of ATG of GLI3. (C) MM6 cells (10 × 106) were treated with 10 μg/ml Poly(I:C) for 1h prior to ChIP and qPCR. (D) Primary human monocytes (D3) were treated with 10μg/ml Poly(I:C) for 1h prior to ChIP and qPCR. (E) Primary human monocytes (D3) were challenged with 10 μg/ml Poly(I:C) for 12h prior to western blot analysis. (F) U937 cells (4 × 106) were transfected with either IRF3 expression construct or empty vector along with pGL4.1_GLI3 promoter construct and pGL4.7 Renilla plasmid as described in the methods section. After 48 hr, cells were harvested and lysed and lysates were used to quantify luciferase activity. These experiments were performed at least 3 times with similar results.
Fig 2: GLI3 is regulated by IRF3 downstream of TRIF.(A) THP-1 cells (5 × 106 cells) were treated with 50μM MEK/Erk inhibitor (PD98059) or DMSO control for 30 min, 500 nM NF-κB inhibitor (QNZ) or DMSO for 2 h, or 1 μM TBK1 inhibitor (BX795) or DMSO for 1 h. Cells were harvested, lysed and lysates were used to determine protein expression by western blot. (B) THP-1 cells (5 × 106 cells) were pretreated with 1 μM BX795 for 1 h, followed by stimulation with 100 ng/ml LPS for 12 h. Cells were lysed and lysates were used to determine protein expression by western blot. (C) U937 cells (5 × 106 cells) were transfected with an IRF3 expression construct or empty vector (EV) for 2 days followed by lysis and western blot. (D) Monocytes (5 × 106 cells) were treated with 1 μg/ml TLR4 agonist MPLA for 12 h followed by western blot to determine GLI3 expression. (E) Monocyte cell lines and primary donor CD14+ cells (D7) (5 × 106 cells) were treated with 10 μg/ml CpG, 10 μg/ml Poly(I:C), 100 ng/ml LPS or left untreated (Ctrl) for 12 h followed by western blot to determine GLI3 expression. Data shown are representative of at least three independent experimental replicates.
Fig 3: LPS-induced GLI3 is regulated by TRIF downstream of TLR4.(A) U937 cells (10 × 106 cells) were transfected with 10 μg of either shMYD88, shTRIF or scrambled controls (shScr). After 2 days, cells were lysed and lysates were used to determine protein expression by western blot. (B) Monocytes (10 × 106 cells) were transfected with a dominant negative form of TRIF (dnTRIF) or empty vector (Ctrl) for 2 days followed by determination of protein expression by western blot. (C) MM6 cells (10 × 106 cells) were transfected with shTRIF or shScr and cultured for 30 h, followed by treatment with 100 ng/ml LPS. After an additional 12 h, cells were lysed and lysates were used for western blot to determine protein expression. These experiments were repeated at least 3 times with similar results.
Fig 4: Functional TLR4 is required for GLI3 expression.(A) Monocytes (5 × 106 cells) were treated with 1 μg/ml TLR4 signaling inhibitor (CLI095) for 12 h followed by determination of GLI3 expression by western blot. (B) Monocytes (5 × 106 cells) were pretreated with CLI095 for 12 h followed by stimulation with 100 ng/ml LPS for an additional 12 h. GLI3 expression was determined by western blot. These experiments were repeated 3 times.
Fig 5: Gli3 mediates TLR-induced inflammation.(A) WT and Irf3−/− MEFs (1.5x105) were treated with transfected with 10 μg/mL Poly(I:C) for the indicated time followed by qPCR to determine GLI3 expression. (B) MM6 cells (4 × 106) were transfected with shScr or shIRF3. After 24 hr, cells were stimulated with 10 μg/mL poly(I:C) for an additional 24 hr. Cells were harvested and used for RNA isolation and qPCR to determine GLI3 expression. (C) IFA-elicited macrophages from M-Gli3−/− or WT mice (n = 14/group) were cultured in the presence or absence of 100 ng/ml LPS for 24 hours. Supernatants were used to quantify cytokine levels by ELISA. (D) WT and Gli3−/− MEFs (1 × 106 cells/ml) were plated in 0.5 ml volume in 12-well plates. Cells were treated with 1 μg/mL MPLA for 24 hr followed by ELISA to quantify cytokine secretion. MEF experiments were repeated twice with similar results.
Supplier Page from Abcam for Anti-Gli3 antibody