Translational control of IRF1 mRNA is independent from Ras/MEK activation.

<p>(A) Polysome profiles of RasV12 cells treated with or without U0126 (20 μM) for 2 hours were determined by recording the optical density (OD) of fractionated gradients at 254 nm. Peaks corresponding to 40S, 60S, 80S, and polysomes are indicated. (B) Ethidium bromide (EtBr) staining of RNA isolated from fraction #1 to #15 (Top panel). RT-PCR analysis of IRF1 (middle panel) and GAPDH (bottom panel) in each fractions. (C) RT-qPCR analysis of IRF1 and GAPDH in pooled fractions representing the sub-polysomes (fraction #1–5), the light polysomes (fraction #6–9), and the heavy polysomes (fraction #10–15). Data was represented as percentage of polysome-associated mRNA/total mRNA (n = 3, 3 independent experiments).</p

IRF1 Downregulation by Ras/MEK Is Independent of Translational Control of IRF1 mRNA - Fig 3

<p><b>Involvement of 5’ and 3’-UTR cis-acting elements in regulation of IRF1 translation by Ras/MEK</b> (A) Illustration of IRF1 3’- and 5’-UTR luciferase reporter constructs. Luciferase activities were measured in cell lysates obtained from RasV12 cells transfected with pGL3-control construct, (B) 5’-UTR or (C) 3’-UTR of IRF1 with or without U0126 treatment (20μM) for 6 hours. RLU were reported as compared with DMSO controls (n = 3, *P<0.05). Result shown is a representative of three independent experiments.</p

IRF1 protein is required for IRF1 promoter activity induced by MEK inhibition.

<p>(A) Control pGL3-Basic plasmid, pGL3-Basic plasmid containing promoter of IRF1 variant 1&3 or IRF1 variant 2 were transfected into RasV12 cells. At 24 hours after transfection, the cells were treated with U0126 (20μM) or IFN-α (500U/ml) for 24 hours. (B) pGL3-Basic plasmid containing IRF1 variant 1&3 promoters or GBP2 promoter was transfected into RasV12 cells, wild-type MEFs or IRF1-deficient MEFs. At 24 hours after transfection, the cells were treated with U0126 (20μM) for 24 hours. Relative luciferase activities (RLU) were reported as compared with DMSO controls (n = 3, *P<0.05, **P<0.01). Result shown is a representative of three independent experiments.</p

IRF1 downregulation by Ras/MEK activation.

<p>NIH3T3 cells were transfected with control pcDNA3 vector or pcDNA3 vector containing RasV12 gene. Western blot analysis was conducted to determine the expression of IRF1, p27^Kip and actin and the phosphorylation of ERK (pERK) at 24, 36 and 48 hours after transfection. Result shown is a representative of two independent experiments.</p

Coordinate downregulation of IFN-γ inducible HLA-II expression by E₂ is reversed by ICI-mediated degradation of ERα in MC2 cells.

<p>VC5 and MC2 cells were cultured in E₂-depleted media, treated with vehicle (ethanol), E₂ (10⁻⁹ M) or/and ICI (10⁻⁶ M) followed by stimulation with IFN-γ (100 U/ml) for 96 hours. HLA-II expression was analyzed by surface flow cytometry using (A) anti-DR, (L243), and intracellular flow cytometry using (B) anti-DM (Map.DM1) and (C) anti-Ii (LN2). Bar graphs represent the MFI ± SEM of three independent experiments. (*p<0.05, **p<0.01). (D) Western blot analysis was performed on whole cell extracts using for HLA-DRα (TAL 1B5), HLA-DM (TAL18.1) and Ii (LN2); GAPDH (Ab8245) is the protein loading control. Bar graphs show the ratio of band intensities, normalized to GAPDH band intensities and represent the mean ± SEM ratio of three independent experiments: (E) HLA-DRα/GAPDH (F) HLA-DM/GAPDH, and (G) Ii/GAPDH (* p<0.05, ** p<0.01).</p

E₂ differentially modulates inducible HLA-DR expression in ERα⁺ and ERα⁻ breast cancer cell lines.

<p>MDA-MB-231, SK-BR-3, MCF-7, BT-474, and T47D were cultured in E₂-depleted media, treated with vehicle (ethanol) or E₂ (10⁻⁹ M) and stimulated or not with IFN-γ (100 U/ml) for 96 hours. (A & E) HLA-DR cell surface expression (L243) was analyzed by flow cytometry: grey line, isotype control; black line, constitutive expression; shaded histogram, IFN-γ induced expression. (B & F) Bar graphs represent the MFI (mean florescence intensity) ± SEM for HLA-DR expression of three independent experiments. (C & G) Western blot analysis was performed on cytoplasmic and nuclear extracts for ERα expression (HC-20) and on cytoplasmic extracts for HLA-DRα (TAL 1B5). Protein loading controls included α-tubulin (B-7) and P84 (5E10) for cytoplasmic and nuclear proteins, respectively. (D & H) Bar graphs show the ratio of band intensity for HLA-DRα, normalized to the α-tubulin band intensity and represent the mean ± SEM of three independent experiments (*p<0.05, **p<0.01, ***p<0.001).</p

E₂-ERα signaling down regulates CIITA protein and mRNA expression in ER⁺ BCCL.

<p>VC5 and MC2 cells were cultured in E₂-depleted media, treated with vehicle (ethanol), E₂ (10⁻⁹ M) or/and ICI (10⁻⁶ M) and stimulated or not with IFN-γ (100 U/ml) for 24 and 4 hours, for CIITA protein and mRNA expression, respectively. (A) Western blot analysis was performed on cytoplasmic and nuclear extracts for CIITA (antiserum #21) and ERα (HC-20). (B) Cytoplasmic CIITA and nuclear CIITA were normalized to GAPDH and P84 respectively; bar graphs represent the mean ± SEM ratio of three independent experiments (**p<0.01). (C) CIITA mRNA was relatively quantified by real time PCR using Taqman gene expression assay. GAPDH was used as an endogenous control and the data were expressed relative to a control B cell line (RAJI). Bar graphs represent the mean ± SEM of three replicate assays (**p<0.01).</p

Representative profiles of IFN sensitive, moderately resistant and completely resistant cell lines.

<p>IFN sensitive (A), moderately resistant (B) and completely resistant cell lines (C) were identified by pretreating cells with IFN (0, 10, 50, 100, 500, 1000 and 5000 U/ml) for 16 hours and then challenged with VSV at a MOI of 1 for 24 hours. Cell viability was determined using crystal violet staining and expressed as average percentage compared to the uninfected control wells (n = 3 wells). One representative experiment is shown.</p

Effect of U0126 treatment on the anti-viral IFN response in moderately resistant and completely resistant cell lines.

<p>(A) Cell lines were infected with VSV (MOI = 1) for 24 hours after treatment with IFN (0–5000 U/ml) with or without U0126 (0–20 µM) for 16 hours. Western blot analysis was used to detect viral protein (VSV-G) levels, the level of phosphorylated ERK (p-ERK) with GAPDH used as a loading control. The samples were analyzed on two membranes simultaneously using identical conditions for incubation and detection. One representative experiment out of 3 is shown. (B) Viral progeny production was determined after infection with VSV (MOI = 1) for 24 hours following treatment with IFN (50 or 2000 U/ml) and with U0126 (0, 5, 10 or 20 µM) for 16 hours.</p

Validation of changes in gene expression by quantitative RT-PCR.

<p>The level of gene expression in HT1080 cells left untreated, treated with IFN (50 U/ml), with U0126 (20µM) or with IFN and U0126 for 6 hours or 12 hours was determined by quantitative RT-PCR. The relative expression level was calculated compared to the 6 hour untreated control after normalization against GAPDH expression levels. (n = 3, * P<0.01 compared to time-matched control, ** P<0.05 compared to all other groups).</p