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

Expression (A) and cellular localization (B) of PTB and unr under 30°C and 37°C culture.

<p>NIH3T3 cells were cultured at 30°C and 37°C for 0, 2, 4, and 6 hours. Western blot analysis was performed using antibodies against PTB, unr, β-actin and Sam68.</p

Temperature sensitivity of HRV IRES-mediated translation in different cell types.

<p>BHK, L929, Huh7 HCT116 and Vero cells were transfected with the HRV IRES construct or a control pRF reporter construct, 24 hours later transfected cells were incubated at 37°C or 30°C for 8 hours. Firefly and <i>Renilla</i> luciferase units were measured using the Dual-Luciferase Reporter Assay System. Firefly/Renilla represents the ratio of viral IRES-mediated translation to cap-dependent translation. Data are expressed as the mean ± SE of 3 independent samples. Statistical analysis was conducted using two-way ANOVA and multiple comparison test. *p<0.01.</p

Temperature-dependent translation initiation by viral IRES elements.

<p><b>(A)</b> Schematic representation of the bicistronic reporter constructs. NIH3T3 cells were transfected with bicistronic reporter constructs containing an IRES element from EMCV, FMDV, HCV, HRV or PV, or a control pRF construct. 24 hours post-transfection, cells were cultured at 30°C <b>(B)</b>, 35°C <b>(C)</b>, or 37°C for 8 hours. Firefly and <i>Renilla</i> luciferase units were measured using the Dual-Luciferase Reporter Assay System. Firefly/Renilla represents the ratio of viral IRES-mediated translation to cap-dependent translation. Data are expressed as the mean ± SE of 3 independent samples. Statistical analysis was conducted using two-way ANOVA and multiple comparison test. *p<0.01.</p

Absence of modifications of reporter construct mRNAs in response to the temperature shift.

<p><b>(A</b>) Schematic representation of the bicistronic reporter construct. Positions of the different regions amplified by qRT-PCR (primer region I and II) or by RT-PCR (amplicons III and IV) are indicated. <b>(B)</b> NIH3T3 cells were transfected with HRV or control pRF reporter construct for 24 hours and, then, incubated at 37°C or 30°C for 8 hours. qRT-PCR analysis of <i>Renilla</i> (primer region I) and firefly luciferase (primer region II) mRNA of PRF and HRV IRES reporter construct (left) and of HRV firefly luciferase, HRV <i>Renilla</i> luciferase, GBP2 and RIG-I mRNA (right). The expression levels were normalized to GAPDH and reported as comparisons to the expression level at 37°C. The <i>Renilla</i> luciferase/firefly luciferase ratio was calculated as 2-[CT (Renilla)—CT (Firefly)]. Data are expressed as the mean ± SE of 3 independent samples. Statistical analyses were performed using a t-test. *p<0.01. <b>(C)</b> RT-PCR analysis for amplicon III, amplicon IV and GAPDH.</p

Temperature sensitivity of cellular IRES-mediated translation.

<p>NIH3T3 cells were transfected with a HRV, Rbm3, Apaf-1, c-myc, BIP, CAT-1 construct or a control pRF reporter construct and 24 hours later were incubated at 37°C (white bar) or 30°C (black bar) for 8 hours. Firefly and <i>Renilla</i> luciferase units were measured using the Dual-Luciferase Reporter Assay System. Firefly/Renilla represents the ratio of viral IRES-mediated translation to cap-dependent translation. Data are expressed as the mean ± SE of 3 independent samples. Statistical analysis was conducted using two-way ANOVA and multiple comparison test. *p<0.01.</p

Time course of the effect of mild hypothermia on promotion of HRV IRES-mediated translation.

<p>NIH3T3 cells transfected with the pRF or HRV IRES reporter constructs were exposed to mild hypothermia (30°C) for 2, 4, 8 and 12 hours. Absolute values of firefly luciferase <b>(A)</b>, absolute values of <i>Renilla</i> luciferase <b>(B)</b> and ratio of Firefly/Renilla luciferase were measured using the Dual-Luciferase Reporter Assay System. Data are expressed as the mean ± SE of 3 independent samples. Statistical analysis was conducted using two-way ANOVA and multiple comparison test. *p<0.01.</p