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

Gene ontology (GO) analysis of genes differentially expressed by IFN (I), U0126 (U) or combined treatment (I+U).

<p>Genes with significantly upregulated or downregulated expression levels compared to control were analyzed for over-representation of GO category compared to that observed in the genome. Significantly over-represented GO categories (FDR <0.05) are shown with the highest FDR = 0 shown in red and categories with FDR ≥0.05 or absent shown in blue.</p

Sensitivity of human cancer cell lines to IFN as measured by antiviral assay.

a<p>IFN concentration that elicits a 50% protection against VSV infection based on analysis of cytopathic effects (CPE).</p

Activation of IFN-induced transcription in control SKOV3 and Ras-transformed SKOV3 cells.

<p>The expression of GBP2, IFIT2, MAP2, RIGI and STAT2 in control SKOV3 and Ras-transformed SKOV3 cells (clone 15) at 12 hours after IFN stimulation (0, 12.5, 50 and 100 U/ml) was determined by quantitative RT-PCR. The relative expression level was calculated compared to the untreated control SKOV3 cells after normalization against GAPDH expression levels. (n = 3, * P<0.05 and ** P<0.01 compared to IFN concentration-matched control).</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