Salubrinal and guanabenz inhibit cytokine-induced Cyto-Src activity.

<p>(A, B) C28/I2 cells transfected with either Cyto-Src or Lyn-Src biosensor were pretreated with TNFα or IL1β for 2 hours before incubating with salubrinal or guanabenz. (A) Effect of Salubrinal and guanabenz on cytokine-induced Cyto-Src activity. (B) Cytokine-induced Lyn-Src activity is not altered by salubrinal or guanabenz. Scale bars, 10 µm. <i>n</i>>7 cells.</p

Salubrinal (Sal) and guanabenze (Gu) increase phosphorylation of eIF2α and decrease Cyto-Src activities.

<p>(A) Western blots showing the elevated level of p-eIF2α by salubrinal and guanabenz. (B) Staining intensity of p-eIF2α, normalized by intensity of eIF2α. * <i>p</i><0.05. (C) Cyto-Src activity by salubrinal and guanabenz. (D) Lyn-Src activity by salubrinal and guanabenz. Scale bars, 10 µm. <i>n</i>>7 cells.</p

Fluid flow magnitude-dependent Lyn-Src activity.

<p>(A) Selective Lyn-Src activities in response to different magnitudes of fluid flow. <i>n</i>>7 cells. (B) Cyto-Src activity is not altered by fluid flow. <i>n</i>>7 cells. (C, D) C28/I2 cells were cotransfected with either Lyn-Src or Cyto-Src biosensor and eIF2α siRNA or NC siRNA, and then subjected to fluid flow (10 dynes/cm²) during FRET imaging. (C) Lyn-Src activity under fluid flow. <i>n</i>>7 cells. (D) Cyto-Src activity under fluid flow. <i>n</i>>7 cells. (E) Lyn-Src activity in cytokine-treated cells under fluid flow(5 dynes/cm²). Cells transfected with a Lyn-Src biosensor were pretreated with cytokines for 2 hour before FRET imaging. <i>n</i>>7 cells. Scale bars, 10 µm.</p

Visual recognition of mirror, video-recorded, and still images in rats

<div><p>Several recent studies have claimed that rodents have good visual recognition abilities. However, the extent to which rats can recognize other rats and distinguish between males and females using visual information alone remains unclear. In the present study, we investigated the ability of rats to visually recognize mirror, video-recorded, and still images and to discriminate between images of males and females. Rats were tested in a place preference apparatus with a mirror, a video-recorded image of a rat, or a still image of a rat at one end. The data were assessed using t-test with Bonferroni correction. Male and female rats spent significantly more time in the mirror chamber and the video-recorded image chamber than in their respective blank chambers (P < 0.05), and male rats also spent more time in the chamber containing a still image. Furthermore, it was found that male rats exhibited significantly more sniffing behavior around the mirror than in the blank chamber (P < 0.05), whereas female rats were no significant differences in the sniffing behaviors in the mirror, moving or still image experiments. Identical results were obtained regardless of whether the rat in the image was the same or opposite sex. These results indicate that rats can process the differences in mirror, video-recorded, and still images as visual information, but are unable to use this information to distinguish between the sexes.</p></div

Involvement of eIF2α in salubrinal-driven Cyto-Src activity.

<p>(A, B) C28/I2 cells were cotransfected with Cyto-Src or Lyn-Src biosensor, and eIF2α or NC siRNA, and then treated with 10 µM salubrinal for 1 hour during imaging. (A) eIF2α siRNA blocks inhibitory effect of salubrinal on Cyto-Src activity. (B) Lyn-Src activity is not altered by eIF2α siRNA. Scale bars, 10 µm. <i>n</i>>7 cells. (C) The basal level of Src activity in C28/I2 cells expressing NC or eIF2α siRNA. <i>n</i>>7 cells. * <i>p</i><0.05 compared to the corresponding NC group.</p

A proposed model of distinctive Src activities at different subcellular locations.

<p>TNFα and IL1β activate Src kinases at the cytoplasm and lipid rafts of the plasma membrane, and actin cytoskeleton and lipid rafts are essential components of the Lyn-Src activation. Salubrinal can inhibit Src kinases in the cytoplasm through phosphorylation of eIF2α, but not in the lipid rafts of the plasma membrane. In contrast, fluid flow at 5 dynes/cm² decreases integrin-mediated Src kinases in the lipid rafts of the plasma membrane, but it did not significantly affect the level of Src activation in the cytoplasm.</p

HSV-1 cell-to-cell spread is limited in PTP1B^-/- MEFs.

<p><b>(A)</b> Virus replication assays were performed in PTP1B+ and PTP1B^-/- MEFs infected with strain 17 (MOI = 5). At 6-hour time points, duplicate samples were collected for measurements of the virus titers (cell lysate + medium), which were averaged and plotted. <b>(B)</b> To measure syncytia formation, PTP1B+ MEFs or PTP1B^-/- MEFs were infected (MOI = 3) with strain 17 and treated with DMSO, 50 μM salubrinal, or 50 μM salubrinal + 30 μM inhibitor XXII. At 24 hpi, cells were immunostained for ZO-1 and DAPI-stained nuclei in syncytia were manually counted. 1000 nuclei were scored per image, and 2 replicates were averaged. <b>(C)</b> To assay for cell-to-cell spread, PTP1B+ MEFs or PTP1B^-/- MEFs were infected (100 PFU/well) with strains KOS or 17, and the cells were incubated in medium containing 5 mg/ml pooled IgG. At 42 hpi, the cells were immunostained for VP5, and the areas of 10–15 plaques per sample were measured. Data are represented as mean ±SD from 3 independent experiments. A student T-test was used to determine statistical significance for the PTP1B^-/- samples compared to the PTP1B+ control. <b>(D and E)</b> PTP1B+ MEFs or PTP1B^-/- MEFs were infected with strain 17 (100 PFU/well) and incubated in medium containing 5 mg/ml of pooled human IgG along with either DMSO or 30 μM inhibitor XXII. At 42 hpi, cells were immunostained for VP5. (D) Representative plaques are shown. (E) To quantify the results, 10–15 plaques per sample were measured, and the mean plaque area was plotted from 2 independent experiments. A student T-test was used to determine statistical significance for samples compared to the PTP1B+ DMSO control. <b>(F)</b> To ascertain their ability to respond to salubrinal, uninfected PTP1B+ MEFs or PTP1B^-/- MEFs were incubated in medium containing DMSO, 50 μM salubrinal, or 1 μM thapsigargin for 2 hours. Cell lysates were harvested in the presence of phosphatase inhibitors and probed via western blotting for total eIF2α and phosphorylated eIF2α. Duplicate samples were analyzed in the same western blot and band intensities were quantified.</p

Parameters influencing salubrinal-induced fusion.

<p><b>(A)</b> Vero cells were infected with strains KOS, F, or 17 at MOIs of 0.5, 1, or 3 and were incubated in medium containing 50 μM salubrinal. At 12 hpi, the fusion ratio (% free nuclei) was determined by flow cytometry. The mean values (±SD) from 3 independent experiments are plotted. <b>(B)</b> After infection with strain 17 (MOI = 3), cells were incubated in medium containing DMSO, and 50 μM salubrinal was added for the indicated time intervals. After a total of 12 hpi, the fusion ratios were measured by flow cytometry. Data are from 2 independent experiments. A student T-test was used to determine statistical significance for samples compared to the DMSO control. <b>(C)</b> After infection with strain 17 (MOI = 3), cells were incubated for the indicated time periods in medium containing 50 μM salubrinal, which was then replaced with control medium. At 12 hpi, the fusion ratio was determined by flow cytometry, and the data were analyzed as in (3B).</p

Differential effects of salubrinal and inhibitor XXII on HSV-1 syncytial variants.

<p><b>(A and B)</b> Vero cells were infected with mutant gB.A855V at MOIs of (A) 1 or (B) 0.1. Since this mutant prefers to drive fusion with neighboring uninfected cells, the baseline level is higher at the lower MOI. Cells were treated with increasing amounts of (A) salubrinal or (B) inhibitor XXII, and at 12 hpi (A) or 24 hpi (B) the fusion ratios were determined by flow cytometry. The data are represented as the mean ±SD from 3 independent experiments, and a student T-test was used to determine statistical significance for samples compared to the DMSO control. <b>(C and D)</b> Cells were infected (MOI = 1) with mutants gK.L118Q or gK.A40V and treated with (C) salubrinal or (D) inhibitor XXII. At 12 hpi, syncytia were analyzed as in (5A). <b>(E and F)</b> Cells were infected (MOI = 1) with mutant UL20.F222A and treated with (E) salubrinal or (F) inhibitor XXII. At 12hpi, syncytia were analyzed as in (5A).</p

Results of control and Ex. 1.

<p>Preference of rats for (A) one of the two blank side chambers under normal conditions (Control) and (B) a chamber containing a mirror (Ex. 1) in terms of the mean time spent in the chamber by (a) males and (b) females, the mean number of times the wall was sniffed by (c) males and (d) females; and the mean time spent sniffing (e) males and (f) females. Error bars represent the SEM. *p < 0.05, **p < 0.01, t-test with Bonferroni correction.</p