mouse embryonic fibroblast cell line nih3t3  (ATCC)

Journal: Life

Article Title: Ubiquitination Regulates Reorganization of the Membrane System During Cytomegalovirus Infection

doi: 10.3390/life15081212

Figure Lengend Snippet: PYR-41-inhibited ubiquitination prevents the establishment of the MCMV infection. ( A ) NIH3T3 fibroblasts were infected with C3X GFP MCMV and treated with DMSO (Ø) or indicated concentrations of PYR-41 at 0, 2 and 4 hpi. GFP expression was determined by flow cytometry after 6 hpi . The upper panel shows GFP intensity normalized to mock-treated cells, and the lower panel shows the percentage of GFP-positive cells. ( B ) pIE1 expression after 6 hpi and ( C ) percentage of pIE1-positive cells (mean ± SD) after infection with MCMV and treatment with DMSO (mock) or different concentrations of PYR-41 at 0, 2 and 4 hpi. Bars—25 μm. ( D ) MCMV-infected cells were treated with 15 μM PYR-41 before (0 hpi →) or 4 h after infection (4 hpi →) and the expression of pIE1 was determined by Western blot analysis. Shown are representative Western blots (complete blots are shown in ) and a quantitative analysis of the signals normalized to β-tubulin. The fold changes represent the signal expression relative to the band with the highest intensity in the mock-treated (Ø) samples, the read bars represent the mean ± SD, and the empty circles are data from an independent experiment. Statistical significance was determined using a two-tailed paired Student t -test (*** p < 0.001; ** p < 0.01; * p < 0.05). The numbers of independent experiments are indicated in parenthesis.

Article Snippet: Murine fibroblast-like cell line NIH3T3 (ATCC CRL-1658) was used for most experiments, and murine embryonic fibroblasts (MEFs), derived from 17-day-old BALB/c mouse embryos, were used for virus production and plaque assays.

Techniques: Ubiquitin Proteomics, Infection, Expressing, Flow Cytometry, Western Blot, Two Tailed Test

Journal: Life

Article Title: Ubiquitination Regulates Reorganization of the Membrane System During Cytomegalovirus Infection

doi: 10.3390/life15081212

Figure Lengend Snippet: pIE1 is ubiquitinated in MCMV-infected cells. Doxycycline-treated (2 μg/mL for 48 h) NIH3T3 HA-Ub cells were infected with MCMV and ( A , B ) were left untreated or ( C , D ) treated with either DMSO (mock) or 15 μM PYR-41 at 4 hpi. At indicated times after infection, the whole cell lysates ( A , C ) and anti-HA immunoprecipitate ( B , D ) were analyzed for pIE1 and β-actin expression by Western blot. Signals were quantified by ImageJ and normalized to β-actin in WCLs. Fold changes represent the signal expression relative to the band with the highest intensity in mock-treated cells. The mean ± SD (red bars) and individual data (empty circles) are shown in the graphs. The number of independent experiments is indicated in parenthesis. The original blots are shown in .

Article Snippet: Murine fibroblast-like cell line NIH3T3 (ATCC CRL-1658) was used for most experiments, and murine embryonic fibroblasts (MEFs), derived from 17-day-old BALB/c mouse embryos, were used for virus production and plaque assays.

Techniques: Infection, Expressing, Western Blot

Journal: Life

Article Title: Ubiquitination Regulates Reorganization of the Membrane System During Cytomegalovirus Infection

doi: 10.3390/life15081212

Figure Lengend Snippet: The effect of PYR-41 on the progression of the MCMV replication cycle. ( A – E ) MCMV-infected NIH3T3 cells were treated with either DMSO (mock) or with 15 μM PYR-41 prior to infection (0 hpi) or 4 hpi. The expression of pE1 ( A , B ), pM57 ( C ) and pM74 ( D , E ) was analyzed by Western blot at the indicated time points after infection. β-tubulin or β-actin were used as loading controls. The original blots are shown in . Signals were normalized to the loading control, and fold changes represent signal expression relative to the band with the highest intensity in the mock-treated samples. The empty circles show the data from independent experiments and the red bars show mean values ± SD. Statistical significance was determined using a two-tailed paired Student t -test (*** p < 0.001; ** p < 0.01; * p < 0.05). The number of independent experiments is indicated in parenthesis. ( F ) Supernatants or cell lysates of PYR-41-treated cells (0 hpi → or 4 hpi →) were harvested at 0, 24, 48, 72 and 96 hpi and the number of infectious units was determined by plaque assay. The mean values ± SD of four independent experiments are shown. Statistical significance was determined using the Mann–Whitney test (* p < 0.05).

Article Snippet: Murine fibroblast-like cell line NIH3T3 (ATCC CRL-1658) was used for most experiments, and murine embryonic fibroblasts (MEFs), derived from 17-day-old BALB/c mouse embryos, were used for virus production and plaque assays.

Techniques: Infection, Expressing, Western Blot, Control, Two Tailed Test, Plaque Assay, MANN-WHITNEY

Journal: Life

Article Title: Ubiquitination Regulates Reorganization of the Membrane System During Cytomegalovirus Infection

doi: 10.3390/life15081212

Figure Lengend Snippet: PYR-41 inhibits the formation of pre-AC. ( A ) NIH3T3 cells were infected with ΔFcR MCMV and analyzed for the expression of GM130 (green), Rab10 (red) and pIE1 (blue) by immunofluorescence after 12 hpi. In the uninfected control, the cell nuclei were stained with DAPI (blue). Arrows indicate extended Golgi, arrowheads indicate condensed Golgi, and asterisks indicate perinuclear Rab10 in pre-AC. The percentage of cells expressing condensed Rab10 and GM130 in pre-AC is shown on the right. Means ± SD (red bars) and individual values (empty circles) are shown. The complete experiment is shown in . ( B ) ΔFcR MCMV-infected cells were treated with 15 μM PYR-41 at 0 and 4 hpi or mock treated. After 12 hpi, the expression of GM130 (green), Rab10 (red) and pIE1 (blue) was analyzed. Arrowheads indicate condensed Golgi and arrows indicate dispersed Golgi. Asterisks indicate Rab10 in pre-AC. ( C – E ) The ratio of pIE1-positive cells ( C ), cells with perinuclear Rab10 ( D ) and condensed/dispersed Golgi patterns ( E ) in PYR-41-treated and mock-treated cells. Shown are the mean ± SD (red bars) and individual values (empty circles). The number of independent experiments is indicated in parenthesis. Statistical significance was determined using a two-tailed paired Student t -test (*** p < 0.001; ** p < 0.01). Bars—10 μm.

Article Snippet: Murine fibroblast-like cell line NIH3T3 (ATCC CRL-1658) was used for most experiments, and murine embryonic fibroblasts (MEFs), derived from 17-day-old BALB/c mouse embryos, were used for virus production and plaque assays.

Techniques: Infection, Expressing, Immunofluorescence, Control, Staining, Two Tailed Test

Journal: Life

Article Title: Ubiquitination Regulates Reorganization of the Membrane System During Cytomegalovirus Infection

doi: 10.3390/life15081212

Figure Lengend Snippet: Treatment with PYR-41 in the late phase of infection inhibits the production of infectious virions. NIH3T3 cells were infected with wt MCMV. After 48 hpi, the cell culture medium was replaced with fresh medium, and cells were treated with 15 μM PYR-41 or mock-treated. The supernatants or cell lysates were harvested after 72 and 96 hpi and infectious virion production was determined using the plaque assay. The mean values of four independent experiments are plotted; the error bars show the standard deviation. Statistical significance was determined using the Mann–Whitney test (* p < 0.05). Ctrl.—control level of virus production in mock-treated cells without changing the cell culture medium 48 hpi.

Article Snippet: Murine fibroblast-like cell line NIH3T3 (ATCC CRL-1658) was used for most experiments, and murine embryonic fibroblasts (MEFs), derived from 17-day-old BALB/c mouse embryos, were used for virus production and plaque assays.

Techniques: Infection, Cell Culture, Plaque Assay, Standard Deviation, MANN-WHITNEY, Control, Virus

Journal: Life

Article Title: Ubiquitination Regulates Reorganization of the Membrane System During Cytomegalovirus Infection

doi: 10.3390/life15081212

Figure Lengend Snippet: PYR-41 disrupts Rab10-associated tubulation in the established AC. NIH3T3 EGFP-Rab10 cells were infected with wt MCMV. At 16 hpi, PYR-41 was injected into the tissue culture medium (15 μM concentration), and cells were imaged live with fluorescence-enhanced DHTM for 1 h. Screenshots at the indicated time points are shown and a full time-lapse in (Mock) and (PYR-41). The arrows indicate expanded Rab10-EGFP-positive tubules in AC.

Article Snippet: Murine fibroblast-like cell line NIH3T3 (ATCC CRL-1658) was used for most experiments, and murine embryonic fibroblasts (MEFs), derived from 17-day-old BALB/c mouse embryos, were used for virus production and plaque assays.

Techniques: Infection, Injection, Concentration Assay, Fluorescence

Journal: Life

Article Title: Ubiquitination Regulates Reorganization of the Membrane System During Cytomegalovirus Infection

doi: 10.3390/life15081212

Figure Lengend Snippet: WASHC1 in MCMV-infected cells. ( A ) NIH 3T3 cells were infected with wt MCMV or left uninfected and analysed 16 h later by immunofluorescence for the expression of pIE1 (green) and WASH1C (red). DAPI (blue) was used to stain the cell nuclei. The percentage of cells with perinuclear WASHC1 (mean ± SD (red bar)) is shown on the right. ( B ) Doxycycline-treated NIH3T3 HA-Ub cells were infected with wt MCMV or left uninfected. After 16 h, 10% of cells were lysed for WCL and 90% of cells were lysed for the immunoprecipitation of ubiquitinated proteins with rabbit anti-HA. The expression of WASHC1, pIE1 and β-actin was visualized with the corresponding primary and secondary antibodies. The fold change represents the signal expression of Ub-WASHC1 relative to the uninfected cells. HC—Heavy chain of anti-Rb IgG pAb. ( C , D ) NIH3T3 cells were transfected with Scr. or WASHC1 siRNA or left untransfected (control). After 48 h, cells were infected with wt MCMV for 16 h or left uninfected and analyzed either by Western blot ( C ) or immunofluorescence ( D ). Signals were normalized to β-actin and fold change represents signal expression relative to uninfected and mock-treated samples. ( D ) Triple staining of Rab10 (red), pIE1 (green) and DAPI (blue) was analyzed by confocal imaging. ( E ) Percentage of MCMV-infected (pIE1-positive) cells with perinuclear accumulation of Rab10 in Scr. and WASH1 siRNA-treated cells at 16 h post-infection, shown as mean ± SD. Ctrl., control the level in non-transfected cells. The number of independent experiments is indicated in parenthesis. ( F ) Cells were treated with Scr., Rab11 or WASHC1 siRNA for 48 h and infected with wt MCMV. Supernatants and cell lysates were harvested at 48 hpi, and the number of infectious units was determined by plaque assay. The number of experiments is indicated in the bars. The error bars show the standard deviation. Statistical significance was determined using the Mann–Whitney test (* p < 0.05, ** p < 0.01, *** p < 0.001). Bars—10 μm.

Article Snippet: Murine fibroblast-like cell line NIH3T3 (ATCC CRL-1658) was used for most experiments, and murine embryonic fibroblasts (MEFs), derived from 17-day-old BALB/c mouse embryos, were used for virus production and plaque assays.

Techniques: Infection, Immunofluorescence, Expressing, Staining, Immunoprecipitation, Transfection, Control, Western Blot, Imaging, Plaque Assay, Standard Deviation, MANN-WHITNEY