human primary bj foreskin fibroblasts  (ATCC)

Journal: bioRxiv

Article Title: Inhibition of MAPK signaling suppresses cytomegalovirus reactivation in CD34 + Kasumi-3 cells

doi: 10.1101/2025.02.13.638080

Figure Lengend Snippet: Kasumi-3 cells were infected (moi = 1.0 TCID 50 /cell) with WT or US28Δ under latent conditions. At 7 dpi, half of each culture was treated with DMSO (-) or 10 μM U0126 (+) for 2 d, after which the cells were washed and co-cultured with naïve fibroblasts to measure the fold change in frequency of infectious centers relative to WT (-U0126, open blue bar) by ELDA. Each data point (circles) denotes the mean of three technical replicates. Error bars indicate the standard deviation (SD) of the three biological replicates ( Figure S3 ). Statistical significance was calculated by two-way ANOVA with Tukey’s test for multiple comparisons. *** p <0.001, ns - not significant

Article Snippet: Primary newborn human foreskin fibroblasts (NuFF-1, passage 13-28; GlobalStem, Rockville, MD, USA) were maintained in Dulbecco’s modified Eagle medium (DMEM), supplemented with 10% FBS, 2 mM L-glutamine, 0.1 mM non-essential amino acids, 10 mM HEPES, and 100 U/ml each of penicillin and streptomycin.

Techniques: Infection, Cell Culture, Standard Deviation

Journal: bioRxiv

Article Title: Inhibition of MAPK signaling suppresses cytomegalovirus reactivation in CD34 + Kasumi-3 cells

doi: 10.1101/2025.02.13.638080

Figure Lengend Snippet: ( A ) NuFF-1 fibroblasts were treated with U0126 for 24 h, then washed with PBS and returned to culture. Percent cell viability is shown relative to cells treated with vehicle (DMSO), assessed at 3 and 6 d post-treatment. Puromycin is shown in Figure S1B as a positive control. N=3, representative assay shown. ( B ) NuFF-1 cells were pretreated for 24 h with DMSO (v/v/) or U0126 at the indicated concentrations. Cells were washed with PBS and infected with TB40/E mCherry (moi = 1 TCID50/cell) for 1 h. Viral inoculum was removed, and cells were washed with 1X PBS. Media containing DMSO (v/v) or U0126 at the indicated concentrations was then replenished. Following 24 h in culture, DMSO- or drug-containing media was removed, cells were washed, and fresh media (without U0126 or DMSO) was replenished. Cells were returned to culture, and media was collected at 96 hpi. Viral titers were quantified by TCID50 assay on naïve NuFF-1 fibroblasts. N=3 biological replicates, each with N=3 technical replicates; representative assay shown.

Article Snippet: Primary newborn human foreskin fibroblasts (NuFF-1, passage 13-28; GlobalStem, Rockville, MD, USA) were maintained in Dulbecco’s modified Eagle medium (DMEM), supplemented with 10% FBS, 2 mM L-glutamine, 0.1 mM non-essential amino acids, 10 mM HEPES, and 100 U/ml each of penicillin and streptomycin.

Techniques: Positive Control, Infection, TCID50 Assay

Journal: bioRxiv

Article Title: Inhibition of MAPK signaling suppresses cytomegalovirus reactivation in CD34 + Kasumi-3 cells

doi: 10.1101/2025.02.13.638080

Figure Lengend Snippet: Kasumi-3 cells were infected (moi = 1.0 TCID 50 /cell) with WT or US28Δ for 7 d and then treated with the indicated compounds for an additional 2 d in the presence of DMSO (-TPA) to maintain latent conditions or TPA (+TPA) to differentiate cells, allowing for reactivation. Cultures were washed to remove the compounds, and the frequency of infectious centers was quantified by co-culturing each cell population with naïve fibroblasts using ELDA software , http://bioinf.wehi.edu.au/software/elda/ . Data is plotted as fold-change in frequency of infectious centers relative to untreated (DMSO, -TPA), WT-infected cells (open gray bar). Each data point (circles) denotes the mean of three technical replicates. Error bars indicate the SD of the three biological replicates (each shown in Figure S3 ). Concentrations for the compounds are as follows: 125 nM SCH772984, 0.5 µM selumetinib, 0.1 µM trametinib). Statistical significance was calculated by two-way ANOVA with Tukey’s test for multiple comparisons. ** p < 0.05, *** p < 0.001.

Article Snippet: Primary newborn human foreskin fibroblasts (NuFF-1, passage 13-28; GlobalStem, Rockville, MD, USA) were maintained in Dulbecco’s modified Eagle medium (DMEM), supplemented with 10% FBS, 2 mM L-glutamine, 0.1 mM non-essential amino acids, 10 mM HEPES, and 100 U/ml each of penicillin and streptomycin.

Techniques: Infection, Software

Journal: bioRxiv

Article Title: Inhibition of MAPK signaling suppresses cytomegalovirus reactivation in CD34 + Kasumi-3 cells

doi: 10.1101/2025.02.13.638080

Figure Lengend Snippet: ( A, C, E ) NuFF-1 fibroblasts were treated DMSO (v/v) or the indicated compound at increasing concentrations for 24 h, washed, and fresh media replenished. Cell viability was determined at 3 or 6 d post-treatment and percent viability is shown relative to cells exposed to vehicle. Puromycin is shown in Figure S1B as a positive control. N=3, representative assay shown for each compound. ( B, D, F ) NuFF-1 cells were pretreated for 24 h with DMSO (v/v/) or the pharmacological compound at increasing concentrations, as indicated. Cells were washed with 1X PBS and infected with TB40/E mCherry (moi = 1.0 TCID 50 /cell) for 1 h. Viral inoculum was removed, cells were washed with 1X PBS, and media containing DMSO (v/v) or the compound at the indicated concentrations was then replenished. Following 24 h in culture, DMSO- or drug-containing media was removed, cells were washed, and fresh media (without compound or DMSO) was replenished. Cells were returned to culture, and media was collected at 96 hpi. Viral titers were quantified by TCID 50 assay on naïve NuFF-1 fibroblasts. Note the DMSO control was used as the control for each inhibitor treatment, as the v/v of DMSO required was the same. N=3 biological replicates, each with N=3 technical replicates; representative assay shown. * p <0.05; ** p <0.01

Article Snippet: Primary newborn human foreskin fibroblasts (NuFF-1, passage 13-28; GlobalStem, Rockville, MD, USA) were maintained in Dulbecco’s modified Eagle medium (DMEM), supplemented with 10% FBS, 2 mM L-glutamine, 0.1 mM non-essential amino acids, 10 mM HEPES, and 100 U/ml each of penicillin and streptomycin.

Techniques: Positive Control, Infection, Control