Journal: bioRxiv

Article Title: Upregulation of ATP Citrate Lyase Phosphorylation and Neutral Lipid Synthesis through Viral Growth Factor Signaling during Vaccinia Virus Infection

doi: 10.1101/2023.09.21.558916

Figure Lengend Snippet: (A) VACV infection induces lipid droplet formation in HFFs in a VGF-dependent manner. HFFs were infected with the indicated viruses at a multiplicity of infection (MOI) of 5 for 8 h. Lipid droplets were stained with HCS Lipidtox Red, and the nuclei were stained with DAPI and imaged under a confocal microscope. The intensity of the red signal that corresponds to the lipid droplets is quantified in the bar graph. (B) VACV infection increases the lipid droplet-associated protein levels in HFFs in a VGF-dependent manner. HFFs were infected with the indicated viruses at an MOI of 5 for the indicated 8 h. Western blotting analysis was performed to measure the levels of perilipin 2. (C) VACV infection-induced lipid droplet formation is cell-type specific. HEPG2 and A549 cells were infected with VACV at an MOI of 5 for 8 h. Lipid droplets were stained with HCS Lipidtox Red, and the nuclei were stained with DAPI and imaged under a confocal microscope. (D) VACV infection does not increase ACLY phosphorylation and lipid droplet-associated protein levels in HEPG2 and A549 cells. HEPG2 and A549 cells were infected with wild-type VACV at a multiplicity of infection (MOI) of 5 for 8 h. Western blotting analysis was performed to measure the levels of ACLY and perilipin 2. (E) EGFR and Akt pathways are important for the formation of lipid droplets during VACV infection. HFFs were infected with wildtype VACV at a MOI of 5 for 8 h in the presence or the absence of 3 µM afatinib (EGFR inhibitor) or 5µM MK-2206 (Akt inhibitor). Lipid droplets were stained with HCS Lipidtox Red imaged under a confocal microscope. The intensity of the red signal that corresponds to the lipid droplets is quantified in the bar graph.

Article Snippet: Primary HFFs, HeLa cells (ATCC CCL-2), and A549 (ATCC CCL-185) were grown in Dulbecco’s modified Eagle medium (DMEM; Fisher Scientific), supplemented with 10% fetal bovine serum (FBS; Peak Serum), 2 mM glutamine (VWR), 100 U/ml of penicillin, and 100 μg/ml streptomycin (VWR) in a humidified incubator at 37 °C with 5% CO 2 .

Techniques: Infection, Staining, Microscopy, Western Blot

Journal: PLOS Pathogens

Article Title: Function and regulation of a steroidogenic CYP450 enzyme in the mitochondrion of Toxoplasma gondii

doi: 10.1371/journal.ppat.1011566

Figure Lengend Snippet: (A) Plaque assays. Analysis of parasite growth for 7 days after fibroblast infection with 150 parasites from the parental and ΔTgMAPR strains showing representative images and quantification of lysed area from 3 independent experiments. Data are means ± SD. (B) Gliding assays. Freshly lysed ΔTgMAPR1 and parental parasites were allowed to glide on FBS-coated glass slides for 30 min before fixation and staining with anti-SAG1 antibody under non-permeabilized conditions. Representative images are shown. Measurement of gliding trails by dot plots, showing means ± SD of 3 independent experiments. (C) Invasion assays. Quantification of invasion of parental and ΔTgMAPR parasites using the red/green invasion assay. Red histograms represent external, attached parasites while green histograms represent internal, penetrated parasites. Data are means ± SEM of 4 independent experiments. (D) [ 3 H]uracil incorporation assays for replication. HFF were infected with parental and ΔTgMAPR parasites for 24 h prior to incubation with tritiated uracil. Data are means ± SD of 3 independent experiments. (E) Induced egress assays. HFF were infected with parental and ΔTgMAPR parasites for 24 h prior to exposure to 2 μM A23187 for time-lapse microscopy views, showing rapid egress for parental parasites that were largely extracellular upon treatment and slower response for the treated mutant (arrows). Quantification of egress time with means in seconds for 3 treated monolayers infected with parental or ΔTgMAPR parasites.

Article Snippet: Human foreskin fibroblasts (HFF), VERO cells and HeLa cells obtained from the American Type Culture Collection (Manassas, VA), and immortalized HFF (hTERT) received from S.N.J.

Techniques: Infection, Staining, Invasion Assay, Incubation, Time-lapse Microscopy, Mutagenesis

Journal: Journal of Virology

Article Title: Human cytomegalovirus mediates APOBEC3B relocalization early during infection through a ribonucleotide reductase-independent mechanism

doi: 10.1128/jvi.00781-23

Figure Lengend Snippet: A3B relocalization occurs early during HCMV infection. ( A and C ) Representative IF microscopy images of HFF-1 cells stably expressing A3B-HA incubated with medium alone (mock) or infected with the indicated HCMV strains for the indicated timepoints (10 µm scale). ( B and D ) Quantification of A3B-HA subcellular localization phenotypes shown in panels A and C. Each histogram bar reports the percentage of cells with whole cell, cytoplasmic, and nuclear A3B-HA ( n > 100 cells per condition; mean ± SD with P values from unpaired Student’s t -tests).

Article Snippet: HFF-1 (ATCC, Manassas, VA, USA), U373 (ATCC), and 293T cells were cultured in Dulbecco's Modified Eagle Medium (DMEM; Cytiva, Marlborough, MA, USA) supplemented with 10% fetal bovine serum (Gibco, Billings, MT, USA) and 1% penicillin/streptomycin (Gibco).

Techniques: Infection, Microscopy, Stable Transfection, Expressing, Incubation

Journal: Journal of Virology

Article Title: Human cytomegalovirus mediates APOBEC3B relocalization early during infection through a ribonucleotide reductase-independent mechanism

doi: 10.1128/jvi.00781-23

Figure Lengend Snippet: A3B relocalization requires de novo translation of HCMV proteins but not viral DNA synthesis. ( A ) Schematic representation of experimental workflows of CHX and PAA treatment of infected cells. Image created with BioRender. ( B ) Representative IF microscopy images of HFF-1 cells stably expressing A3B-HA incubated with medium alone (mock) or infected with AD169-GFP and treated with DMSO or CHX for 24 h (10 µm scale). ( C ) Representative IF microscopy images of U373 cells stably expressing A3B-HA incubated with medium alone (mock) or infected with AD169-GFP or AD169-GFP ΔIE1 for 72 h (10 µm scale). ( D ) Representative IF microscopy images of HFF-1 cells stably expressing A3B-HA incubated with medium alone (mock) or infected with AD169-GFP and treated with DMSO or PAA for 48 h (10 µm scale). ( E ) Quantification of A3B-HA subcellular localization phenotypes shown in panels B and D. Each histogram bar reports the percentage of cells with cytoplasmic A3B-HA ( n > 80 cells per condition; mean ± SD with P values from unpaired Student’s t -tests).

Article Snippet: HFF-1 (ATCC, Manassas, VA, USA), U373 (ATCC), and 293T cells were cultured in Dulbecco's Modified Eagle Medium (DMEM; Cytiva, Marlborough, MA, USA) supplemented with 10% fetal bovine serum (Gibco, Billings, MT, USA) and 1% penicillin/streptomycin (Gibco).

Techniques: DNA Synthesis, Infection, Microscopy, Stable Transfection, Expressing, Incubation