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primary hffs (ATCC)

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ATCC primary hffs

VACV stimulates neutral lipid droplet formation in primary <t>HFFs.</t> ( A ) Role of neutral lipids in cellular metabolism. The enzyme ACLY converts cytoplasmic citrate, generated by the TCA cycle from glucose or glutamine, into acetyl-CoA and oxaloacetate. Acetyl-CoA can be further utilized for lipid synthesis, and neutral lipid synthesis via a series of reactions catalyzed by the enzymes ACC, FASN, ACAT, and DGAT. The fig was created with BioRender.com. ( B ) VACV infection stimulates the formation of neutral lipid droplets. HFFs 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 ( n ≥ 3). ( C ) The intensities of the red signals corresponding to the lipid droplets in were quantified in the bar graph using ImageJ. ( D ) VACV infection increases the lipid droplet-associated protein PLIN2 levels in HFFs. HFFs were infected with WT VACV at an MOI of 5 for 8 h. Western blotting analysis was performed to measure the levels of PLIN2. ( E ) VACV infection-induced lipid droplet formation is cell-type specific. HepG2 <t>and</t> <t>A549</t> 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 ( n ≥ 3). ( F ) The intensities of the signals corresponding to the lipid droplets in were quantified in the bar graph. ( G ) VACV infection does not alter the levels of PLIN2 in HepG2 and A549 cells. The cells were infected with WT VACV at an MOI of 5 for 8 h. Western blotting analysis was performed to measure the levels of PLIN2. For P values, ns, P > 0.05; ** P ≤ 0.01; *** P ≤ 0.001. **** P ≤ 0.0001. In D and G, representative images of multiple biological replicates were shown. The numbers below each band indicate the average intensities of respective proteins as calculated by ImageJ. The relative average intensities ± standard deviation of PLIN2/GAPDH, normalized to mock, are shown in the tables below the images ( n ≥ 3 for D, n ≥ 2 for G). n indicates the number of biological replicates.

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1) Product Images from "Stimulation of neutral lipid synthesis via viral growth factor signaling and ATP citrate lyase during vaccinia virus infection"

Article Title: Stimulation of neutral lipid synthesis via viral growth factor signaling and ATP citrate lyase during vaccinia virus infection

Journal: Journal of Virology

doi: 10.1128/jvi.01103-24

VACV stimulates neutral lipid droplet formation in primary HFFs. ( A ) Role of neutral lipids in cellular metabolism. The enzyme ACLY converts cytoplasmic citrate, generated by the TCA cycle from glucose or glutamine, into acetyl-CoA and oxaloacetate. Acetyl-CoA can be further utilized for lipid synthesis, and neutral lipid synthesis via a series of reactions catalyzed by the enzymes ACC, FASN, ACAT, and DGAT. The fig was created with BioRender.com. ( B ) VACV infection stimulates the formation of neutral lipid droplets. HFFs 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 ( n ≥ 3). ( C ) The intensities of the red signals corresponding to the lipid droplets in were quantified in the bar graph using ImageJ. ( D ) VACV infection increases the lipid droplet-associated protein PLIN2 levels in HFFs. HFFs were infected with WT VACV at an MOI of 5 for 8 h. Western blotting analysis was performed to measure the levels of PLIN2. ( E ) 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 ( n ≥ 3). ( F ) The intensities of the signals corresponding to the lipid droplets in were quantified in the bar graph. ( G ) VACV infection does not alter the levels of PLIN2 in HepG2 and A549 cells. The cells were infected with WT VACV at an MOI of 5 for 8 h. Western blotting analysis was performed to measure the levels of PLIN2. For P values, ns, P > 0.05; ** P ≤ 0.01; *** P ≤ 0.001. **** P ≤ 0.0001. In D and G, representative images of multiple biological replicates were shown. The numbers below each band indicate the average intensities of respective proteins as calculated by ImageJ. The relative average intensities ± standard deviation of PLIN2/GAPDH, normalized to mock, are shown in the tables below the images ( n ≥ 3 for D, n ≥ 2 for G). n indicates the number of biological replicates.

Figure Legend Snippet: VACV stimulates neutral lipid droplet formation in primary HFFs. ( A ) Role of neutral lipids in cellular metabolism. The enzyme ACLY converts cytoplasmic citrate, generated by the TCA cycle from glucose or glutamine, into acetyl-CoA and oxaloacetate. Acetyl-CoA can be further utilized for lipid synthesis, and neutral lipid synthesis via a series of reactions catalyzed by the enzymes ACC, FASN, ACAT, and DGAT. The fig was created with BioRender.com. ( B ) VACV infection stimulates the formation of neutral lipid droplets. HFFs 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 ( n ≥ 3). ( C ) The intensities of the red signals corresponding to the lipid droplets in were quantified in the bar graph using ImageJ. ( D ) VACV infection increases the lipid droplet-associated protein PLIN2 levels in HFFs. HFFs were infected with WT VACV at an MOI of 5 for 8 h. Western blotting analysis was performed to measure the levels of PLIN2. ( E ) 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 ( n ≥ 3). ( F ) The intensities of the signals corresponding to the lipid droplets in were quantified in the bar graph. ( G ) VACV infection does not alter the levels of PLIN2 in HepG2 and A549 cells. The cells were infected with WT VACV at an MOI of 5 for 8 h. Western blotting analysis was performed to measure the levels of PLIN2. For P values, ns, P > 0.05; ** P ≤ 0.01; *** P ≤ 0.001. **** P ≤ 0.0001. In D and G, representative images of multiple biological replicates were shown. The numbers below each band indicate the average intensities of respective proteins as calculated by ImageJ. The relative average intensities ± standard deviation of PLIN2/GAPDH, normalized to mock, are shown in the tables below the images ( n ≥ 3 for D, n ≥ 2 for G). n indicates the number of biological replicates.

Techniques Used: Generated, Infection, Staining, Microscopy, Western Blot, Standard Deviation

Neutral lipid synthesis is important for efficient VACV replication. ( A, B ) Chemical inhibition of lipid droplet synthesis suppresses VACV replication. HFFs were infected with VACV in the presence or absence of 30 µM avasimibe, 125 µM T-863, or 125 µM PF-06424439. Virus titers were measured by a plaque assay at ( A ) 24 hpi (MOI = 2) and ( B ) 48 hpi (MOI = 0.01). The numbers above the bars indicate the fold reduction in VACV titers compared to vehicle ( n ≥ 3). ( C ) The inhibition of lipid droplet synthesis does not alter HFF viability. HFFs were grown in the presence or absence of 30 µM avasimibe, 125 µM T-863, or 125 µM PF-06424439 for 48 h. Cell viability was determined by trypan blue exclusion assay using a hemocytometer ( n ≥ 3). ( D-F ) Lipid droplet synthesis is important for the expression of VACV late genes. HFFs were infected with VACV (MOI = 2) that expressed Gaussia luciferase under ( D ) early (vEGLuc), ( E ) intermediate (vIGLuc), and ( F ) late (vLGLuc) promoters. A Gaussia luciferase assay was performed to measure the early, intermediate, and late gene expression at 6, 8, and 16 hpi, respectively. AraC, which inhibits VACV DNA replication and intermediate and late gene expression, was used at a concentration of 40 µg/mL as a control ( n ≥ 3). ( G, H ) Chemical inhibition of lipid droplet synthesis differentially suppresses VACV replication in transformed cells. HepG2 ( G ) and A549 ( H ) cells were infected with MOI of 0.01 of vLGLuc in the presence or absence of 30 µM avasimibe, 125 µM T-863, or 125 µM PF-06424439. A Gaussia luciferase assay was performed to measure the late gene expression at 24 hpi. AraC was used at a concentration of 40 µg/mL as a control ( n ≥ 3). ( I, J ) the inhibition of lipid droplet synthesis does not alter the viability of the transformed cells. HepG2 ( I ) and A549 ( J ) cells were grown in the presence or absence of 30 µM avasimibe, 125 µM T-863, or 125 µM PF-06424439 for 48 h. Cell viability was determined by trypan blue exclusion assay using an automated cell counter ( n ≥ 3). n indicates the number of biological replicates. For P -values, ns, P > 0.05; * P ≤ 0.05; **** P ≤ 0.0001.

Figure Legend Snippet: Neutral lipid synthesis is important for efficient VACV replication. ( A, B ) Chemical inhibition of lipid droplet synthesis suppresses VACV replication. HFFs were infected with VACV in the presence or absence of 30 µM avasimibe, 125 µM T-863, or 125 µM PF-06424439. Virus titers were measured by a plaque assay at ( A ) 24 hpi (MOI = 2) and ( B ) 48 hpi (MOI = 0.01). The numbers above the bars indicate the fold reduction in VACV titers compared to vehicle ( n ≥ 3). ( C ) The inhibition of lipid droplet synthesis does not alter HFF viability. HFFs were grown in the presence or absence of 30 µM avasimibe, 125 µM T-863, or 125 µM PF-06424439 for 48 h. Cell viability was determined by trypan blue exclusion assay using a hemocytometer ( n ≥ 3). ( D-F ) Lipid droplet synthesis is important for the expression of VACV late genes. HFFs were infected with VACV (MOI = 2) that expressed Gaussia luciferase under ( D ) early (vEGLuc), ( E ) intermediate (vIGLuc), and ( F ) late (vLGLuc) promoters. A Gaussia luciferase assay was performed to measure the early, intermediate, and late gene expression at 6, 8, and 16 hpi, respectively. AraC, which inhibits VACV DNA replication and intermediate and late gene expression, was used at a concentration of 40 µg/mL as a control ( n ≥ 3). ( G, H ) Chemical inhibition of lipid droplet synthesis differentially suppresses VACV replication in transformed cells. HepG2 ( G ) and A549 ( H ) cells were infected with MOI of 0.01 of vLGLuc in the presence or absence of 30 µM avasimibe, 125 µM T-863, or 125 µM PF-06424439. A Gaussia luciferase assay was performed to measure the late gene expression at 24 hpi. AraC was used at a concentration of 40 µg/mL as a control ( n ≥ 3). ( I, J ) the inhibition of lipid droplet synthesis does not alter the viability of the transformed cells. HepG2 ( I ) and A549 ( J ) cells were grown in the presence or absence of 30 µM avasimibe, 125 µM T-863, or 125 µM PF-06424439 for 48 h. Cell viability was determined by trypan blue exclusion assay using an automated cell counter ( n ≥ 3). n indicates the number of biological replicates. For P -values, ns, P > 0.05; * P ≤ 0.05; **** P ≤ 0.0001.

Techniques Used: Inhibition, Infection, Virus, Plaque Assay, Trypan Blue Exclusion Assay, Expressing, Luciferase, Concentration Assay, Control, Transformation Assay

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Journal: Journal of Virology

Article Title: Stimulation of neutral lipid synthesis via viral growth factor signaling and ATP citrate lyase during vaccinia virus infection

doi: 10.1128/jvi.01103-24

Figure Lengend Snippet: VACV stimulates neutral lipid droplet formation in primary HFFs. ( A ) Role of neutral lipids in cellular metabolism. The enzyme ACLY converts cytoplasmic citrate, generated by the TCA cycle from glucose or glutamine, into acetyl-CoA and oxaloacetate. Acetyl-CoA can be further utilized for lipid synthesis, and neutral lipid synthesis via a series of reactions catalyzed by the enzymes ACC, FASN, ACAT, and DGAT. The fig was created with BioRender.com. ( B ) VACV infection stimulates the formation of neutral lipid droplets. HFFs 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 ( n ≥ 3). ( C ) The intensities of the red signals corresponding to the lipid droplets in were quantified in the bar graph using ImageJ. ( D ) VACV infection increases the lipid droplet-associated protein PLIN2 levels in HFFs. HFFs were infected with WT VACV at an MOI of 5 for 8 h. Western blotting analysis was performed to measure the levels of PLIN2. ( E ) 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 ( n ≥ 3). ( F ) The intensities of the signals corresponding to the lipid droplets in were quantified in the bar graph. ( G ) VACV infection does not alter the levels of PLIN2 in HepG2 and A549 cells. The cells were infected with WT VACV at an MOI of 5 for 8 h. Western blotting analysis was performed to measure the levels of PLIN2. For P values, ns, P > 0.05; ** P ≤ 0.01; *** P ≤ 0.001. **** P ≤ 0.0001. In D and G, representative images of multiple biological replicates were shown. The numbers below each band indicate the average intensities of respective proteins as calculated by ImageJ. The relative average intensities ± standard deviation of PLIN2/GAPDH, normalized to mock, are shown in the tables below the images ( n ≥ 3 for D, n ≥ 2 for G). n indicates the number of biological replicates.

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: Generated, Infection, Staining, Microscopy, Western Blot, Standard Deviation

Journal: Journal of Virology

Article Title: Stimulation of neutral lipid synthesis via viral growth factor signaling and ATP citrate lyase during vaccinia virus infection

doi: 10.1128/jvi.01103-24

Figure Lengend Snippet: Neutral lipid synthesis is important for efficient VACV replication. ( A, B ) Chemical inhibition of lipid droplet synthesis suppresses VACV replication. HFFs were infected with VACV in the presence or absence of 30 µM avasimibe, 125 µM T-863, or 125 µM PF-06424439. Virus titers were measured by a plaque assay at ( A ) 24 hpi (MOI = 2) and ( B ) 48 hpi (MOI = 0.01). The numbers above the bars indicate the fold reduction in VACV titers compared to vehicle ( n ≥ 3). ( C ) The inhibition of lipid droplet synthesis does not alter HFF viability. HFFs were grown in the presence or absence of 30 µM avasimibe, 125 µM T-863, or 125 µM PF-06424439 for 48 h. Cell viability was determined by trypan blue exclusion assay using a hemocytometer ( n ≥ 3). ( D-F ) Lipid droplet synthesis is important for the expression of VACV late genes. HFFs were infected with VACV (MOI = 2) that expressed Gaussia luciferase under ( D ) early (vEGLuc), ( E ) intermediate (vIGLuc), and ( F ) late (vLGLuc) promoters. A Gaussia luciferase assay was performed to measure the early, intermediate, and late gene expression at 6, 8, and 16 hpi, respectively. AraC, which inhibits VACV DNA replication and intermediate and late gene expression, was used at a concentration of 40 µg/mL as a control ( n ≥ 3). ( G, H ) Chemical inhibition of lipid droplet synthesis differentially suppresses VACV replication in transformed cells. HepG2 ( G ) and A549 ( H ) cells were infected with MOI of 0.01 of vLGLuc in the presence or absence of 30 µM avasimibe, 125 µM T-863, or 125 µM PF-06424439. A Gaussia luciferase assay was performed to measure the late gene expression at 24 hpi. AraC was used at a concentration of 40 µg/mL as a control ( n ≥ 3). ( I, J ) the inhibition of lipid droplet synthesis does not alter the viability of the transformed cells. HepG2 ( I ) and A549 ( J ) cells were grown in the presence or absence of 30 µM avasimibe, 125 µM T-863, or 125 µM PF-06424439 for 48 h. Cell viability was determined by trypan blue exclusion assay using an automated cell counter ( n ≥ 3). n indicates the number of biological replicates. For P -values, ns, P > 0.05; * P ≤ 0.05; **** P ≤ 0.0001.

Techniques: Inhibition, Infection, Virus, Plaque Assay, Trypan Blue Exclusion Assay, Expressing, Luciferase, Concentration Assay, Control, Transformation Assay

Fibroblasts (NuFF-1) were infected (MOI= 0.5 TCID50/cell) with WT, UL33-3xF, UL33Δ1, or UL33Δ2 and analyzed at 96 hpi. (A) Whole cell lysates were probed by immunoblot with the indicated antibodies. IE1, marker of lytic infection; actin, loading control. N = 3, representative blots shown. (B) Cells were fixed, permeabilized, and UL33 localization was assessed using an antibody against the FLAG epitopes (green). mCherry (red) is shown as a marker of infection, and DAPI (blue) was used to visualize nuclei. Images were acquired at 63X magnification. N= 3, representative images shown.

Journal: bioRxiv

Article Title: The Human Cytomegalovirus vGPCR UL33 is Essential for Efficient Lytic Replication in Epithelial Cells

doi: 10.1101/2024.09.18.609710

Figure Lengend Snippet: Fibroblasts (NuFF-1) were infected (MOI= 0.5 TCID50/cell) with WT, UL33-3xF, UL33Δ1, or UL33Δ2 and analyzed at 96 hpi. (A) Whole cell lysates were probed by immunoblot with the indicated antibodies. IE1, marker of lytic infection; actin, loading control. N = 3, representative blots shown. (B) Cells were fixed, permeabilized, and UL33 localization was assessed using an antibody against the FLAG epitopes (green). mCherry (red) is shown as a marker of infection, and DAPI (blue) was used to visualize nuclei. Images were acquired at 63X magnification. N= 3, representative images shown.

Article Snippet: Primary newborn human foreskin fibroblasts (NuFF-1) were obtained from GlobalStem.

Techniques: Infection, Western Blot, Marker, Control

(A) Fibroblasts (NuFF-1) were infected (MOI= 0.5 TCID50/cell) with TB40/E- mCherry -UL33-3xF (UL33-3xF). Cells were next fixed and permeabilized over a 96 h time course, and UL33 was visualized via its FLAG epitopes (green). mCherry (red) is a marker of infection, and DAPI (blue) was used to visualize host cell nuclei. Images were acquired at 40X magnification. N = 3, representative images shown (B) Cells were infected as in (A) , with an additional, parallel culture treated with PAA post-adsorption, which was collected at 72 hpi. Cells were otherwise collected over a 96 h time course, and whole-cell lysates were immunoblotted for the FLAG epitope tag to detect pUL33-3xF. IE1 is shown as a marker of lytic infection, and actin is shown as a loading control. M, mock-infected cells, harvested at 96 hpi. N = 3, representative blots shown.

Journal: bioRxiv

Article Title: The Human Cytomegalovirus vGPCR UL33 is Essential for Efficient Lytic Replication in Epithelial Cells

doi: 10.1101/2024.09.18.609710

Figure Lengend Snippet: (A) Fibroblasts (NuFF-1) were infected (MOI= 0.5 TCID50/cell) with TB40/E- mCherry -UL33-3xF (UL33-3xF). Cells were next fixed and permeabilized over a 96 h time course, and UL33 was visualized via its FLAG epitopes (green). mCherry (red) is a marker of infection, and DAPI (blue) was used to visualize host cell nuclei. Images were acquired at 40X magnification. N = 3, representative images shown (B) Cells were infected as in (A) , with an additional, parallel culture treated with PAA post-adsorption, which was collected at 72 hpi. Cells were otherwise collected over a 96 h time course, and whole-cell lysates were immunoblotted for the FLAG epitope tag to detect pUL33-3xF. IE1 is shown as a marker of lytic infection, and actin is shown as a loading control. M, mock-infected cells, harvested at 96 hpi. N = 3, representative blots shown.

Article Snippet: Primary newborn human foreskin fibroblasts (NuFF-1) were obtained from GlobalStem.

Techniques: Infection, Marker, Adsorption, FLAG-tag, Control

Multistep growth analyses were performed in epithelial cells (ARPE-19 cells) infected (MOI= 0.1 TCID50/cell) with the indicated viruses over a 30-day time course. Infected cells were collected at the indicated times, and viral titers of cell-associated virus were quantified by TCID50 using naïve NuFF-1 fibroblasts. N = 3 biological replicates; graph depicts one biological replicate, with 3 technical replicates per time point. Error is shown as Standard deviation and significance was calculated using unpaired t-tests. * p <0.05, ** p <0.01, *** p <0.005

Journal: bioRxiv

Article Title: The Human Cytomegalovirus vGPCR UL33 is Essential for Efficient Lytic Replication in Epithelial Cells

doi: 10.1101/2024.09.18.609710

Figure Lengend Snippet: Multistep growth analyses were performed in epithelial cells (ARPE-19 cells) infected (MOI= 0.1 TCID50/cell) with the indicated viruses over a 30-day time course. Infected cells were collected at the indicated times, and viral titers of cell-associated virus were quantified by TCID50 using naïve NuFF-1 fibroblasts. N = 3 biological replicates; graph depicts one biological replicate, with 3 technical replicates per time point. Error is shown as Standard deviation and significance was calculated using unpaired t-tests. * p <0.05, ** p <0.01, *** p <0.005

Article Snippet: Primary newborn human foreskin fibroblasts (NuFF-1) were obtained from GlobalStem.

Techniques: Infection, Virus, Standard Deviation

(A) Fibroblasts (NuFF-1) or (B) epithelial cells (ARPE-19) were mock-, WT-, or UL33Δ-infected (MOI= 0.5 TCID50/cell) for the times indicated. Whole cell lysates were prepared and representative viral proteins from the IE (IE1), E (pUL44), and L (pp28) kinetic classes were assessed by immunoblot. Actin was assayed as a loading control. N=3, representative blots shown.

Journal: bioRxiv

Article Title: The Human Cytomegalovirus vGPCR UL33 is Essential for Efficient Lytic Replication in Epithelial Cells

doi: 10.1101/2024.09.18.609710

Figure Lengend Snippet: (A) Fibroblasts (NuFF-1) or (B) epithelial cells (ARPE-19) were mock-, WT-, or UL33Δ-infected (MOI= 0.5 TCID50/cell) for the times indicated. Whole cell lysates were prepared and representative viral proteins from the IE (IE1), E (pUL44), and L (pp28) kinetic classes were assessed by immunoblot. Actin was assayed as a loading control. N=3, representative blots shown.

Article Snippet: Primary newborn human foreskin fibroblasts (NuFF-1) were obtained from GlobalStem.

Techniques: Infection, Western Blot, Control

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