Journal: Cells

Article Title: Merkel Cell Polyomavirus Large T Antigen Induces Cellular Senescence for Host Growth Arrest and Viral Genome Persistence through Its Unique Domain

doi: 10.3390/cells12030380

Figure Lengend Snippet: MCPyV LT induces cellular senescence. ( A ) Lentiviral constructs for LT expression. ( B ) Senescence assay. Normal BJ−hTERT human fibroblast cells were transduced with either eGFP empty vector or LT constructs and were selected with puromycin at two days post−transduction. A colorimetric senescence-associated β−galactosidase assay (SA−β−gal) was conducted 14 days post- transduction. MCPyV LT−expressing cells with blue staining were positive for SA−β−gal, indicated by white arrows. Images are representative of 3 experiments. ( C ) Fluorescent intensity of SA−β−gal activity (SA−β−gal + ) was measured in GFP + (LT−expressing) cells by flow cytometry. MCPyV LT expression significantly increased the number of senescent cells. One−way ANOVA test was used to determine statistical significance (*** p < 0.001, **** p < 0.0001, ns = not significant). Standard error bars represent standard error value, n = 3. ( D ) MCPyV LT promotes senescent phenotypes. Nuclear area (µm 2 ) of empty vector or LT−transduced BJ−hTERT cells. One−way ANOVA test (**** p < 0.0001, ns = not significant). Standard error bars represent mean value with standard deviation, n = 50 cells per construct. ( E ) SASP activation by MCPyV LT. Quantitative reverse transcription polymerase chain reaction (RT−qPCR) analysis of SASP cytokines. Abbreviations— ANKRD1 : Ankyrin repeat domain 1; CSF2 : Colony-stimulating factor 2; CXCL : chemokine (C−X−C motif) ligand; EDN1 : Endothelin 1; IL : Interleukin. SASPs mRNA levels were substantially upregulated by LT expression except IL−7 . One−way ANOVA test was used to determine statistical significance (**** p < 0.0001, * p < 0.05, ns = not significant). Standard error bars represent standard error value, n = 3. ( F ) MCPyV LT activates p21. Growth arrest genes were upregulated in MCPyV LT−expressing cells. Immunoblot of cell cycle−related genes in BJ−hTERT cells transduced with empty vector or LT constructs. β−Actin was used as a loading control.

Article Snippet: The detection of SA-β-Gal activity was performed using a colorimetric senescence β-Galactosidase histochemical staining kit (G-Biosciences, St. Louis, MO, USA) or a fluorescent Cell MeterTM Cellular Senescence Activity Assay Kit with XiteTM Red beta-D-galactopyranoside (AAT Bioquest, Pleasanton, CA, USA) according to the manufacturer’s instructions.

Techniques: Construct, Expressing, Transduction, Plasmid Preparation, Staining, Activity Assay, Flow Cytometry, Standard Deviation, Activation Assay, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Control

Journal: Cells

Article Title: Merkel Cell Polyomavirus Large T Antigen Induces Cellular Senescence for Host Growth Arrest and Viral Genome Persistence through Its Unique Domain

doi: 10.3390/cells12030380

Figure Lengend Snippet: MCPyV−induced senescence is required for viral genome maintenance. ( A ) p21 knockdown decreases the percentage of senescent SA−β−gal + cells. Schematic representing whole MCPyV genome construct encoding origin (Ori), VP1, VP2, T antigens (TAg), and eGFP−P2A (left). BJ−hTERT cells were reversely transfected with the MCPyV genome and then transduced with shp21.1 for p21 knockdown at 14 days post−transfection. At 21 days post−transfection, MCPyV−transfected cells were subjected to a senescence assay. Histogram representing GFP + /SA−β−gal + (MCPyV + , senescent cells) for transfected MCPyV or MCPyV+p21KD cells (middle). Percentage of GFP + /SA−β−gal + in population (right). Statistical significance was determined using unpaired Student’s t -test (** p < 0.01). Standard error bars represent mean value with standard error: n = 3; 10,000 cells recorded per replicate. ( B ) MCPyV−induced senescence is p21−dependent. Colorimetric SA−β−gal staining of MCPyV−transfected cells at 21 days post−transfection. ( C ) Cellular senescence maintains MCPyV genome. BJ−hTERT cells were co−transfected with MCPyV (GFP+) and mCherry−shScr or mCherry−shp21.1 (p21KD, mCherry + ). Cells were harvested at the stated days post−transfection and were analyzed by flow cytometry. Transfected cells were gated for mCherry + (p21KD) cells and then examined for G2−arrested GFP + cells (MCPyV + ). Unpaired Student’s t -test (ns = not significant). Standard error bars represent mean value with standard error, n = 3. ( D ) qPCR was conducted on cells reversely co−transfected with mCherry−shScr or mCherry−shp21.1, and MCPyV genome levels were reported at the given time points. Multiple unpaired Student’s t -test (* p < 0.05). Standard error bars represent mean value with standard error, n = 3. ( E ) Senolytic treatment selectively clears MCPyV−induced senescent cells. At 14 days post−transfection, empty vector− or MCPyV−transfected cells were treated with navitoclax (250 nM), dasatinib and quercetin (D+Q, 25 nM dasatinib and 250 nM quercetin), or DMSO for two days, and then subjected to a SA−β−gal assay or qPCR ( F ). Scale bar = 180 μm. One−way ANOVA (*** p < 0.001). Standard error bars represent mean value with standard error, n = 3. ( G ) MCPyV induces senescence to sustain viral genome maintenance. MCPyV LT activates p53 and, subsequently, p21 through nucleolar stress responses. Activation of p21 induces G2 cell cycle arrest, leading to changes in growth arrest and senescent gene expression, as well as the promotion of cellular senescence. Cellular senescence establishes MCPyV infection in fibroblast cells, enabling it to extend its lifecycle within the host. Figure created with BioRender.com.

Article Snippet: The detection of SA-β-Gal activity was performed using a colorimetric senescence β-Galactosidase histochemical staining kit (G-Biosciences, St. Louis, MO, USA) or a fluorescent Cell MeterTM Cellular Senescence Activity Assay Kit with XiteTM Red beta-D-galactopyranoside (AAT Bioquest, Pleasanton, CA, USA) according to the manufacturer’s instructions.

Techniques: Knockdown, Construct, Transfection, Transduction, Staining, Flow Cytometry, Plasmid Preparation, Activation Assay, Gene Expression, Infection