Journal: Molecular cancer research : MCR

Article Title: Signals from the metastatic niche regulate early and advanced ovarian cancer metastasis through miR-4454 downregulation

doi: 10.1158/1541-7786.MCR-19-1162

Figure Lengend Snippet: (A) Overview of the strategy for analysis of primary vs. metastatic OC patient tumor microRNA sequencing data and OC on organotypic 3D omental culture microRNA profiling. RNA isolated from frozen tumor tissue was used for microRNAseq. GFP-tagged OC cells (Kuramochi, OVCAR4 and OVCAR8) were grown on 3D omental culture for 48 h, isolated by FACS, RNA was isolated and microRNA expression was compared to the sham treated controls using NanoString. (B) Schematic representation of the organotypic omental 3D culture model. Mesothelial cells and fibroblasts were isolated from omentum obtained from female donors. The fibroblasts were mixed with type I collagen and seeded in a 10 cm dish. Once attached, they were overlaid with a confluent monolayer of mesothelial cells. Together they mimic the basement membrane and the mesothelium on the surface of the omentum. They were allowed to grow and for 24h so that the secreted factors from the cells could generate a complex microenvironment. Thereafter, GFP labeled OC cells (Kuramochi/OVCAR4/OVCAR8) were seeded on the 3D omentum culture and allowed to grow. After 2 days, the OC cells were separated using fluorescence activated cell sorting (FACS) and used for RNA isolation and microRNA profiling with NanoString nCounter miRNA Expression Assay (800 microRNAs). Controls were seeded directly on the 10 cm culture dishes and were sham treated. (C) microRNA-target interaction networks for the 26 common downregulated microRNAs in patient metastasis and 3D omental culture using miRNet. microRNA hubs are depicted as blue dots and targets as red dots. (D) Top: qRT-PCR validation of miR-4454 in a set of OC patient primary and metastatic tumors. A line connects the expression data for each pair of primary and metastasis. Bottom: qRT-PCR validation of miR-4454 expression in OC cells (Kuramochi, OVCAR4 and OVCAR8) seeded on the 3D omental culture. * p<0.01, Students t-test. (E) OC cells isolated from ascites of 2 OC patients (ASC1 and 2) were grown on 3D omental culture for 48 h, separated by FACS and used for RNA isolation and qRT-PCR for miR-4454 expression. Mean ± SD from 3 independent experiments. * p<0.01, Students t-test. (F) Kaplan–Meier plot comparing patient survival when miR-4454 is upregulated or downregulated 1.5 fold in metastasis (Mets) vs. primary tumors in the OC patient cohort.

Article Snippet: RNA was isolated from the cells using miRNeasy Kit (Qiagen Cat# 217004) and used for reverse transcription with Applied Biosystems High Capacity Reverse Transcription Kit (Cat# 4368813) or TaqMan ® MicroRNA Reverse Transcription Kit (Cat# 4366597). qRT-PCR was performed for miR-4454 using Taqman miRNA assay (Assay ID 461830_mat) according to the manufacturer’s protocol using a Roche LightCycler 96 system with U6 (Assay ID 001973) as endogenous control.

Techniques: Sequencing, Isolation, Expressing, Membrane, Labeling, Fluorescence, FACS, Quantitative RT-PCR, Biomarker Discovery

Journal: Molecular cancer research : MCR

Article Title: Signals from the metastatic niche regulate early and advanced ovarian cancer metastasis through miR-4454 downregulation

doi: 10.1158/1541-7786.MCR-19-1162

Figure Lengend Snippet: (A) Overexpression of miR-4454 inhibits motility and growth of OC cells. Kuramochi, OVCAR3, OVCAR4 and OVCAR8 cells were transfected with pre-miR-4454 or scrambled control oligos (Scr) and used for the functional assays. Migration: Transfected cells were seeded in transwell inserts with 8 μm pores and allowed to migrate towards medium containing 10% FBS. Migrated cells were fixed at the optimal time points, imaged and quantified using image-J software (mean ± SD; 3 independents experiments). Proliferation: Transfected cells were seeded in 96-well plates and allowed to grow for 5 days. Cell proliferation was determined by MTT assay (mean ± SD; 3 independents experiments). Colony formation: Transfected cells were seeded in 6-well plates for a colony formation assay. Once visible colonies were formed, the colonies were fixed, stained, imaged and counted using image-J software (mean ± SD; 3 independents experiments). (B) Inhibition of miR-4454 increases the motility and growth of metastatic OC cells. Kuramochi, OVCAR3, OVCAR4 and OVCAR8 cells were transfected with anti-miR-4454 or scrambled control oligos (Scr) and used for the migration, proliferation and colony formation assays as described above (mean ± SD; 3 independents experiments). * p<0.01, ** p<0.05, Students t-test. (C) Invasion through the 3D omentum culture. Schematic representation of the 3D invasion model mimicking the invasion of metastasizing OC cells through the outer layers of the omentum. The 3D omentum culture was assembled in fluoroblock transwell inserts with 8 μm pores. GFP labeled OC cells were seeded on the 3D culture and allowed to invade though it towards the lower chamber containing medium with 10% FBS. (D) Top: Kuramochi and OVCAR4 cells were transfected with pre-miR-4454 or scrambled control oligo (Scr) and evaluated for their ability to invade through the 3D omental culture. Invasion was stopped at the optimal time. The invaded GFP labeled OC cells were imaged using the EVOS FL auto microscope and quantified using image-J software (mean ± SD; 3 independent experiments). Bottom: Kuramochi and OVCAR4 cells were transfected with anti-miR-4454 or scrambled control oligo (Scr) and their ability to invade through the 3D omental culture was evaluated. The invaded GFP cells were imaged and quantified using image-J software (mean ± SD; 3 independent experiments). Representative images of OC cells that have invaded through the 3D omentum culture, for each of these treatment groups are shown above respective graphs. (E). OVCAR8 GFP cells transfected with scrambled control oligos, anti-miR-193b or pre-miR-193b, were seeded on 3D omental culture and allowed to form colonies. The fluorescent colonies were imaged on a Syngene G-Box imaging system and counted using image-J software (mean ± SD; 3 independents experiments). * p<0.01, Students t-test compared to control.

Article Snippet: RNA was isolated from the cells using miRNeasy Kit (Qiagen Cat# 217004) and used for reverse transcription with Applied Biosystems High Capacity Reverse Transcription Kit (Cat# 4368813) or TaqMan ® MicroRNA Reverse Transcription Kit (Cat# 4366597). qRT-PCR was performed for miR-4454 using Taqman miRNA assay (Assay ID 461830_mat) according to the manufacturer’s protocol using a Roche LightCycler 96 system with U6 (Assay ID 001973) as endogenous control.

Techniques: Over Expression, Transfection, Control, Functional Assay, Migration, Software, MTT Assay, Colony Assay, Staining, Inhibition, Labeling, Microscopy, Imaging

Journal: Molecular cancer research : MCR

Article Title: Signals from the metastatic niche regulate early and advanced ovarian cancer metastasis through miR-4454 downregulation

doi: 10.1158/1541-7786.MCR-19-1162

Figure Lengend Snippet: Kuramochi cells were transfected with anti-miR-4454/BAG5-siRNA/SPARC-siRNA or both anti-miR-4454 and siRNA or scrambled control oligos (Scr). The transfected cells were then used for the functional assays. (A) Migration: Transfected cells were seeded in transwell inserts with 8 μm pores in and allowed to migrate towards medium containing 10% FBS. Migrated cells were fixed, imaged and quantified using image-J software (mean ± SD; 3 independents experiments). (B) Transfected cells were seeded in 96-wella plates and allowed to grow for 5 days. Cell proliferation was determined by MTT assay (mean ± SD; 3 independents experiments). (C) Colony formation: Transfected cells were seeded in 6-well plates and allowed to form colonies. Colonies were fixed after 2–3 weeks, stained (0.005% crystal violet) and counted using image-J software (mean ± SD; 3 independents experiments). * p<0.01, Students t-test. (D) Kaplan–Meier plot for effect of BAG5 (Affy ID:202985_s_at) and SPARC (Affy ID: 212667_at) expression levels on 5-year progression free survival in ovarian cancer patients using KM Plotter. Red: High expression; Black: Low expression.

Article Snippet: RNA was isolated from the cells using miRNeasy Kit (Qiagen Cat# 217004) and used for reverse transcription with Applied Biosystems High Capacity Reverse Transcription Kit (Cat# 4368813) or TaqMan ® MicroRNA Reverse Transcription Kit (Cat# 4366597). qRT-PCR was performed for miR-4454 using Taqman miRNA assay (Assay ID 461830_mat) according to the manufacturer’s protocol using a Roche LightCycler 96 system with U6 (Assay ID 001973) as endogenous control.

Techniques: Transfection, Control, Functional Assay, Migration, Software, MTT Assay, Staining, Expressing

Journal: Molecular cancer research : MCR

Article Title: Signals from the metastatic niche regulate early and advanced ovarian cancer metastasis through miR-4454 downregulation

doi: 10.1158/1541-7786.MCR-19-1162

Figure Lengend Snippet: OVCAR8 cells stably overexpressing miR-4454 were generated using a lentiviral pre-miR-4454 vector. The effect of stable expression of miR-4454 in OVCAR8 cells on their ability to form metastasis in mouse xenografts was tested by injecting 5×106 cells intraperitoneally in female, athymic nude mice and compared to vector controls. Mice were euthanized after 35 days and the peritoneal metastasis were counted, surgically resected and weighed. (A) Graphical representation of the number of metastasis. (B) Graphical representation total tumor weight of OVCAR8 tumors stably expressing miR-4454 compared to control vector (mean ± SD; n=10 mice/group; *p<0.01). (C) Representative images of the peritoneal metastasis in each group with the tumors outlined in black (D) Validation of miR-4454 overexpression in the omental tumors. Omental tumors (n=3/group) were homogenized, RNA was isolated, and qRT-PCR was performed for miR-4454. (E) FFPE tumor sections (n=5/group) were stained for cleaved caspase-3. A representative image is shown (left) and the quantification of the cleaved caspase-3 staining is plotted (right). (F) Left: Representative images of FFPE tumor sections (n=5/group) stained for Ki-67. Right: The quantification of the Ki-67 staining (mean ± SD; *p<0.01, Students t-test).

Article Snippet: RNA was isolated from the cells using miRNeasy Kit (Qiagen Cat# 217004) and used for reverse transcription with Applied Biosystems High Capacity Reverse Transcription Kit (Cat# 4368813) or TaqMan ® MicroRNA Reverse Transcription Kit (Cat# 4366597). qRT-PCR was performed for miR-4454 using Taqman miRNA assay (Assay ID 461830_mat) according to the manufacturer’s protocol using a Roche LightCycler 96 system with U6 (Assay ID 001973) as endogenous control.

Techniques: Stable Transfection, Generated, Plasmid Preparation, Expressing, Control, Biomarker Discovery, Over Expression, Isolation, Quantitative RT-PCR, Staining

Journal: Molecular cancer research : MCR

Article Title: Signals from the metastatic niche regulate early and advanced ovarian cancer metastasis through miR-4454 downregulation

doi: 10.1158/1541-7786.MCR-19-1162

Figure Lengend Snippet: (A) GFP-tagged Kuramochi cells were seeded on a confluent monolayer of human primary mesothelial cells (HPMC), normal omental fibroblasts (NOF) and cancer associated fibroblasts (CAF) for a period of 48 h. Thereafter, the Kuramochi cells were separated by FACS, RNA was isolated and qRT-PCR was performed for the expression of miR-4454. A significant reduction in miR-4454 expression was observed upon coculture with NOFs and CAFs (mean ± SD from 3 independent experiments). (B) Kuramochi cells were grown in condition medium (CM) collected from normal omental fibroblasts (NOF) or cancer associated fibroblasts (CAF) isolated from OC patients. qRT-PCR was done to test the effect on expression of miR-4454 in OC cells grown on CM compared with corresponding controls grown in serum free medium. (C): Proximal culture overview. Step 1 OC cells were seeded on the lower surface of a transwell insert with 0.4μm pores and allowed to attach. Step 2 Then the insert was flipped and placed in a well containing growth medium. Step 3 The fibroblasts were seeded on the upper surface of the insert and the two cell types were allowed to grow in close proximity of each other but separated by a 10μm thick membrane having 0.4μm pores. This allowed the dynamic exchange of paracrine factors (magnified inset) at localized high concentrations while inhibiting juxtacrine signaling. (D) Kuramochi cells in proximal culture with NOFs, CAFs or dermal fibroblasts (D.Fibroblast) for 48 h were used for RNA isolation and qRT-PCR for miR-4454 expression compared corresponding controls, which did not have the fibroblasts seeded on the opposite surface. Mean ± SD from 3 independent experiments; * p<0.01, Students t test.

Article Snippet: RNA was isolated from the cells using miRNeasy Kit (Qiagen Cat# 217004) and used for reverse transcription with Applied Biosystems High Capacity Reverse Transcription Kit (Cat# 4368813) or TaqMan ® MicroRNA Reverse Transcription Kit (Cat# 4366597). qRT-PCR was performed for miR-4454 using Taqman miRNA assay (Assay ID 461830_mat) according to the manufacturer’s protocol using a Roche LightCycler 96 system with U6 (Assay ID 001973) as endogenous control.

Techniques: Isolation, Quantitative RT-PCR, Expressing, Membrane

Journal: Molecular cancer research : MCR

Article Title: Signals from the metastatic niche regulate early and advanced ovarian cancer metastasis through miR-4454 downregulation

doi: 10.1158/1541-7786.MCR-19-1162

Figure Lengend Snippet: (A) RNAseq was done on Kuramochi and OVCAR4 cells transfected with pre-miR-4454 or scrambled control oligos (n=3/group). Venn diagram showing the number of genes uniquely and commonly downregulated in Kuramochi and OVCAR4 cells overexpressing miR-4454 compared to the respective controls. (B) The 811 common downregulated genes were analyzed for predicted miR-4454 seed matches in their 3’UTR using microRNA target prediction software TargetScan, miRDB and TargetMiner. Heat map showing the extent of downregulation of the 68 genes, with predicted miR-4454 seed matches, in miR-4454 overexpressing Kuramochi and OVCAR4 cells compared to the corresponding controls. (C) qRT-PCR validation of selected targets in OC cells overexpressing miR-4454. qRT-PCR for SPARC, BAG5 and SOX4 was done using RNA from Kuramochi and OVCAR4 cells transfected with pre-miR-4454 or scrambled control oligos. The baseline SOX4 expression was too low and it was not detected. (mean ± SD from 3 independent experiments). * p<0.01, ** p<0.05, Students t-test. (D) 3’UTR luciferase reporter assay for the binding of miR-4454 to the 3’UTR of BAG5 or SPARC. Wild type (WT) or miR-4454 seed match mutated (Mut) BAG5 or SPARC 3’UTR luciferase constructs were co-transfected with pre-miR-4454 (pre-4454) or scrambled control (Scr) in 293T cells, the conditioned medium collected, and the secreted gaussia luciferase and alkaline phosphatase activity was measured. Luciferase activity was normalized to the alkaline phosphatase activity for transfection normalization (mean±SD; 3 independent experiments). (E-F) Functional effects of miR-4454 targets. Kuramochi, OVCAR4 and OVCAR8 cells were transfected with BAG5 siRNA (E) or SPARC siRNA (F) or scrambled control oligo (Scr) and the effects of silencing them were evaluated on the ability of the cells to migrate, proliferate and form colonies. (E) and (F) left panel - Migration: Transfected cells were seeded in transwell inserts with 8 μm pores in and allowed to migrate towards medium containing 10% FBS in the lower chamber. Migrated cells were fixed, imaged and quantified using image-J software (mean ± SD; 3 independents experiments). Representative images of migrated cells are provided over each treatment group. (E) and (F) middle panel - Proliferation: Transfected cells were seeded in 96-well plates and allowed to grow for 5 days. Cell proliferation was determined by MTT assay (mean ± SD; 3 independents experiments). (E) and (F) right panel - Colony formation: Transfected cells were seeded in 6 well plates and allowed to form colonies. Colonies were fixed after 2–3 weeks, stained (0.005% crystal violet) and counted using image-J software (mean ± SD; 3 independents experiments). Representative images of colonies are provided. * p<0.01, Students t-test.

Article Snippet: RNA was isolated from the cells using miRNeasy Kit (Qiagen Cat# 217004) and used for reverse transcription with Applied Biosystems High Capacity Reverse Transcription Kit (Cat# 4368813) or TaqMan ® MicroRNA Reverse Transcription Kit (Cat# 4366597). qRT-PCR was performed for miR-4454 using Taqman miRNA assay (Assay ID 461830_mat) according to the manufacturer’s protocol using a Roche LightCycler 96 system with U6 (Assay ID 001973) as endogenous control.

Techniques: Transfection, Control, Software, Quantitative RT-PCR, Biomarker Discovery, Expressing, Luciferase, Reporter Assay, Binding Assay, Construct, Activity Assay, Functional Assay, Migration, MTT Assay, Staining

Journal: Molecular cancer research : MCR

Article Title: Signals from the metastatic niche regulate early and advanced ovarian cancer metastasis through miR-4454 downregulation

doi: 10.1158/1541-7786.MCR-19-1162

Figure Lengend Snippet: (A) The OC cells (OVCAR3, OVCAR4 and Kuramochi) were transfected with pre-miR-4454 or scrambled control oligo (Scr) (left panel) or with BAG5 siRNA or scrambled control oligos (Scr) (right panel). Cells were allowed to grow for 48 h and then lysed in RIPA buffer. Proteins were separated by 4–20% gradient SDS-PAGE and transferred to nitrocellulose membrane, probed with BAG5, cleaved PARP, cleaved caspase-3 and p53 antibodies. Representative blots from 3 independent experiments are shown. (B) OVCAR3, OVCAR4 or Kuramochi cells were transfected with pre-miR-4454 or scrambled control oligo (Scr) (left panel) or with SPARC siRNA or scrambled control oligos (Scr) (right panel). Cells were allowed to grow for 48 h and then lysed in RIPA buffer. Proteins were separated by 4–20% gradient SDS-PAGE and transferred to nitrocellulose membrane, probed with SPARC, phosphorylated FAK at Tyr397 (pFAK) and total FAK antibodies. Representative blots from 3 independent experiments are shown. (C) Schematic overview of findings. Paracrine signals from microenvironmental fibroblasts lead to downregulation of miR-4454 in metastasizing OC cells. The downregulation of miR-4454 promotes metastatic colonization through the concomitant increased expression of its targets SPARC and BAG5, which help by activating FAK, promoting mutant p53 gain of function by its stabilization and inhibiting apoptosis respectively.

Article Snippet: RNA was isolated from the cells using miRNeasy Kit (Qiagen Cat# 217004) and used for reverse transcription with Applied Biosystems High Capacity Reverse Transcription Kit (Cat# 4368813) or TaqMan ® MicroRNA Reverse Transcription Kit (Cat# 4366597). qRT-PCR was performed for miR-4454 using Taqman miRNA assay (Assay ID 461830_mat) according to the manufacturer’s protocol using a Roche LightCycler 96 system with U6 (Assay ID 001973) as endogenous control.

Techniques: Transfection, Control, SDS Page, Membrane, Expressing, Mutagenesis

Journal: Journal of Biomedicine and Biotechnology

Article Title: Efficient siRNA Delivery by the Cationic Liposome DOTAP in Human Hematopoietic Stem Cells Differentiating into Dendritic Cells

doi: 10.1155/2009/410260

Figure Lengend Snippet: Effects of siRNA delivery agents on cytotoxicity and transfection rates . Freshly isolated HSCs were subjected to agent-specific transfection protocols using a fluorescein-labeled, nontargeting siRNA control as described in the Materials and Methods. After 24 hours incubation, cytotoxicity was assessed through the trypan blue dye exclusion test (a), while transfection rates were calculated from the ratio between fluorescein-labeled and total number of DAPI-stained cells (b). Data shown are representative of triplicate determinations.

Article Snippet: Cells subjected to mock- and scrambled-siRNA transfection (ID-46183G, Ambion) under the same experimental conditions were employed for control purposes.

Techniques: Transfection, Isolation, Labeling, Incubation, Staining

Journal: Journal of Biomedicine and Biotechnology

Article Title: Efficient siRNA Delivery by the Cationic Liposome DOTAP in Human Hematopoietic Stem Cells Differentiating into Dendritic Cells

doi: 10.1155/2009/410260

Figure Lengend Snippet: Cell viability during repeated, DOTAP-mediated transfection rounds in differentiating HSCs . Freshly isolated HSCs were induced to differentiate into immunogenic dendritic cells as described in the Materials and Methods. Immediately after treatment, cultures were split into two cell populations, of which one, used as the control, was grown in absence of manipulations ( white bars ), while the other was subjected to two and three DOTAP-based, scrambled siRNA transfection rounds performed at day 0 and day 3 ( black bars ), and day 0, day 3 and day 9 ( grey bars ). Cell viability was monitored by trypan blue staining at different time points as indicated.

Article Snippet: Cells subjected to mock- and scrambled-siRNA transfection (ID-46183G, Ambion) under the same experimental conditions were employed for control purposes.

Techniques: Transfection, Isolation, Staining

Journal: Journal of Biomedicine and Biotechnology

Article Title: Efficient siRNA Delivery by the Cationic Liposome DOTAP in Human Hematopoietic Stem Cells Differentiating into Dendritic Cells

doi: 10.1155/2009/410260

Figure Lengend Snippet: Expression of phenotypic markers throughout HSC differentiation into immunogenic dendritic cells in presence of multiple, DOTAP-mediated transfection rounds . HSCs were subjected to differentiation agents, and then immediately split into two parallel cultures, one of which was maintained in the absence of manipulations, while the other was subjected to DOTAP-based, scrambled siRNA transfection rounds. Phenotypic antigens typically modulated during HSC differentiation into immunogenic dendritic cells were analyzed in HSCs at day 0 ( white bars ), day 3 ( grey bars ), and day 14 ( black bars ). In all instances, analyses were performed through a FACScan flow cytometer using monoclonal antibodies labeled with fluorescein isothiocyanate or phycoerythrin. Data shown are representative of triplicate determinations.

Article Snippet: Cells subjected to mock- and scrambled-siRNA transfection (ID-46183G, Ambion) under the same experimental conditions were employed for control purposes.

Techniques: Expressing, Transfection, Flow Cytometry, Labeling

Journal: Journal of Biomedicine and Biotechnology

Article Title: Efficient siRNA Delivery by the Cationic Liposome DOTAP in Human Hematopoietic Stem Cells Differentiating into Dendritic Cells

doi: 10.1155/2009/410260

Figure Lengend Snippet: Silencing of cathepsin S during HSC differentiation . (a) Levels of cathepsin S were assessed by Western blotting in dendritic cells generated from the differentiation in vitro of HSCs. During the 14-day differentiation process, cells were subjected to DOTAP-based transfection rounds on day 0, 3, and 9 using a CTSS-targeting siRNA as described in the Materials and Methods. Untreated and scrambled siRNA-transfected cells were employed for control purposes. Levels of precursor (66 KDa) and mature (26 KDa) forms of cathepsin S at different time points (i.e., days 7 and 14) are indicated. β -actin was used to normalize the signals. (b) Shown is the same analysis as in panel A, except for the number of transfections, that were performed on day 0 and 3 only. Graphs are representative of experiments consistently reproduced using at least 20 different populations of HSCs.

Article Snippet: Cells subjected to mock- and scrambled-siRNA transfection (ID-46183G, Ambion) under the same experimental conditions were employed for control purposes.

Techniques: Western Blot, Generated, In Vitro, Transfection

Journal: PLoS Pathogens

Article Title: SAMHD1 phosphorylation and cytoplasmic relocalization after human cytomegalovirus infection limits its antiviral activity

doi: 10.1371/journal.ppat.1008855

Figure Lengend Snippet: a) SAMHD1 levels were analyzed by immunoblotting in cell lysates of HFFs not infected (n.i.) or infected with AD169 at an MOI of 1 for 3 days. Expression of IE1/IE2 viral antigens was used as control for infection, while the p85 subunit of PI3K was used as loading control. A representative experiment out of six is shown. b) The relative amount of SAMHD1 protein, normalized to that of p85, was determined by densitometric analysis and is relative to that of n.i. cells, which was arbitrarily set as 1. Data are expressed as mean ± SE of eight independent experiments. c) SAMHD1 expression in HFFs not infected (n.i.) or infected with HCMV clinical isolates collected from infants affected by congenital HCMV infection (P1, P6, P7 and P10), or with Merlin. Data derive from one representative experiment out of two. d) SAMHD1 expression in HFFs, ARPE-19 and HMVEC cells infected with the indicated HCMV strains. Data derive from one representative experiment out of two, performed at the same time with all conditions. Numbers indicate the relative amount of SAMHD1 protein, determined as in panel b). e) The relative amount of SAMHD1 protein from HFFs infected with the indicated clinical strains was determined as in panel b). Data are expressed as mean ± SE of eight independent experiments. All data derive from cells infected at an MOI of 1 and harvested at 3 dpi. f) PMA-differentiated THP-1 cells not infected or infected with TR at an MOI of 1 for the indicated times. Expression of SAMHD1, viral antigens and p85 was evaluated as above. A representative experiment out of two is shown. g) SAMHD1 expression in HFFs induced to reach confluence (Con), serum-starved (Starv), proliferating (Prol), or infected with AD169 at an MOI of 1 for 3 days. Data derive from one representative experiment out of two. h) Antibody validation of the anti-SAMHD1 antibody by immunocapture MS. Proteins enriched by the antibody towards SAMHD1 ( y -axis) are plotted versus non-specific enrichment by one Rabbit IgG pool ( x- axis). Enriched proteins identified are quantified by label-free quantification and plotted as intensities (log 10 ). **, p < 0.01; ***, p < 0.001; ns, not significant.

Article Snippet: The following antibodies were used in immunoblot: anti-SAMHD1 rabbit polyclonal antibody (Ab) (Proteintech); anti-SAMHD1 rabbit polyclonal Ab specific for the phosphorylated T592 residue (ProSci); rabbit polyclonal anti-p85 subunit of PI3K (N-SH2 domain) and mouse monoclonal antibody (mAb) anti-IE1/IE2 viral proteins (MAB810R; Merck Millipore); mAb anti-UL97 (kindly provided by Thomas Mertens, Ulm University Medical Center, Germany); rabbit anti-Cdk1 (#77055) and mAb anti-tubulin (both from Cell Signaling); rabbit anti-Cdk2 (sc-163) and mAb anti-lamin A (C-3) (both from Santa Cruz).

Techniques: Western Blot, Infection, Expressing, Control, Biomarker Discovery, Quantitative Proteomics

Journal: PLoS Pathogens

Article Title: SAMHD1 phosphorylation and cytoplasmic relocalization after human cytomegalovirus infection limits its antiviral activity

doi: 10.1371/journal.ppat.1008855

Figure Lengend Snippet: a-h) HFFs were transfected with SAMHD1 siRNA or non-targeting siRNA (ctrl). Two days later, cells were either infected with AD169 at an MOI of 1 and harvested at 3 dpi (a-e) or infected at an MOI of 0.1 or 0.05 and harvested at 6 dpi (f-h). a) Levels of SAMHD1 and IE1/IE2 viral protein expression were assayed by immunoblotting. The p85 subunit of PI3K was used as loading control. One representative experiment is shown. b) The relative amount of SAMHD1 protein, normalized to that of p85, was determined by densitometric analysis and is relative to that of n.i./siCtrl cells, which was arbitrarily set as 1. Data are expressed as mean ± SE. c) The percentage of IE+ cells was analyzed by FACS after intracellular staining with a specific anti-IE1/IE2 mAb. FACS plots derive from one representative experiment. d) The percentage of IE+ cells is expressed as mean ± SD, as detected by FACS. e) Cell culture supernatants were assayed for infectious virus production by plaque assays. Results are expressed as mean ± SE. All data in panels a-e derive from five independent experiments. f) Immunoblotting at 6 dpi was performed as described in panel a). One representative experiment out of two is shown. g) The relative amount of SAMHD1 protein was determined as in panel b). Data are expressed as mean ± SE of two independent experiments with cells infected at both MOI. h) Viral titers were measured and expressed as in panel e) and derive from cell culture supernatants of two independent experiments, were HFFs were infected at an MOI of 0.1 or MOI of 0.05 for 6 days. i-j) HFFs were infected with single-cycle VLPs loaded with Vpx (Vpx) or unloaded (ΔVpx) at an MOI of 1, and then the same cells were infected with AD169 at an MOI of 1. At different dpi, cells and supernatants were harvested and subjected to immunoblotting and plaque assays, respectively. i) Immunoblotting of SAMHD1 protein expression. One representative experiment out of three is shown. j) Cell culture supernatants were assayed for infectious virus production by plaque assays. Results are expressed as mean ± SE of four experiments at 2–3 dpi. nt, not treated; n.i., not infected cells; *, p < 0.05; ***, p < 0.001; ns, not significant.

Article Snippet: The following antibodies were used in immunoblot: anti-SAMHD1 rabbit polyclonal antibody (Ab) (Proteintech); anti-SAMHD1 rabbit polyclonal Ab specific for the phosphorylated T592 residue (ProSci); rabbit polyclonal anti-p85 subunit of PI3K (N-SH2 domain) and mouse monoclonal antibody (mAb) anti-IE1/IE2 viral proteins (MAB810R; Merck Millipore); mAb anti-UL97 (kindly provided by Thomas Mertens, Ulm University Medical Center, Germany); rabbit anti-Cdk1 (#77055) and mAb anti-tubulin (both from Cell Signaling); rabbit anti-Cdk2 (sc-163) and mAb anti-lamin A (C-3) (both from Santa Cruz).

Techniques: Transfection, Infection, Expressing, Western Blot, Control, Staining, Cell Culture, Virus