Journal: bioRxiv

Article Title: Potent synthetic lethality between PLK1 and EYA-family inhibitors in tumours of the central and peripheral nervous system

doi: 10.1101/2025.01.19.633804

Figure Lengend Snippet: (A) Representative immunofluorescence images showing G2 and M-phase cells stained with p-S46 TCTP, p-S10 H3 and DAPI. (B) Representative quantitative image-based cytometry plot showing G2 and M-phase cell cycle gates as determined by staining intensity of p-S10 H3 and DAPI. (C) Cellular quantitation of p-S46 TCTP intensity in G2-phase following treatment with DMSO, Benzarone (7.5 µM), Bi2536 (10 nM), or Benzarone + Bi2536 for 5.5 hrs (one-way ANOVA with Tukey’s multiple comparison test, ** p ≤ 0.01, **** p ≤ 0.0001). (D) Cellular quantitation of p-S46 TCTP intensity in M-phase following treatment with DMSO, Benzarone (7.5 µM), Bi2536 (10 nM), or Benzarone + Bi2536 for 5.5 hrs (one-way ANOVA with Tukey’s multiple comparison test, * p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.0001). (E) Representative immunofluorescence images showing an S-phase cell stained with RAD51, EdU, and DAPI. Individual RAD51 foci are indicated with red outline. (F) Representative quantitative image-based cytometry plot showing S-phase cell cycle gate as determined by staining intensity of EdU and DAPI. (G) Cellular quantitation of RAD51 foci number in S-phase following treatment with DMSO, Benzarone (7.5 µM), Bi2536 (10 nM), or Benzarone + Bi2536 for 5.5 hrs (one-way ANOVA with Tukey’s multiple comparison test, ** p ≤ 0.01). (H) Quantitation of RAD51 foci intensity in S-phase following treatment with DMSO, Benzarone (7.5 µM), Bi2536 (10 nM), or Benzarone + Bi2536 for 5.5 hrs (one-way ANOVA with Tukey’s multiple comparison test, * p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.0001).

Article Snippet: Cells were incubated with anti-p-S46 TCTP (Cell Signalling 5251) and anti-p-S10 H3 primary antibodies (Cell Signalling 9701) overnight at 4 ° C followed by 3 x 10 minute washes in PBS.

Techniques: Immunofluorescence, Staining, Cytometry, Quantitation Assay, Comparison

Journal: bioRxiv

Article Title: Potent synthetic lethality between PLK1 and EYA-family inhibitors in tumours of the central and peripheral nervous system

doi: 10.1101/2025.01.19.633804

Figure Lengend Snippet: (A) Cellular quantitation of p-S46 TCTP intensity in M-phase following treatment with DMSO, Benzarone (7.5 µM), Bi2536 (10 nM), or Benzarone + Bi2536 for 5.5 hrs (two-tailed t test, **** p ≤ 0.0001). (B) Representative quantitative imaged based cytometry plot showing G2-phase cell cycle gate as determined by staining intensity of EdU and DAPI. (C) Quantitation of RAD51 foci number in G2-phase following treatment with DMSO, Benzarone (7.5 µM), Bi2536 (10 nM), or Benzarone + Bi2536 for 5.5 hrs (one-way ANOVA with Tukey’s multiple comparison test, ** p ≤ 0.01). (D) Quantitation of RAD51 foci intensity in G2-phase following treatment with DMSO, Benzarone (7.5 µM), Bi2536 (10 nM), or Benzarone + Bi2536 for 5.5 hrs (one-way ANOVA with Tukey’s multiple comparison test, * p ≤ 0.05). (E) Representative image of chromosome spread with SCEs indicated with red arrows. (F) Quantitation of SCEs per chromosome following treatment with DMSO, Benzarone (7.5 µM), Bi2536 (10 nM), or Benzarone + Bi2536 for 24 hrs (one-way ANOVA with Tukey’s multiple comparison test, ** p ≤ 0.01, **** p ≤ 0.0001).

Article Snippet: Cells were incubated with anti-p-S46 TCTP (Cell Signalling 5251) and anti-p-S10 H3 primary antibodies (Cell Signalling 9701) overnight at 4 ° C followed by 3 x 10 minute washes in PBS.

Techniques: Quantitation Assay, Two Tailed Test, Cytometry, Staining, Comparison

Journal: The Journal of Biological Chemistry

Article Title: Elimination of Aicardi Goutières Syndrome Protein SAMHD1 Activates Cellular Innate Immunity and Suppresses SARS-CoV-2 Replication

doi: 10.1016/j.jbc.2022.101635

Figure Lengend Snippet: SAMHD1 loss suppresses SARS-CoV-2 and HCoV-OC43 replication in 293T cells. (A) SAMHD1 expression in SAMHD1 WT and KO 293T cells were confirmed via western blot using anti-human SAMHD1 antibody. GAPDH was used as a loading control. Separately, using 96-well plates, SAMHD1 WT (blue) and KO (red) 293T cells were infected with either (B & C) SARS CoV-2 or (D & E) HCoV-OC43 at MOI 0.1 in triplicates. Extracellular (B & D) and intracellular (C & E) RNAs were isolated from collected media and harvested cells, respectively, on Day 2 post-infection for qRT-PCR analyses. The data are presented as means of triplicates, and the standard deviations from the means are represented as error bars.

Article Snippet: Proteins of interest in this study were detected via primary antibodies specific for human SAMHD1 (Abcam # 67820), human STAT1 (Abcam # 234400), human pSTAT1 (Abcam # 109461), human RNaseH2 (Abcam # 83943), SARS-CoV-2 nucleocapsid (ACROBiosystems # NUN-S47) and human GAPDH (Cell Signaling # 2118).

Techniques: Expressing, Western Blot, Infection, Isolation, Quantitative RT-PCR

Journal: The Journal of Biological Chemistry

Article Title: Elimination of Aicardi Goutières Syndrome Protein SAMHD1 Activates Cellular Innate Immunity and Suppresses SARS-CoV-2 Replication

doi: 10.1016/j.jbc.2022.101635

Figure Lengend Snippet: SAMHD1 loss downregulates SARS-CoV-2 and HCoV-OC43 RNA levels in differentiated THP-1 macrophages. (A) SAMHD1 expression in PMA-differentiated SAMHD1 WT and KO THP-1 macrophages were confirmed via western blot using anti-human SAMHD1 antibody. GAPDH was used as a loading control. Separately, using 96-well plates, differentiated SAMHD1 WT and KO THP-1 cells were infected with either (B) SARS CoV-2 or (C) HCoV-OC43 at MOI 0.1 in triplicates, and intracellular RNAs were isolated on Days 2 (blue), 3 (red) and 6 (green) post-infection for qRT-PCR analyses. The data are presented as means of triplicates, and the standard deviations from the means are represented as error bars.

Article Snippet: Proteins of interest in this study were detected via primary antibodies specific for human SAMHD1 (Abcam # 67820), human STAT1 (Abcam # 234400), human pSTAT1 (Abcam # 109461), human RNaseH2 (Abcam # 83943), SARS-CoV-2 nucleocapsid (ACROBiosystems # NUN-S47) and human GAPDH (Cell Signaling # 2118).

Techniques: Expressing, Western Blot, Infection, Isolation, Quantitative RT-PCR

Journal: The Journal of Biological Chemistry

Article Title: Elimination of Aicardi Goutières Syndrome Protein SAMHD1 Activates Cellular Innate Immunity and Suppresses SARS-CoV-2 Replication

doi: 10.1016/j.jbc.2022.101635

Figure Lengend Snippet: Vpx treatment suppresses SARS-CoV-2 replication in primary human monocyte-derived macrophages. (A) SAMHD1 expression in primary human MDMs treated with either VLP Vpx (-) or Vpx (+) were confirmed via western blot using anti-human SAMHD1 antibody. GAPDH was used as the loading control. MDMs were prepared from GM-CSF-mediated 7-day differentiation of human primary monocytes pooled from 5 healthy donors. (B) Separately, using 96-well plates, primary MDMs were treated with VLP Vpx (-) or Vpx (+) for 12 h in triplicates, before the cells were infected with SARS-CoV-2 (MOI 0.1). On Days 2 (blue) and 3 (red) post-infection, intracellular RNAs were isolated from the infected MDMs for SARS-CoV-2 RNA copy numbers quantification via qRT-PCR. The data are presented as means of triplicates, and the standard deviations from the means are represented as error bars.

Article Snippet: Proteins of interest in this study were detected via primary antibodies specific for human SAMHD1 (Abcam # 67820), human STAT1 (Abcam # 234400), human pSTAT1 (Abcam # 109461), human RNaseH2 (Abcam # 83943), SARS-CoV-2 nucleocapsid (ACROBiosystems # NUN-S47) and human GAPDH (Cell Signaling # 2118).

Techniques: Derivative Assay, Expressing, Western Blot, Infection, Isolation, Quantitative RT-PCR

Journal: The Journal of Biological Chemistry

Article Title: Elimination of Aicardi Goutières Syndrome Protein SAMHD1 Activates Cellular Innate Immunity and Suppresses SARS-CoV-2 Replication

doi: 10.1016/j.jbc.2022.101635

Figure Lengend Snippet: Baricitinib treatment overrides the anti-SARS-CoV-2 effects of SAMHD1 KO. (A) STAT1 and pSTAT1 protein expressions in SAMHD1 WT 293T cells following treatment with different concentrations of baricitinib as indicated, were evaluated via western blots using anti-human STAT1 and pSTAT1 antibodies. SARS-CoV-2 nucleocapsid and human GAPDH were presented as infection and loading controls, respectively. The relative STAT1, pSTAT1 and nucleocapsid proteins levels were normalized with GAPDH and the ratios of each protein between respective treatment groups with the mock-infected or mock-treated cells were calculated. (B) SAMHD1 WT (blue) and KO (red) 293T cells were pre-treated with different concentrations of baricitinib as indicated for 1 h, and the cells were infected with SARS-CoV-2 (MOI 0.1). On Day 2 post-infection, extracellular (B) and intracellular (C) RNA in the media and cells, respectively, were isolated for SARS-CoV-2 RNA copy numbers quantification via qRT-PCR. The data are presented as means of triplicates, and the standard deviations from the means are represented as error bars.

Article Snippet: Proteins of interest in this study were detected via primary antibodies specific for human SAMHD1 (Abcam # 67820), human STAT1 (Abcam # 234400), human pSTAT1 (Abcam # 109461), human RNaseH2 (Abcam # 83943), SARS-CoV-2 nucleocapsid (ACROBiosystems # NUN-S47) and human GAPDH (Cell Signaling # 2118).

Techniques: Western Blot, Infection, Isolation, Quantitative RT-PCR

Journal: Oncoscience

Article Title: Non-selective beta blockers inhibit angiosarcoma cell viability and increase progression free- and overall-survival in patients diagnosed with metastatic angiosarcoma

doi: 10.18632/oncoscience.413

Figure Lengend Snippet: (A) Angiosarcoma cell lines were treated with increasing concentrations of the non-selective beta blocker propranolol (0 to 200 µM). Cell viability was measured after 48 hours. (B) SVR cells were treated with a DMSO control or 25 µM propranolol for 24 hours. Protein lysates were collected and subjected to immunoblotting to detect levels of key cell cycle regulators. β-Actin was used as a loading control. (C) SVR cells were treated with 25 µM propranolol for 1 hour. Protein lysates were subjected to duplicate antibody arrays that simultaneously detected the relative phosphorylation of 24 kinases. The normalized levels of the phosphorylated proteins across the treatments are visualized via a heat map (red=increased phosphorylation, green=decreased phosphorylation) . (D) Angiosarcoma xenografts were injected with an isotonic saline control or propranolol (15 mg/kg). Cell lysates were collected at 15 minutes post-treatment and subjected to immunoblotting for key mitogenic and survival signaling regulators. β-actin was used as a control to ensure equal loading of the samples. (E) Tumor sections from an angiosarcoma patient collected at diagnostic biopsy (before propranolol administration, pre-treatment) and at one week after administration of propranolol (120 mg/day) were analyzed by IHC to determine the levels of p-p44/42 (Thr202/Tyr204), p-SAPK/JNK (Thr183/Tyr185), p-p38 (T180/Y182), and p-p53 (S46). Brown coloration indicates positive antigen staining.

Article Snippet: The following antibodies were used: anti-p-p44/42 (Thr202/Tyr204) (Cell Signaling #4370), anti-p-SAPK/JNK (Thr183/Tyr185) (Cell Signaling #4668), anti-p-p38 (T180/Y182) (Abcam #4822), and anti-phospho-p53 (S46) (Cell Signaling #2521).

Techniques: Control, Western Blot, Phospho-proteomics, Injection, Saline, Diagnostic Assay, Staining

Journal: medRxiv

Article Title: Novel SARS-CoV-2 specific antibody and neutralization assays reveal wide range of humoral immune response during COVID-19

doi: 10.1101/2020.07.07.20148106

Figure Lengend Snippet: a . Schematic illustration of Spike protein expression on the cell surface and soluble ACE2-Fc staining followed by an anti-Fc antibody staining. b . 293 cells transfected with Spike protein with or without endoplasmic reticulum retention signal (ERRS) or with VSV-G as a negative control. The cells were stained with ACE2-Fc and anti-Fc-APC secondary antibody, flow cytometry data overlays are shown. c . Schematic representation of Spike protein pseudovirus generation and subsequent infection of ACE2-expressing host cells. A lentivector plasmid and a Spike protein over-expressing envelope plasmid are used to co-transfect 293 cells to generate Spike pseudovirus that in turn infect engineered cells over-expressing wild type ACE2 or ACE2-mKO2 fusion. d . Infection of wild type 293 cells with either bald lentiviruses generated without envelope plasmid or Spike protein pseudovirus. e . Infection of 293-ACE2 cells with bald and Spike lentiviruses. GFP and mKO2 markers are used to determine ACE2 over-expressing cells in ACE2-IRES-GFP and ACE2-mKO2, respectively. f . The titrations of SARS-CoV-2 and SARS-CoV Spike protein pseudoviruses encoding RFP. ACE2-IRES-GFP expressing 293 cells were incubated with 3-fold serial dilutions of virus supernatant, stored for several hours at 4°C or serially frozen and thawed for 1, 2 and 3 cycles, and analyzed for RFP expression by flow cytometry on day 3 post-infection. Percent infection is % RFP+ cells after gating on GFP+ cells (i.e. ACE2+). Titration experiments were replicated twice except for the “1 freeze/thaw cycle” for which titrations were replicated 4 times. Error bars represent 1 standard deviation of mean values.

Article Snippet: Three-fold serially diluted monoclonal antibodies including anti-SARS-CoV-2 Neutralizing human IgG1 Antibody from Acro Biosystems, NAb#1 , GenScript clone ID 6D11F2, NAb#2 ( ) and GenScript clone ID 10G6H5, NAb#3 , Invitrogen clone ID MA5-35939 Nab#4 , recombinant human ACE2-Fc (Acro Biosystems, sACE2#1 and GenScript, sACE2#2 ( ) or plasma from COVID-19 convalescent individuals and healthy donors were incubated with RFP-encoding SARS-CoV-2 or GFP-encoding SARS-CoV pseudotyped virus with 0.2 multiplicity of infection (MOI) for 1 hour at 37 ° C degrees.

Techniques: Expressing, Staining, Transfection, Negative Control, Flow Cytometry, Infection, Plasmid Preparation, Generated, Incubation, Titration, Standard Deviation

Journal: medRxiv

Article Title: Novel SARS-CoV-2 specific antibody and neutralization assays reveal wide range of humoral immune response during COVID-19

doi: 10.1101/2020.07.07.20148106

Figure Lengend Snippet: a . Wild type or ACE2-IRES-GFP over-expressing 293 cells were stained with SARS-CoV-2 S1 protein fused to mouse Fc, and anti-mouse Fc secondary antibody. B . ACE2 expression, detected as in a , in wild type and ACE2 overexpressing 293 cells compared in an overlay of flow data.

Article Snippet: Three-fold serially diluted monoclonal antibodies including anti-SARS-CoV-2 Neutralizing human IgG1 Antibody from Acro Biosystems, NAb#1 , GenScript clone ID 6D11F2, NAb#2 ( ) and GenScript clone ID 10G6H5, NAb#3 , Invitrogen clone ID MA5-35939 Nab#4 , recombinant human ACE2-Fc (Acro Biosystems, sACE2#1 and GenScript, sACE2#2 ( ) or plasma from COVID-19 convalescent individuals and healthy donors were incubated with RFP-encoding SARS-CoV-2 or GFP-encoding SARS-CoV pseudotyped virus with 0.2 multiplicity of infection (MOI) for 1 hour at 37 ° C degrees.

Techniques: Expressing, Staining

Journal: medRxiv

Article Title: Novel SARS-CoV-2 specific antibody and neutralization assays reveal wide range of humoral immune response during COVID-19

doi: 10.1101/2020.07.07.20148106

Figure Lengend Snippet: a . Illustration of Spike protein pseudovirus blocked by soluble ACE2 or neutralizing antibodies. B . SARS-CoV-2 and SARS-CoV pseudovirus neutralization with soluble ACE2. SARS-CoV-2 RFP and SARS-CoV GFP pseudoviruses were pre-incubated with soluble ACE2 for 1 hour and added to 293 cells expressing ACE2-IRES-GFP or ACE2-mKO2 fusion, respectively. c . Neutralization of SARS-CoV-2 and SARS-CoV with S-RBD specific antibodies and soluble ACE2 (sACE2). Viruses were pre-incubated with antibodies (NAb#1 and SARS-CoV-2 S-RBD non-Nab) or soluble ACE2 (sACE2) proteins for 1 hour at the concentrations shown and subsequently added to target cells. Expression of RFP was determined at day 3 post-infection. Infection percentages were normalized to negative controls which are the infection conditions with no blocking agent. d . Neutralization of SARS-CoV-2 pseudoviruses using 4 different S-RBD NAbs and 2 different soluble ACE2 proteins. NAb #1 and #4 were human antibodies whereas NAb #2 and #3 were mouse. Graphs in figures c and d represent 3 replicates of the experiments. Error bars indicate one standard deviation of mean values.

Article Snippet: Three-fold serially diluted monoclonal antibodies including anti-SARS-CoV-2 Neutralizing human IgG1 Antibody from Acro Biosystems, NAb#1 , GenScript clone ID 6D11F2, NAb#2 ( ) and GenScript clone ID 10G6H5, NAb#3 , Invitrogen clone ID MA5-35939 Nab#4 , recombinant human ACE2-Fc (Acro Biosystems, sACE2#1 and GenScript, sACE2#2 ( ) or plasma from COVID-19 convalescent individuals and healthy donors were incubated with RFP-encoding SARS-CoV-2 or GFP-encoding SARS-CoV pseudotyped virus with 0.2 multiplicity of infection (MOI) for 1 hour at 37 ° C degrees.

Techniques: Neutralization, Incubation, Expressing, Infection, Blocking Assay, Standard Deviation

Journal: medRxiv

Article Title: Novel SARS-CoV-2 specific antibody and neutralization assays reveal wide range of humoral immune response during COVID-19

doi: 10.1101/2020.07.07.20148106

Figure Lengend Snippet: a . Normalized percent infection levels of SARS-CoV-2 and SARS-CoV pseudoviruses in neutralization assay using soluble ACE2 at 1 μ/mL, 3 μ/mL and 10 μ/mL concentrations. Significance was determined using two-tailed Mann-Whitney U test.

Article Snippet: Three-fold serially diluted monoclonal antibodies including anti-SARS-CoV-2 Neutralizing human IgG1 Antibody from Acro Biosystems, NAb#1 , GenScript clone ID 6D11F2, NAb#2 ( ) and GenScript clone ID 10G6H5, NAb#3 , Invitrogen clone ID MA5-35939 Nab#4 , recombinant human ACE2-Fc (Acro Biosystems, sACE2#1 and GenScript, sACE2#2 ( ) or plasma from COVID-19 convalescent individuals and healthy donors were incubated with RFP-encoding SARS-CoV-2 or GFP-encoding SARS-CoV pseudotyped virus with 0.2 multiplicity of infection (MOI) for 1 hour at 37 ° C degrees.

Techniques: Infection, Neutralization, Two Tailed Test, MANN-WHITNEY

Journal: medRxiv

Article Title: Novel SARS-CoV-2 specific antibody and neutralization assays reveal wide range of humoral immune response during COVID-19

doi: 10.1101/2020.07.07.20148106

Figure Lengend Snippet: a . Neutralization assay with S-RBD specific NAb, healthy control plasma, and a COVID-19 patient plasma. 3-fold serial dilutions of NAb from 10 μ/ml to 1 ng/ml or the plasma from 1:10 to 1:10,000 were pre-incubated with Spike protein pseudovirus and added to 293-ACE2 cells. GFP expression was analyzed by flow cytometry 3 days post infection. b . SARS-CoV-2 neutralization titers (NT50) of COVID-19 plasma grouped as outpatient, hospitalized, ICU or deceased and convalescent plasma donor groups. c . NT50 of COVID-19 patient and plasma donor groups subdivided into males and females. d . Comparison of NT50 of COVID-19 plasma for SARS-CoV-2 and SARS-CoV neutralization. SARS-CoV-2 or SARS-CoV pseudoviruses were pre-incubated with COVID-19 plasma from all severity groups (n=104), 293-ACE2 cells were infected and RFP expression was determined at day 3 using flow cytometry. e . Graph of SARS-CoV-2 NT50 values from hospitalized subjects plotted against SARS-CoV. Two-tailed Mann-Whitney U test was used to determine the statistical significances in figures b, c and d and two-tailed Spearman’s was used for figure e . Horizontal bars in b, c and d indicate mean values.

Article Snippet: Three-fold serially diluted monoclonal antibodies including anti-SARS-CoV-2 Neutralizing human IgG1 Antibody from Acro Biosystems, NAb#1 , GenScript clone ID 6D11F2, NAb#2 ( ) and GenScript clone ID 10G6H5, NAb#3 , Invitrogen clone ID MA5-35939 Nab#4 , recombinant human ACE2-Fc (Acro Biosystems, sACE2#1 and GenScript, sACE2#2 ( ) or plasma from COVID-19 convalescent individuals and healthy donors were incubated with RFP-encoding SARS-CoV-2 or GFP-encoding SARS-CoV pseudotyped virus with 0.2 multiplicity of infection (MOI) for 1 hour at 37 ° C degrees.

Techniques: Neutralization, Incubation, Expressing, Flow Cytometry, Infection, Two Tailed Test, MANN-WHITNEY

Journal: medRxiv

Article Title: Novel SARS-CoV-2 specific antibody and neutralization assays reveal wide range of humoral immune response during COVID-19

doi: 10.1101/2020.07.07.20148106

Figure Lengend Snippet: a . Correlation of SARS-CoV-2 NT50 with SARS-CoV when all severity groups were combined, or only outpatient and plasma donor subjects were combined. b . SARS-CoV neutralization titers (NT50) of COVID-19 plasma grouped as outpatient, hospitalized, ICU or deceased and convalescent plasma donor groups. Two-tailed Spearman’s was used for statistical analysis.

Article Snippet: Three-fold serially diluted monoclonal antibodies including anti-SARS-CoV-2 Neutralizing human IgG1 Antibody from Acro Biosystems, NAb#1 , GenScript clone ID 6D11F2, NAb#2 ( ) and GenScript clone ID 10G6H5, NAb#3 , Invitrogen clone ID MA5-35939 Nab#4 , recombinant human ACE2-Fc (Acro Biosystems, sACE2#1 and GenScript, sACE2#2 ( ) or plasma from COVID-19 convalescent individuals and healthy donors were incubated with RFP-encoding SARS-CoV-2 or GFP-encoding SARS-CoV pseudotyped virus with 0.2 multiplicity of infection (MOI) for 1 hour at 37 ° C degrees.

Techniques: Neutralization, Two Tailed Test