Journal:

Article Title: Tat Protein of Human Immunodeficiency Virus Type 1 Induces Interleukin-10 in Human Peripheral Blood Monocytes: Implication of Protein Kinase C-Dependent Pathway

doi:

Figure Lengend Snippet: Production of IL-10 by monocytes treated with Tat. (A) Monocytes (106) were incubated in the absence or presence of Tat (1, 10, 100, or 500 nM) or LPS (100 ng/ml) for 24 h. (B) Monocytes were identically treated with LPS (100 ng/ml) or Tat (10 or 100 nM) but for 24, 48, or 72 h. (C) Specificity of Tat. Monocytes (106) were incubated in the absence or presence of native Tat (10 or 100 nM), oxidized Tat (1 h at 25°C in PBS plus 3% H2O2 at 10 and 100 nM), or LPS (100 ng/ml) for 24 h. (D) PBMC were depleted of monocytes by three successive adherence steps (1°, 2°, and 3°) in 24-well plates. After each adherence step, cells remaining in suspension (106) were incubated in the absence or presence of LPS at 100 ng/ml or Tat at 1, 10, or 100 nM. Culture supernatants were recovered, and the presence of IL-10 was determined by ELISA. For A, B, and D, the values are the means ± standard deviation (SD) of three experiments with cells from one donor. Similar results were obtained with cells from three different donors. For C, the values are from results obtained with three different donors. Ctr, control.

Article Snippet: Cytokines were quantified from a standard curve generated by using various concentrations of recombinant human IL-10 (R & D Systems).

Techniques: Incubation, Suspension, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal:

Article Title: Tat Protein of Human Immunodeficiency Virus Type 1 Induces Interleukin-10 in Human Peripheral Blood Monocytes: Implication of Protein Kinase C-Dependent Pathway

doi:

Figure Lengend Snippet: Specificity of Tat-induced IL-10 production and mapping of the active domain. (A) Monocytes (106) were incubated with wild-type GST-Tat 1-101 (1 or 10 nM) or with recombinant mutants GST-Tat 1-72, GST-Tat 1-55, GST-Tat 1-45, GST-Tat 20-72, or GST-Tat 30-72 (1 or 10 nM) or with GST as a negative control for 24 h. Culture supernatants were recovered, and the presence of IL-10 was determined by ELISA. The values are the means ± SD of three experiments with cells from one donor. Similar results were obtained with cells from three different donors. (B) Tat (10 nM) was incubated for 1 h or not with MAbs directed against Tat epitopes 1 to 15, 46 to 60, or 74 to 86 (2.5 or 0.025 μg/ml) or the mixture of the three MAbs. After 24 h, culture supernatants were recovered, and the presence of IL-10 was determined by ELISA. As control, an MAb directed against gp140 of simian immunodeficiency virus was used as a control in the same conditions. Results represent the ratio of production of IL-10 by monocytes stimulated by Tat (10 nM) incubated with MAb and production of IL-10 produced after stimulation by Tat (10 nM) alone (P/P0). On the right, the percent inhibition of IL-10 production induced by Tat (10 nM) is represented. Mouse MAbs were obtained from the Agence Nationale de la Recherche sur le SIDA (Paris, France).

Article Snippet: Cytokines were quantified from a standard curve generated by using various concentrations of recombinant human IL-10 (R & D Systems).

Techniques: Incubation, Recombinant, Negative Control, Enzyme-linked Immunosorbent Assay, Virus, Produced, Inhibition

Journal:

Article Title: Tat Protein of Human Immunodeficiency Virus Type 1 Induces Interleukin-10 in Human Peripheral Blood Monocytes: Implication of Protein Kinase C-Dependent Pathway

doi:

Figure Lengend Snippet: Production of IL-10 by monocytes stimulated by Tat immobilized in wells. (A) Tat was immobilized in wells by incubation for 2 h at 37°C. After two washes to eliminate nonfixed Tat, monocytes (106) were added and cultured for 24 h with different concentrations of Tat (5, 50, or 500 nM). Culture supernatants were recovered, and the presence of IL-10 was determined by ELISA. (B) HeLa P4 cells were incubated with soluble (solid bars) or immobilized (shaded bars) Tat, and a Tat-dependent-transactivating test was done as described in Materials and Methods. The values are the means ± SD of three experiments. For A, similar results were obtained with cells from two different donors.

Article Snippet: Cytokines were quantified from a standard curve generated by using various concentrations of recombinant human IL-10 (R & D Systems).

Techniques: Incubation, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal:

Article Title: Tat Protein of Human Immunodeficiency Virus Type 1 Induces Interleukin-10 in Human Peripheral Blood Monocytes: Implication of Protein Kinase C-Dependent Pathway

doi:

Figure Lengend Snippet: Effect of PLC inhibitor U73122 on Tat-induced production of IL-10. Monocytes (106) were treated or not with U73122 (2.5 or 7.5 μM) for 30 min. Tat (10 nM) was then added for 24 h. Culture supernatants were recovered, and the presence of IL-10 was determined by ELISA. The values are the means ± SD of three experiments. Similar results were obtained with cells from three different donors.

Article Snippet: Cytokines were quantified from a standard curve generated by using various concentrations of recombinant human IL-10 (R & D Systems).

Techniques: Enzyme-linked Immunosorbent Assay

Journal:

Article Title: Tat Protein of Human Immunodeficiency Virus Type 1 Induces Interleukin-10 in Human Peripheral Blood Monocytes: Implication of Protein Kinase C-Dependent Pathway

doi:

Figure Lengend Snippet: Involvement of the calcium pathway. (A) Effect of calcineurin inhibitor cyclosporin A (Cs A) and of the chelator of intracellular calcium BAPTA/AM on the Tat-induced production of IL-10. Monocytes were treated or not for 30 min as indicated and then treated or not with Tat at 10 nM. Culture supernatants were recovered, and the presence of IL-10 was determined by ELISA. (B) Positive control of inhibition by cyclosporin A and BAPTA/AM on the ionomycin-induced production of TNF-α. Monocytes were treated or not for 30 min as indicated and then treated or not with ionomycin (1 μM) for 24 h. Culture supernatants were then collected, and the presence of TNF-α was determined by ELISA. These results are representative of three independent experiments done on cells from two donors. (C and D) Variations in intracellular calcium concentrations determined by microspectrofluorimetry using fluo-3AM as the probe. Cells were incubated for 30 min in the presence of fluo-3 (5 μM) and observed microscopically after two washes. Monocytes were stimulated by 100 nM Tat (C) or 1 μM ionomycin (D). The curves are the results obtained from the means for 11 different cells from a single donor. Similar results were obtained with cells from two different donors.

Article Snippet: Cytokines were quantified from a standard curve generated by using various concentrations of recombinant human IL-10 (R & D Systems).

Techniques: Enzyme-linked Immunosorbent Assay, Positive Control, Inhibition, Incubation

Journal:

Article Title: Tat Protein of Human Immunodeficiency Virus Type 1 Induces Interleukin-10 in Human Peripheral Blood Monocytes: Implication of Protein Kinase C-Dependent Pathway

doi:

Figure Lengend Snippet: Involvement of PKC in Tat-induced production of IL-10. (A) Monocytes (106) were treated or not with the PKC inhibitor RO318220 (RO) at the concentrations indicated (2.5 and 5 μM) or by the PKA inhibitor H89 (50 and 100 μM) for 30 min. Tat was then added for 24 h. These results are representative of three independent experiments done on cells from three donors. (B) Control of the specificity of the kinase inhibitors H89 and RO318220. Monocytes (106) were treated or not with the PKC inhibitor RO318220 (2.5 and 5 μM) or by the PKA inhibitor H89 (50 and 100 μM) for 30 min. Rolipram was then added for 24 h. Culture supernatants were then collected, and the presence of IL-10 was determined by ELISA. (C and D) Monocytes were treated or not with PMA (50 or 100 ng/ml), a PKC activator, for 24 h (C) or 48 h (D), and Tat (10 nM) was then added. After 24 h, culture supernatants were recovered, and the presence of IL-10 was determined by ELISA. The values in C are the means ± SD of three experiments. The results in C and D are representative of three independent experiments done on cells from three donors.

Article Snippet: Cytokines were quantified from a standard curve generated by using various concentrations of recombinant human IL-10 (R & D Systems).

Techniques: Enzyme-linked Immunosorbent Assay

Journal:

Article Title: Tat Protein of Human Immunodeficiency Virus Type 1 Induces Interleukin-10 in Human Peripheral Blood Monocytes: Implication of Protein Kinase C-Dependent Pathway

doi:

Figure Lengend Snippet: Role of NF-κB in Tat-induced IL-10 production. (A) Effect of the serine protease inhibitor TLCK (50 and 100 μM) on NF-κB nuclear translocation induced by Tat (10 nM) (lanes 7 and 8). Lanes 3 and 4 correspond to the activation of NF-κB with Tat (10 and 1 nM, respectively). Ctr, control. (B) Effect of treatment with TLCK (TL) at 50 and 100 μM on IL-10 production by monocytes (106) treated with 10 nM Tat (T). The values are the means ± SD of three experiments. Similar results were obtained with cells from three different donors.

Article Snippet: Cytokines were quantified from a standard curve generated by using various concentrations of recombinant human IL-10 (R & D Systems).

Techniques: Protease Inhibitor, Translocation Assay, Activation Assay

Journal:

Article Title: Tat Protein of Human Immunodeficiency Virus Type 1 Induces Interleukin-10 in Human Peripheral Blood Monocytes: Implication of Protein Kinase C-Dependent Pathway

doi:

Figure Lengend Snippet: Involvement of MAP kinases ERK1/2 in the Tat-induced production of IL-10. (A) Western blot analysis of the activation of MAP kinases ERK1/2 by Tat. Cytoplasmic protein extracts were prepared from monocytes (2 × 106) treated with PMA at 50 ng/ml (lanes 2 and 6) or 10 nM (lanes 3 and 7) or 100 nM (lanes 4 and 8) Tat for 15 or 30 min. Visualization was done with an antibody recognizing total ERK1/2 or only phosphorylated ERK1/2 (pERK1/2). These results are representative of two independent experiments done on cells from two different donors. Ctr, control. (B) Effect of PD 98 059 (PD) at 10 or 100 μM, an inhibitor of MAP kinases ERK1/2, on monocytes (106) treated with 10 nM Tat (T). Similar results were obtained with cells from three different donors.

Article Snippet: Cytokines were quantified from a standard curve generated by using various concentrations of recombinant human IL-10 (R & D Systems).

Techniques: Western Blot, Activation Assay

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Personalized cardiovascular risk assessment in Rheumatoid Arthritis patients using circulating molecular profiles and their modulation by TNFi, IL6Ri, and JAKinibs.

doi: 10.1016/j.biopha.2024.116357

Figure Lengend Snippet: Fig. 5. Serum from RA patients with high CVD risk induced the upregulation of CVD-related markers in endothelial cells (ECs) and monocytes, which was prevented by biological and targeted synthetic DMARDs.- (A and C) Heat map showing the changes promoted in CV- related proteins on endothelial cells (A) and monocytes (C) treated in vitro with serum from RA patients belonging to cluster 3 (showing the highest inflammatory profile) in comparison with serum from HD. Levels of inflammatory proteins are expressed as NPX (normalized protein expression, arbitrary units on a Log 2 scale), and have undergone a clustering analysis to aid interpretation. Proteins showing significant alterations (FDR-adjusted p value <0.05) are detached in bold. (B and D) Predicted and validated protein-protein interactions among proteins potentially modulated by RA-C3 serum, using the STRING platform. Protein networks showing the relationship between differen tially expressed proteins are displayed. Below are displayed tables containing functional enrichment analysis of biological processes by using the Gene ontology platform. (E and G) Changes in cardiovascular mediators’ levels in cell lysates of endothelial cells and monocytes cultured with HD or RA-C3 serum, either, in the presence or in the absence of TNFi, IL6Ri or JAKinibs. *p<0.05, **p<0.01, ***p<0.001. (F and H) Venn diagram showing the common and differential proteins modulated in endothelial cells and monocytes by TNFi, IL6Ri or JAKinibs.

Article Snippet: Neutrophils and monocytes purified from HD and primary human umbilical vein endothelial cells (HUVECs) were subjected to a 12-h treatment (neutrophils) or 24-h treatment (monocytes and HUVECs)at 37 ◦C. with the serum from HD, the serum of RA patients that had suffered previous CV events (RA-CVD), or the serum from patients belonging to the cluster 3 (RA-C3), either in the presence or absence of TNFi (Etanercept, - European Pharmacopoeia reference standard, Strasbourg, France- 10 ug/mL), IL6Ri (Sarilumab, -Selleckchem, Planegg, Germany- 10 ug/mL,) or JAKinibs (Baricitinib, -Tocris Bioscience, Bristol, UK- 10 ug/mL).

Techniques: In Vitro, Comparison, Expressing, Protein-Protein interactions, Functional Assay, Cell Culture

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Personalized cardiovascular risk assessment in Rheumatoid Arthritis patients using circulating molecular profiles and their modulation by TNFi, IL6Ri, and JAKinibs.

doi: 10.1016/j.biopha.2024.116357

Figure Lengend Snippet: Fig. 4. Changes in clinical and molecular profiles of rheumatoid arthritis patients treated with b-DMARDs or ts-DMARDs. (A) RA patients’ (n = 83) changes in clinical features at 6 months of TNFi, IL6Ri and JAKinibs therapies, including Disease Activity Score (DAS28), Clinical Disease Activity Index (CDAI), and Simple Disease Activity index (SDAI). At the initial timepoint (t0), each data point corresponds to an individual patient, with a subsequent connection established to the respective measurement obtained at the 6-month interval. (B) Heat map showing differential levels of circulating inflammatory proteins in plasma of patients with RA after 6 months of TNFi, IL6Ri and JAKinibs therapies (Δ T6-T0). Levels of inflammatory proteins are expressed as log 2,and have undergone a clustering analysis to aid interpretation. (C) RA patients’ changes in NETosis bioproducts (elastase and nucleosomes) and lipoperoxides after 6 months of TNFi, IL6Ri and JAKinibs therapies. * p ≤0.05, ** p ≤0.01, *** p ≤0.001, **** p ≤0.0001.

Article Snippet: Neutrophils and monocytes purified from HD and primary human umbilical vein endothelial cells (HUVECs) were subjected to a 12-h treatment (neutrophils) or 24-h treatment (monocytes and HUVECs)at 37 ◦C. with the serum from HD, the serum of RA patients that had suffered previous CV events (RA-CVD), or the serum from patients belonging to the cluster 3 (RA-C3), either in the presence or absence of TNFi (Etanercept, - European Pharmacopoeia reference standard, Strasbourg, France- 10 ug/mL), IL6Ri (Sarilumab, -Selleckchem, Planegg, Germany- 10 ug/mL,) or JAKinibs (Baricitinib, -Tocris Bioscience, Bristol, UK- 10 ug/mL).

Techniques: Activity Assay, Clinical Proteomics

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Personalized cardiovascular risk assessment in Rheumatoid Arthritis patients using circulating molecular profiles and their modulation by TNFi, IL6Ri, and JAKinibs.

doi: 10.1016/j.biopha.2024.116357

Figure Lengend Snippet: Fig. 6. Serum from RA patients with high CVD risk induced NETosis and neutrophils activation, which was prevented by b-DMARDs and ts-DMARDs.- (A- B) Bar graphs showing the changes promoted in gene expression levels of several activation markers on neutrophils treated in vitro with serum from RA patients belonging to cluster 3 (showing the highest inflammatory profile) either in the presence or in the absence of TNFi, IL6Ri or JAKinibs inhibitors. All experiments were compared with neutrophils treated with HD serum, set at 100% in each panel. (Significance at p<0.05, a, vs neutrophils treated with HD serum; b, vs neutrophils treated with RA-C3 serum). (C) Representative micrographs of neutrophil extracellular traps (NETs) from HD neutrophils treated in vitro with serum from RA patients belonging to cluster 3 (showing the highest inflammatory profile) either in the presence or in the absence of TNFi, IL6Ri or JAKinibs inhibitors. NETs were visualized by using a Nikon Eclipse-Ti-S fluorescence microscope 20x objective. Scale bar 10 micrometers. (D) Concentration of cell-free nucleosomes and cell-free elastase in supernatants from cell cultures of HD neutrophils performed as above described (Significance at p<0.05, a, vs neutrophils treated with HD serum; b, vs neutrophils treated with RA-C3 serum).

Article Snippet: Neutrophils and monocytes purified from HD and primary human umbilical vein endothelial cells (HUVECs) were subjected to a 12-h treatment (neutrophils) or 24-h treatment (monocytes and HUVECs)at 37 ◦C. with the serum from HD, the serum of RA patients that had suffered previous CV events (RA-CVD), or the serum from patients belonging to the cluster 3 (RA-C3), either in the presence or absence of TNFi (Etanercept, - European Pharmacopoeia reference standard, Strasbourg, France- 10 ug/mL), IL6Ri (Sarilumab, -Selleckchem, Planegg, Germany- 10 ug/mL,) or JAKinibs (Baricitinib, -Tocris Bioscience, Bristol, UK- 10 ug/mL).

Techniques: Activation Assay, Gene Expression, In Vitro, Fluorescence, Microscopy, Concentration Assay

Journal: Cellular Microbiology

Article Title: Enteropathogenic Escherichia coli (EPEC) inactivate innate immune responses prior to compromising epithelial barrier function

doi: 10.1111/j.1462-5822.2007.00923.x

Figure Lengend Snippet: EPEC inhibits phosphorylation-associated activation of IKK, ERK, p38 and PI3-K pathways. Differentiated Caco-2 cells were (B–E) left untreated or (A) pretreated, or not, with the proteasome inhibitor MG132 (50 μM final concentration) for 1 h prior to 3 h apical inoculations with pre-activated eae (Intimin-deficient) or espA (effector delivery-defective) mutants. Infections were stopped by 1 h treatment with bactericidal levels of gentamicin (100 μg ml −1 final concentration) prior to adding TNFα (10 ng ml −1 final concentration) for 30 min to the basolateral compartment of uninfected (NI) or infected cells followed by the isolation of Triton X-100 (1% v/v) soluble extracts as described in Experimental procedures . Equal protein loadings were resolved by SDS-PAGE (12%), transferred to nitrocellulose and probed with antibodies specific to (A) IκB or its phosphorylation on serine 32, P-IκB, (B) p65 isoform of NF-κB or its phosphorylation on serine 536, P-p65, (C) p38 Map kinase or its phosphorylation on threonine180/tyrosine182, P-p38, (D) ERK1/2 MAP kinase or phosphorylation on threonine202/tyrosine204 (P-Erk) and MEK MAP kinase kinase or phosphorylation on serine 221 (P-MEK) respectively, and (E) Akt or its phosphorylation on serine 473 (P-Akt). Linked graphs show relative amplitude of phosphorylated bands (arbitrary units) compared with those of the corresponding uninfected untreated cells obtained by densitometry examination of data from three independent experiments with standard deviation indicated by error bars. Statistical significance evaluated using Student's t -test comparing data with that of the corresponding uninfected untreated cells. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Blots were incubated overnight at 4°C with gentle rocking in a 5% bovine serum albumin/PBS solution containing antibodies (from Santa Cruz) against IκBα, p38, NF-κB (p65 isoform) or (from Cell Signalling) phospho-NF-κB (p65 isoform; Ser 536), phospho-IκBα (Ser 32), phospho-p38 (Thr180/Tyr182), ERK, phospho-ERK (Thr202/Tyr204), MEK and phospho-MEK (Ser221).

Techniques: Activation Assay, Concentration Assay, Infection, Isolation, SDS Page, Standard Deviation

Journal: bioRxiv

Article Title: Bni5 tethers myosin-II to septins to enhance retrograde actin flow and the robustness of cytokinesis

doi: 10.1101/2023.11.07.566094

Figure Lengend Snippet: (A) Myo1 accumulation at the budding site depends on the CC1 of Bni5. Montages of GFP-tagged Bni5 fragments with respect to Myo1-mScarlet were created from selected frames of time-lapse series taken with a 2.5-min interval. Strains used are as follows: YEF11029 ( bni5Δ MYO1-mScarlet [ GFP-BNI5(FL) ], YEF11027 ( bni5Δ MYO1-mScarlet [ GFP-bni5(41-448) ], and YEF11033 ( bni5Δ MYO1-mScarlet [ GFP ]). See also Figure S4 . (B) Bud neck localization of Myo1 in bni5(ΔCC1) cells. Myo1 localization was scored from the imaging data in (A). (C) Bni5(CC1) localization at the division site during cytokinesis. Montages of GFP-Bni5(CC1) with respect to Myo1-mScarlet were created from time-lapse series taken with a 2.5-min interval. Strain used is YEF11058 ( bni5Δ MYO1-mScarlet [ GFP-bni5(1-40) ]). (D) Distinct patterns of bud neck localization of Bni5 FL vs its CC1 fragment. Localization of GFP-Bni5(FL) and GFP-Bni5(CC1) at the bud neck during anaphase and cytokinesis was scored from the imaging data in (C). See also Figure S4 . (E) The bud neck localization of Bni5(CC1) depends on Myo1. Montages of GFP-Bni5(CC1) with respect to Cdc3-mCherry in WT and myo1Δ cells were created from time-lapse series taken with a 2.5-min interval. Strains used are as follows: YEF11038 ( bni5Δ CDC3-mCherry [ GFP-bni5(1-40) ] and YEF11114 ( myo1Δ CDC3-mCherry [ GFP-bni5(1-40) ]). (F) Quantitative analysis of Bni5(CC1) localization at the bud neck in WT and myo1Δ cells. The imaging data in (E) were used for this analysis. (G) Diagram of putative and demonstrated interactions among Bni5, Myo1, and Iqg1. Minimal targeting domain 1 (mTD1) and targeting domain 2 (TD2) as the binding sites for Bni5 and Iqg1, respectively, were determined in . Motor, IQ, and CC represent the motor domain, IQ motifs, and coiled-coil domain, respectively. (H) Time-lapse analysis of Bni5(CC1) localization at the bud neck in myo1-mTD1Δ cells. Montages were created from time-lapse series taken with a 2.5-min interval. Strain used is YEF11757 ( myo1-mTD1Δ CDC3-mCherry [ GFP-bni5(1-40) ]). (I) Quantitative analysis of Bni5(CC1) localization at the bud neck in myo1-mTD1Δ cells. The imaging data in (H) were used for this analysis. See also Figure S4 . (J) Forced tethering of Bni5(CC1) to the septin hourglass restores Myo1 localization at the bud neck. Cells in the exponential growth phase were subjected to imaging. Strains used are as follows: YEF11212 ( CDC11-GFP MYO1-mScarlet bni5(1-40)-GBP ), YEF11211 ( CDC11-GFP MYO1-mScarlet bni5Δ::GBP ), YEF11088 ( CDC11-GFP MYO1-mScarlet ), and YEF11248 ( CDC11-GFP MYO1-mScarlet bni5Δ ). (K) Quantitative analysis of the Bni5(CC1)-restored Myo1 at the bud neck. Myo1-mScarlet intensity at the bud neck in cells with small- and medium-bud (S/G2 cells) was scored from the imaging data in (J). Magenta circles, blue bars, and gray error bars represent all data points, mean values, and SD values, respectively. See also Figure S4 . (L) Bni5 binds to the mTD1 of Myo1 via CC1. Top left: SDS-PAGE stained with Coomassie Blue depicting amounts of each indicated purified protein used as the input for the in vitro binding assay. Bottom left: In vitro binding assay results (Coomassie Blue stained gel) for the indicated GST-tagged proteins bound to glutathione resin and their ability to pull down MBP-mTD1. Green boxes represent GST and GST-tagged Bni5 fragments as described on the top right. Bottom right: immunoblotted membrane with antibody against MBP; kDa = kiloDalton. This experiment was repeated three times, and a representative immunoblot is shown.

Article Snippet: Protein concentrations were determined by standard curve intensity measurements from Coomassie blue-stained bovine serum albumin (A7617, Sigma Aldrich, St. Louis, MO, USA) of known concentrations.

Techniques: Imaging, Binding Assay, SDS Page, Staining, Purification, In Vitro, Membrane, Western Blot

Journal: bioRxiv

Article Title: Bni5 tethers myosin-II to septins to enhance retrograde actin flow and the robustness of cytokinesis

doi: 10.1101/2023.11.07.566094

Figure Lengend Snippet: (A) Summary of colocalization between different Bni5 fragments and septins. Selected and modified data from the results of are presented. Alleles in green and black indicate colocalization and non-colocalization with septins (Cdc3-mCherry), respectively. Bold lines and dashed lines indicate the presence and absence of the region of Bni5 in relevant alleles. An asterisk represents variants of Bni5. Strains used are as follows: YEF11053 ( bni5Δ CDC3-mCherry [ GFP-BNI5(FL) ]), YEF11038 ( bni5Δ CDC3-mCherry [ GFP-bni5(1-40) ]), YEF11521 ( bni5Δ CDC3-mCherry [ GFP-bni5(41-305) ]), YEF11181 ( bni5Δ CDC3-mCherry [ GFP-bni5(306-339) ]), YEF11041 ( bni5Δ CDC3-mCherry [ GFP-bni5(340-393) ]), YEF11042 ( bni5Δ CDC3-mCherry [ GFP-bni5(394-448) ]), YEF11182 ( bni5Δ CDC3-mCherry [ GFP-bni5(306-393) ]), YEF11040 ( bni5Δ CDC3-mCherry [ GFP-bni5(340-448) ]), YEF11039 ( bni5Δ CDC3-mCherry [ GFP-bni5(306-448) ]), YEF11184 ( bni5Δ CDC3-mCherry [ GFP-bni5(Δ306-339) ]), YEF11049 ( bni5Δ CDC3-mCherry [ GFP-bni5(Δ340-393) ]), YEF11051 ( bni5Δ CDC3-mCherry [ GFP-bni5(1-393) ]), and YEF11055 ( bni5Δ CDC3-mCherry [ GFP-bni5(1-305) ]). (B) Colocalization of indicated Bni5 fragments with septins at the bud neck. Cells in the exponential growth phase were subjected to imaging. Asterisks represent variants of Bni5. Strains used are as follows: YEF11053, YEF11181, YEF11040, and YEF11039. (C) Time-lapse analysis of colocalization between Bni5 fragments and septins at the bud neck. Montages of Bni5 fragments with respect to Cdc3-mCherry were created from time-lapse series taken with a 2.5-min interval. Arrowhead indicates the onset of GFP-CC2-CC3 localization at the budding site. An asterisk represents variants of Bni5. Strains used are the same as in (B). (D) Localization of Bni5 fragments in elm1Δ cells. Cells in the exponential growth phase were subjected to imaging. Strains used are as follows: YEF11190 (b ni5Δ elm1Δ CDC3-mCherry [ GFP-BNI5(FL) ]), YEF11197 ( bni5Δ elm1Δ CDC3-mCherry [ GFP-bni5(306-393) ]), YEF11194 ( bni5Δ elm1Δ CDC3-mCherry [ GFP-bni5(340-448) ]), and YEF11193 ( bni5Δ elm1Δ CDC3-mCherry [ GFP-bni5(306-448) ]). (E) Quantitative analysis of Bni5 fragment localization in elm1Δ cells during bud formation. Localization of the indicated GFP-Bni5 fragments in elm1Δ cells with a small or medium bud (S/G2 cells) was scored from the imaging data in (D). An asterisk represents variants of Bni5. (F) The CC2-containing fragments of Bni5 interact with Elm1. Top left: SDS-PAGE stained with Coomassie Blue depicting amounts of each indicated purified protein used as the input for the in vitro binding assay. Bottom left: In vitro binding assay results (Coomassie Blue stained gel) for the indicated GST-tagged proteins bound to glutathione resin and their ability to pull down His-SUMO-Elm1(421-640). Green boxes represent GST and GST-tagged Bni5 fragments as described on the top right. Bottom right: immunoblotted membrane with antibody against His; kDa = kiloDalton. An asterisk represents variants of Bni5. This experiment was repeated three times, and a representative immunoblot is shown. (G) Localization of Bni5(Ext-CC2-CC3) in different septin mutants. Cells in the exponential growth phase were subjected to imaging. Strains used are as follows: YEF11039 ( bni5Δ CDC3-mCherry [ GFP-bni5(306-448) ]), YEF11121 ( shs1Δ CDC3-mCherry [ GFP-bni5(306-448) ]), YEF11310 ( cdc11Δ CDC3-mCherry [ GFP-bni5(306-448) ]), YEF11790 ( shs1Δ cdc11Δ CDC3-mCherry [ GFP-bni5(306-448) ]), and YEF11117 ( cdc10Δ CDC3-mCherry [ GFP-bni5(306-448) ]). (H) Quantitative analysis of Bni5(Ext-CC2-CC3) localization in different septin mutants. Localization of the indicated GFP-Bni5 fragments in cells with a small or medium bud (S/G2 cells) was scored from the imaging data in (G). (I) Bni5 binds to septin filaments via Ext-CC2. Left: SDS-PAGE stained with Coomassie Blue depicting the amount of each indicated purified protein used as the input for the in vitro binding assay. Right: In vitro binding assay results (Coomassie Blue stained gel) for indicated GST-tagged proteins co-sedimented with septin filaments by ultracentrifugation; kDa = kiloDalton. Green boxes represent GST-tagged proteins. An asterisk represents variants of Bni5. This experiment was repeated three times, and a representative immunoblot is displayed. See also Figure S4 .

Article Snippet: Protein concentrations were determined by standard curve intensity measurements from Coomassie blue-stained bovine serum albumin (A7617, Sigma Aldrich, St. Louis, MO, USA) of known concentrations.

Techniques: Modification, Imaging, SDS Page, Staining, Purification, In Vitro, Binding Assay, Membrane, Western Blot

Journal: bioRxiv

Article Title: Discovery of a Novel Inhibitor of Coronavirus 3CL Protease for the Potential Treatment of COVID-19

doi: 10.1101/2020.09.12.293498

Figure Lengend Snippet: X-ray structures of SARS CoV-2 3CLpro apoenzyme (left) and SARS CoV-2 3CLpro in complex with PF-00835231 (right).

Article Snippet: As PF-00835231 and remdesivir, a nucleoside RNA-dependent RNA polymerase inhibitor, target different steps in the virus replication cycle, the antiviral activity of the two compounds was evaluated alone and in combination using HeLa-ACE2 cells .

Techniques:

Journal: bioRxiv

Article Title: Discovery of a Novel Inhibitor of Coronavirus 3CL Protease for the Potential Treatment of COVID-19

doi: 10.1101/2020.09.12.293498

Figure Lengend Snippet: ( A ) In vitro antiviral activity, and cytotoxicity for PF-00835231 and PF-07304814 with and without the P-gp efflux inhibitor, CP-100356. ( B ) EC 50 values with PF-00835231 with increasing P-gp inhibitor in human lung and monkey kidney cell lines. A549-ACE2 human lung carcinoma data (red) as reported in .

Article Snippet: As PF-00835231 and remdesivir, a nucleoside RNA-dependent RNA polymerase inhibitor, target different steps in the virus replication cycle, the antiviral activity of the two compounds was evaluated alone and in combination using HeLa-ACE2 cells .

Techniques: In Vitro, Activity Assay

Journal: bioRxiv

Article Title: Discovery of a Novel Inhibitor of Coronavirus 3CL Protease for the Potential Treatment of COVID-19

doi: 10.1101/2020.09.12.293498

Figure Lengend Snippet: ( A ) (Top) Representative antiviral dose response curves of PF-00835231 in combination with remdesivir against SARS-CoV-2. Serial dilutions of PF-00835231 with a range of fixed concentration of remdesivir. (Bottom) In vitro absolute antiviral activity shift in 50% and 90% antiviral activity with fixed concentrations of remdesivir. ( B ) (Top) A representative 3-dimensional drug interaction landscape plotting synergy scores analyzed using Synergyfinder (median scores of 3 replicates). (Bottom) Average in vitro combination synergy scores from the 3 experiments using 2 different patients’ sera (shown separately).

Article Snippet: As PF-00835231 and remdesivir, a nucleoside RNA-dependent RNA polymerase inhibitor, target different steps in the virus replication cycle, the antiviral activity of the two compounds was evaluated alone and in combination using HeLa-ACE2 cells .

Techniques: Concentration Assay, In Vitro, Activity Assay

Journal: bioRxiv

Article Title: Discovery of a Novel Inhibitor of Coronavirus 3CL Protease for the Potential Treatment of COVID-19

doi: 10.1101/2020.09.12.293498

Figure Lengend Snippet: A . Study design for in vivo experiments. Infection with SARS-CoV-MA15 was always on day 0. Treatment began on day 0 (panel C) or day 0, day 1, or day 2 post-infection (panel B). Lungs were harvested on day 4 post-infection for viral titers and lung histopathology (n=5). B . Lung viral titers for PF-00835231-treated (100 mg/kg, S.C. BID) mice when treatment was started on day 0 or delayed to day 1 and day 2 post-infection. C . Lung viral titers for mice treated in a dose-response of PF-00835231 at 30, 100, 300 mg/kg, S.C. BID starting on day 0. D . Change in body weight of the mice from the experiment shown in panel C. E . Representative photomicrographs of lung sections stained with hematoxylin and eosin. Lungs from infected/untreated mice (top row) displayed perivascular and interstitial inflammation (top, left), degeneration and desquamation of the bronchiolar epithelium (top, middle) and proteinaceous exudate in the alveolar space with interstitial inflammatory cells (top, right), all of which were not observed in uninfected/untreated mice (middle row), and in the infected mice treated with PF-00835231 at 300 mg/kg (bottom row). The scale bars represent 50 μm. LOD, limit of detection; I, infected, untreated; LOQ, limit of quantification; U, uninfected; V, vehicle-treated; Viral Titers error bars represent standard deviation; Weight change error bars represent SEM.

Article Snippet: As PF-00835231 and remdesivir, a nucleoside RNA-dependent RNA polymerase inhibitor, target different steps in the virus replication cycle, the antiviral activity of the two compounds was evaluated alone and in combination using HeLa-ACE2 cells .

Techniques: In Vivo, Infection, Histopathology, Staining, Standard Deviation

Journal: bioRxiv

Article Title: Discovery of a Novel Inhibitor of Coronavirus 3CL Protease for the Potential Treatment of COVID-19

doi: 10.1101/2020.09.12.293498

Figure Lengend Snippet: ( A ) Chemical structure of conversion of prodrug PF-07304814 to the active moiety PF-00835231 by alkaline phosphatase. ( B) Dose feasibility matrix illustrating the ability to achieve higher target exposures with increasing solubility and the limitations of dosing PF-00835231. with dosing either aqueous PF-00835231, clinically formulated PF-00835231, or aqueous PF-07304814 (prodrug). The infeasible limit (red) is assumed to be 1 L per day with a 2x potential benefit with a Cyp inhibitor (orange). Any dose under that is considered feasible (green).

Article Snippet: As PF-00835231 and remdesivir, a nucleoside RNA-dependent RNA polymerase inhibitor, target different steps in the virus replication cycle, the antiviral activity of the two compounds was evaluated alone and in combination using HeLa-ACE2 cells .

Techniques: Solubility

Journal: bioRxiv

Article Title: Discovery of a Novel Inhibitor of Coronavirus 3CL Protease for the Potential Treatment of COVID-19

doi: 10.1101/2020.09.12.293498

Figure Lengend Snippet: ( A) Substrate saturation plot of the metabolism of phosphate prodrug PF-07304814 to the active drug PF-00835231 in human liver S9 fraction. ( B ) HPLC-UV chromatograms of extracts of an incubation of phosphate prodrug PF-07304814 (Rt 6.3 min) in human liver S9 demonstrating complete conversion to the active entity PF-00835231 (Rt 6.7 min).

Article Snippet: As PF-00835231 and remdesivir, a nucleoside RNA-dependent RNA polymerase inhibitor, target different steps in the virus replication cycle, the antiviral activity of the two compounds was evaluated alone and in combination using HeLa-ACE2 cells .

Techniques: Incubation

Journal: bioRxiv

Article Title: Discovery of a Novel Inhibitor of Coronavirus 3CL Protease for the Potential Treatment of COVID-19

doi: 10.1101/2020.09.12.293498

Figure Lengend Snippet: ( A ) Rat, dog and monkey PK following IV administration of PF-07304814 (1.17mg/kg) or PF-00835231 (2mg/kg rat, 1mg/kg dog and monkey) demonstrating high levels of PF-00835231 formed in vivo . (n=2) ( B ) Predicted human PK parameters and measured protein binding for PF-07304814 and PF-00835231 used for human dose prediction. ( C ) Projected human systemic exposure profiles at the minimally efficacious dose of 500mg/day of PF-07304814 delivered as a continuous IV infusion. The predicted unbound steady state concentrations for the prodrug PF-07304814 (purple) and the active moiety PF-00835231 (blue) are 0.17μM and 0.5μM respectively. (NOAEL= No Observed Adverse Effect Level; C eff = projected minimally efficacious concentration, Fu= unbound fraction; error bars= range of replicates)

Article Snippet: As PF-00835231 and remdesivir, a nucleoside RNA-dependent RNA polymerase inhibitor, target different steps in the virus replication cycle, the antiviral activity of the two compounds was evaluated alone and in combination using HeLa-ACE2 cells .

Techniques: In Vivo, Protein Binding, Concentration Assay