Journal: Cell death & disease

Article Title: Selective cell death of latently HIV-infected CD4 + T cells mediated by autosis inducing nanopeptides.

doi: 10.1038/s41419-019-1661-7

Figure Lengend Snippet: Fig. 7 HIV infection increases Na+/K+-ATPase in CD4+ resting memory T cells. a ATP1A1 expression level was measured by western blot of cell lysates of CD4+ T memory cells. b CD4+ T cells were pretreated with 50 nM digoxin for 2 h followed by infection with HIVNL-43 (M.O.I = 0.1). The infected CD4+ T cells were incubated with 50 nM digoxin for 12 days. The cell culture supernatants were tested for HIVp24 by ELISA. c At day 12 p.i, digoxin treated CD4+ T cells under went magnetic negative selection to enrich for central memory CD4+ T cells. The purified CD4+ T memory cells were treated with 50 nM digoxin, 100 nM atazanavir, and 200 nM tenofovir for another 20 days. The harvested cells were measured for integrated HIV DNA using Alu-gag QPCR. All analyses are summarized from four different donors and normalized to loading control ACTB with mean. *P < 0.05, ***P < 0.001

Article Snippet: Then, the central memory CD4+ T cells were purified from HIV-infected CD4+ T cells using negative magnetic isolation, and further cultured with 1 ng/mL IL7 (PeproTech, Rocky Hill, NJ), 100 nM atazanavir and 200 nM tenofovir (Selleckchem, Houston, TX) for another 20 days.

Techniques: Infection, Expressing, Western Blot, Incubation, Cell Culture, Enzyme-linked Immunosorbent Assay, Selection, Control

Journal: bioRxiv

Article Title: Large-Scale Analysis of Cell Death Phenotypic Heterogeneity

doi: 10.1101/2020.02.28.970079

Figure Lengend Snippet: (A) Outline of key RAS/MAPK pathway components highlighting inhibitors (orange) of MEK1/2 (i.e. MEKis) and ERK1/2. (B) Cell death in A549 N cells summarized over time and MEKi concentration. (C) Cell death kinetic parameters for the data in B summarized over compound concentration. Note: parameters can only be computed with confidence for compound concentrations with lethal fraction > ∼0.2. (D) Western blot for phosphorylated and total ERK1/2 in A549 N cells. (E) Quantification of phospho-ERK1/2 and total ERK1/2 at different concentrations of pimasertib at two timepoints. (F) Data for ERK kinase translocation reporter (KTR) in A549 N cells in response to MEKi treatment over time. ERK KTR response to an unrelated lethal compound, bortezomib, is shown as a control. Death onset for lethal conditions (from B,C for MEKis and not shown for Btz) is shown by a dashed line (D O ) (G) Cell death in A549 N cells summarized over time and concentration for the ERK inhibitor (ERKi) SCH772984. Cell death kinetic parameter values are shown for cells treated with 5 µM SCH772984. (H) Expression of the MAPK pathway target DUSP4 in response to SCH772984. (I) Quantification of experiment outlined in H, for three individual experiments. (J) Normalized live cell (mKate2 + objects) counts within the same population of cells over two cycles of pimasertib (Pim, 5 µM, grey shaded area) addition with an intervening period of regrowth in the absence of drug (white area). All data are from at least three independent experiments, and represented as the mean (B,G), mean ± 95% C.I. (C), or mean ± SD (E,F,I,J).

Article Snippet: Camptothecin (Cat# S1288), vinblastine (Cat# S1248), pimasertib (Cat# S1457), trametinib (Cat# S2673), ABT-737 (Cat# S1002), A-1155463 (Cat# S7800), Nutlin-3 (Cat# S1061) were purchased from Selleck Chemicals (Houston, TX).

Techniques: Concentration Assay, Western Blot, Translocation Assay, Control, Expressing

Journal: bioRxiv

Article Title: Large-Scale Analysis of Cell Death Phenotypic Heterogeneity

doi: 10.1101/2020.02.28.970079

Figure Lengend Snippet: (A) Cell death summarized over time and MEKi concentration. Data are mean values from three independent experiments. Cell death kinetic parameters for select concentrations indicated by the symbols are shown at right, with 95% confidence intervals in brackets. nd: not determined. (B) Cell death summarized over time and SCH772984 concentration. Data are mean values from three independent experiments. Cell death kinetic parameters for one concentration indicated by the black circle are shown, with 95% confidence intervals in brackets. nd: not determined. (C) Phospho-ERK1/2 (pERK), p53 and p21 levels in A549 N cells ± nutlin-3 (10 µM, 48 h). (D) Cell death over time following pretreatment (2 day) ± nutlin-3 (10 µM). +Cmpd indicates the time of pimasertib (Pim, 5 µM), trametinib (Tram, 5 µM) or camptothecin (Cpt, 10 µM) addition. p53 stabilization has no effect on the kinetics of MEKi-induced cell death. (E) Analysis of cell cycle phase by PI/FACS ± nutlin-3 (10 µM) for the indicated times. (F) Cell death ± pimasertib ± vehicle (ethanol, EtOH), 2-deoxyglucose (2-DG, 10 mM) or oligomycin (Olig, 100 nM). Disruption of bioenergetics has no effect on FK. (G) Oxygen consumption (OCR) and extracellular acidification (ECAR) determined using a Seahorse assay in A549 N cells ± 2-DG or Olig as in F. (H) Outline of FACS-based scheme to isolate cells with high or low intracellular ROS as assessed using H 2 DCFDA. (I) Cell death over time for isolated H 2 DCFDA High and H 2 DCFDA Low sub-populations exposed to pimasertib (5 µM). (J) Cell death over time in A549 N cells ± MEKis ± N-acetylcysteine (NAC, 1 mM). Erastin2 is a positive control compound that induces oxidative cell death. Data in D-G, I and J are mean ± SD from at least three independent experiments.

Article Snippet: Camptothecin (Cat# S1288), vinblastine (Cat# S1248), pimasertib (Cat# S1457), trametinib (Cat# S2673), ABT-737 (Cat# S1002), A-1155463 (Cat# S7800), Nutlin-3 (Cat# S1061) were purchased from Selleck Chemicals (Houston, TX).

Techniques: Concentration Assay, Disruption, Isolation, Positive Control

Journal: bioRxiv

Article Title: Large-Scale Analysis of Cell Death Phenotypic Heterogeneity

doi: 10.1101/2020.02.28.970079

Figure Lengend Snippet: (A) Cell death in response to treatment with pimasertib (Pim, 5 µM), trametinib (Tram, 5 µM), bortezomib (Btz, 50 nM), camptothecin (Cpt, 1 µM) ± cycloheximide (CHX). (B) Cell death ± Q-VD-OPh (50 µM). Pim, Tram and Btz treatments are as in A. (C) Protein expression ± pimasertib (5 µM, 48 h). (D) Expression of BIM in response to doxycycline (Dox). (E) Cell death over time ± Pim combined with Dox-inducible expression of wild-type BIM. (F) Cell death kinetic parameters computed from the data in E. (G) Expression of the inactive BIM G156E mutant + Dox. (H) Cell death over time in response to Dox-inducible expression of mutant BIM G156E + Pim. (I) Cell death in Calu-6 N cells in response to compound treatment ± the selective MCL1 inhibitor S63845 (5 µM). (J) Cell death kinetic parameters computed from the data in I. (K) Expression of MCL1 in A549 N;Cas9 cells transduced with short guide RNAs against GFP or MCL1 . (L) Cell death over time in the cells lines from K, treated as indicated. D R values computed from each curve are indicated for MEKis. Data are from at least three independent experiments and represent mean ± SD (A,B,E,H,I,L) or mean ± 95% C.I (F,J).

Article Snippet: Camptothecin (Cat# S1288), vinblastine (Cat# S1248), pimasertib (Cat# S1457), trametinib (Cat# S2673), ABT-737 (Cat# S1002), A-1155463 (Cat# S7800), Nutlin-3 (Cat# S1061) were purchased from Selleck Chemicals (Houston, TX).

Techniques: Expressing, Mutagenesis, Transduction

Journal: bioRxiv

Article Title: Large-Scale Analysis of Cell Death Phenotypic Heterogeneity

doi: 10.1101/2020.02.28.970079

Figure Lengend Snippet: (A) Summary of RNA sequencing analysis of cells treated with pimasertib at cytotoxic (5 µM, A549 and Calu-6) and cytostatic (non-cytotoxic) (0.5 µM in A549) doses. RNA was obtained for analysis from two independent experiments and FKPM values for each gene were averaged prior to further analysis. (B) GO analysis for the 555 significantly altered death-associated genes. (C) Inhibitors (bold text) block the function of BCL-2-family proteins to induce apoptosis. (D) Cell death over time in A549 N cells co-treated with MEKis and the BCL-xL inhibitors ABT-737 or A-1155463 (both 5 µM). (E) Cell death kinetic parameters for treatments in D. (F) Cell death over time in A549 N cells co-treated with MEKis or bortezomib (Btz) and the MCL1 inhibitor S63845 (5 µM). (G) Cell death kinetic parameters for treatments in F. (H) Mean single cell fluorescence intensity (M.F.I.) for MCL1 and BCL-xL in individual A549 N cells determined by immunofluorescence at 48 h ± Pim (5 µM). Coefficient of variation (CV) is indicated. (I) MCL1 M.F.I. over time in A549 N cells treated as indicated. Coefficient of variation (CV) is shown for all conditions, and ranges (min-max) are shown for the 96 h sample. For H and I, at least 115 individual cells are quantified per condition from multiple independent microscopic fields, and median and interquartile ranges are indicated. (J) Expression of MCL1 in A549 N cells transduced with CMV-Empty and CMV-MCL1 lentivirus. (K) Cell death over time ± MCL1 overexpression, as in J. (L) D R values computed from lethal fraction curves in K for MEKis. Results are from three independent experiments and represent the mean ± SD (D,F,K) or the mean ± 95% C.I. (E, G, L).

Article Snippet: Camptothecin (Cat# S1288), vinblastine (Cat# S1248), pimasertib (Cat# S1457), trametinib (Cat# S2673), ABT-737 (Cat# S1002), A-1155463 (Cat# S7800), Nutlin-3 (Cat# S1061) were purchased from Selleck Chemicals (Houston, TX).

Techniques: RNA Sequencing, Blocking Assay, Fluorescence, Immunofluorescence, Expressing, Transduction, Over Expression

Journal: Frontiers in Cardiovascular Medicine

Article Title: Antiretroviral Drugs Regulate Epigenetic Modification of Cardiac Cells Through Modulation of H3K9 and H3K27 Acetylation

doi: 10.3389/fcvm.2021.634774

Figure Lengend Snippet: ARV drugs treatment suppresses active histone marks. (A,B) Western blots show acetylation of histone at H3K9 and H3K27 and graphs show its quantification ( *** p < 0.001). NRVCs were treated with ARV drugs (5 μM of Ritonavir, Abacavir, Atazanavir and Lamivudine) for 4, 12, and 24 h and western blots were done with total protein lysate. (C,D) Representative images show immunofluorescence staining of NRVCs stained with H3K9ac (green), actinin (red), and nucleus stained with DAPI (blue). Cells were treated with ARV drugs for 12 h and fixed with 4% PFA. (D) Graph shows quantification of microscopic images ( n = 50 cells); ( * p < 0.05). (E,F) Representative images show immunofluorescence staining of NRVCs stained with H3K27ac (green), actinin (red), and nucleus stained with DAPI (blue). Cells were treated with ARV drugs for 12 h and fixed with 4% PFA. (D) Graph shows quantification of microscopic images ( n = 50 cells); ( * p < 0.05). (G,H) Western blot shows acetylation of NRVCs after drugs treatment. NRVCs were treated with individual ARV drug for 12 h and western blot was done with total protein lysate. Graphs show quantification of western blot ( *** p < 0.001; ** p < 0.01, ns, not significant).

Article Snippet: The cells were then treated with ARV drugs (5 μM of Ritonavir, Abacavir, Atazanavir and Lamivudine, (Selleck Chemicals Llc, Pittsburgh, PA) for 24 h. Cellular viability was detected using the CellTiter-Glo luminescent cell viability assay (Promega).

Techniques: Western Blot, Immunofluorescence, Staining

Journal: Frontiers in Cardiovascular Medicine

Article Title: Antiretroviral Drugs Regulate Epigenetic Modification of Cardiac Cells Through Modulation of H3K9 and H3K27 Acetylation

doi: 10.3389/fcvm.2021.634774

Figure Lengend Snippet: ARV drugs treatment promotes repressive histone marks. (A,B) Western blot shows antiretroviral drug treatment increase the methylation of H3K27me3 and H3K9me3. NRVCs were treated with ARV drugs (5 μM of Ritonavir, Abacavir, Atazanavir and Lamivudine) for 4, 12, and 24 h and western blots were done with total protein lysate. Graph shows quantification of western blots ( *** p < 0.001; ** p < 0.01, ns, not significant) (C) Representative images show immunofluorescence staining of NRVCs stained with H3K9me3 (green), actinin (red), and nucleus stained with DAPI (blue). Cells were treated with ARV drugs for 12 h and fixed with 4% PFA. (D) Graph shows quantification of microscopic images ( n = 50 cells); ( * p < 0.05). (E) Representative images show immunofluorescence staining of NRVCs stained with H3K27me3 (green), actinin (red), and nucleus stained with DAPI (blue). Cells were treated with ARV drugs for 12 h and fixed with 4% PFA. (F) Graph shows quantification of microscopic images ( n = 50 cells); ( *** p < 0.001).

Article Snippet: The cells were then treated with ARV drugs (5 μM of Ritonavir, Abacavir, Atazanavir and Lamivudine, (Selleck Chemicals Llc, Pittsburgh, PA) for 24 h. Cellular viability was detected using the CellTiter-Glo luminescent cell viability assay (Promega).

Techniques: Western Blot, Methylation, Immunofluorescence, Staining

Journal: Frontiers in Cardiovascular Medicine

Article Title: Antiretroviral Drugs Regulate Epigenetic Modification of Cardiac Cells Through Modulation of H3K9 and H3K27 Acetylation

doi: 10.3389/fcvm.2021.634774

Figure Lengend Snippet: PCR array analysis of differentially expressed epigenetic chromatin modifying enzymes on ARV drugs treatment.

Article Snippet: The cells were then treated with ARV drugs (5 μM of Ritonavir, Abacavir, Atazanavir and Lamivudine, (Selleck Chemicals Llc, Pittsburgh, PA) for 24 h. Cellular viability was detected using the CellTiter-Glo luminescent cell viability assay (Promega).

Techniques:

Journal: Frontiers in Cardiovascular Medicine

Article Title: Antiretroviral Drugs Regulate Epigenetic Modification of Cardiac Cells Through Modulation of H3K9 and H3K27 Acetylation

doi: 10.3389/fcvm.2021.634774

Figure Lengend Snippet: ARV drugs treatment modulates expression of epigenetic regulating enzyme. NRVCs were treated with ARV drugs (5 μM of Ritonavir, Abacavir, Atazanavir, and Lamivudine) for 4, 12, and 24 h and expression of epigenetic enzyme was measured by RT 2 PCR array profiling. (A) Clustergram showing differentially expressed genes ( p < 0.05, Fold Change >1.2) obtained on RT 2 PCR array profiling, green represent minimum and red represent maximum magnitude of expression. (B) Scatter Plot representing normalized expression of genes between 4, 12, and 24 h ARV drugs treated vs. control group. Central diagonal line represents no change, whereas, outer diagonal lines indicate the fold regulation threshold (>1.2). Genes with data points beyond the outer lines in the upper left and lower right corners are up-regulated or down-regulated, respectively. (C) Graphical representation of differentially expressed chromatin modifying enzymes against expression regulation on drug treatment at 4 h (red), 12 h (green), and 24 h (blue).

Article Snippet: The cells were then treated with ARV drugs (5 μM of Ritonavir, Abacavir, Atazanavir and Lamivudine, (Selleck Chemicals Llc, Pittsburgh, PA) for 24 h. Cellular viability was detected using the CellTiter-Glo luminescent cell viability assay (Promega).

Techniques: Expressing, Control

Journal: Frontiers in Cardiovascular Medicine

Article Title: Antiretroviral Drugs Regulate Epigenetic Modification of Cardiac Cells Through Modulation of H3K9 and H3K27 Acetylation

doi: 10.3389/fcvm.2021.634774

Figure Lengend Snippet: Expression of epigenetic chromatin enzymes affected by ARV drugs treatment. (A) Graphs showing mRNA expression of Sirt1, Ezh2, and Suv39h1 in NRVCs treated with drugs. Cells were treated with ARV drugs (5 μM of Ritonavir, Abacavir, Atazanavir and Lamivudine) for different time point (4, 12, and 24 h) and expression was checked by qRT-PCR. (B,C) Western blots showing protein expression of SIRT1, SUV39H1, and EZH2 enzymes and graph show quantification. NRVCs were treated with ARV drugs (5 μM of Ritonavir, Abacavir, Atazanavir, and Lamivudine) for 4, 12, and 24 h and expression was checked in total protein lysate by western blot with respective antibody ( * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns, not significant).

Article Snippet: The cells were then treated with ARV drugs (5 μM of Ritonavir, Abacavir, Atazanavir and Lamivudine, (Selleck Chemicals Llc, Pittsburgh, PA) for 24 h. Cellular viability was detected using the CellTiter-Glo luminescent cell viability assay (Promega).

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: Frontiers in Cardiovascular Medicine

Article Title: Antiretroviral Drugs Regulate Epigenetic Modification of Cardiac Cells Through Modulation of H3K9 and H3K27 Acetylation

doi: 10.3389/fcvm.2021.634774

Figure Lengend Snippet: Expression of SIRT1 is critical in ARV drugs mediated modulation of cellular acetylation. (A,B) Western blots show that knockdown of SIRT1 upregulate acetylation of histone at H3K9. NRVCs were treated with SIRT1 siRNA for 48 h and followed by ARV drugs treatment (5 μM of Ritonavir, Abacavir, Atazanavir, and Lamivudine) for 12 h. Graph shows quantification of western blot. ( *** p < 0.001; ** p < 0.01, * p < 0.05, ns, not significant). (C,D) Western blots show that over expression of SIRT1 can significantly decreases the H3K9ac in rat cardiomyocytes. SIRT1 and GFP protein were over expressed in the cardiomyocytes for 48 h by adenoviral transduction and treated with the ARV drugs for another 24 h. Graph shows the quantification of H3K9ac ( * p < 0.05). (E) Drug treatment upregulates the SIRT1 enzyme activity. NRVCs were treated with ARV drugs for 4–24 h and enzyme activity was determined in total protein lysate. ( *** p < 0.001; **** p < 0.0001, ns, not significant). (F) ARV drugs treatment reduces cellular viability in SIRT1 knockdown cells. NRVCs were treated with SIRT1 siRNA and ARV drugs and viability was measure by CellTiter-Glo ( ** p < 0.01; * p < 0.05, ns, not significant). (G) Graph shows that overexpression of SIRT1 improves cellular viability during ARV mediated cellular stress. NRVCs were transfected with adenovirus for 48 h and then treated with ARV drugs for another 24 h. Cellular viability was determined by the CellTiter-Glo ( *** p < 0.001; ** p < 0.01, * p < 0.05, ns, not significant). (H,I) Representative microscopy images show that ARV drugs treatment induces cellular hypertrophy and SIRT1 over expression significantly reduces the cellular hypertrophy in ARV drugs treated cells ( * p < 0.05). NRVCs were transfected with adenovirus for 48 h and then treated with ARV drugs for another 24 h. Drug treated cells were fixed with 4% PFA and stained with actinin antibody (red) and DAPI for nucleus. Cell size was determined by Image J software (National Institute of Health, USA). Graph shows the measurement of cell size ( *** p < 0.001, ns, not significant). (J,K) Representative images show that ARV drugs treatment induces cellular ROS level and SIRT1 over expression significantly reduces the ROS level of drug treated cells. Graph shows the quantification of ROS. NRVCs were transfected with adenovirus for 48 h and then treated with ARV drugs for another 24 h. ROS level of the cells were determined by DHE staining. Live imaging was done under fluorescence microscope ( *** p < 0.001, ns, not significant).

Article Snippet: The cells were then treated with ARV drugs (5 μM of Ritonavir, Abacavir, Atazanavir and Lamivudine, (Selleck Chemicals Llc, Pittsburgh, PA) for 24 h. Cellular viability was detected using the CellTiter-Glo luminescent cell viability assay (Promega).

Techniques: Expressing, Western Blot, Knockdown, Over Expression, Transduction, Activity Assay, Transfection, Microscopy, Staining, Software, Imaging, Fluorescence

Journal: Aging Cell

Article Title: Antiretroviral protease inhibitors induce features of cellular senescence that are reversible upon drug removal

doi: 10.1111/acel.13750

Figure Lengend Snippet: The anti‐HIV PI cocktail atazanavir + ritonavir (ATV/r) induces senescence in cultured cells. IMR‐90 primary human fibroblasts were cultured in the presence of ATV/r for 14 days. (a). RNA was isolated from untreated (DMSO) and ATV/r‐treated cells, and p16 INK4a and p21 WAF1 mRNA levels, normalized for Actin mRNA, were measured by qPCR. (b). mRNA levels of LMNB1 in DMSO‐ and ATV/r‐treated cells were similarly measured. (c). SASP component mRNA levels were measured using qPCR. (d). Representative images of SA‐β‐gal positivity in control cells (left panel) and cells induced to senesce by ATV/r (right panel). (e). Intracellular levels of proteins prelamin a, activated (P‐ser37) p53, p21 WAF1 , HMGB1 and beta‐Actin (control) using western analysis. (f). Cells were analyzed for HMGB1 release from the nucleus, proliferation (EdU), and nuclei morphology (DAPI) by microscopy.

Article Snippet: Atazanavir, ritonavir and darunavir were from Medchem Express.

Techniques: Cell Culture, Isolation, Control, Western Blot, Microscopy

Journal: Aging Cell

Article Title: Antiretroviral protease inhibitors induce features of cellular senescence that are reversible upon drug removal

doi: 10.1111/acel.13750

Figure Lengend Snippet: ATV/r treatment accelerates aging phenotypes. Young adult mice at 5 months of age were treated with 62 mg/kg atazanavir and 21 mg/kg ritonavir in drinking water for 8 weeks. (a). Schematic of the experimental setup. (b). Representative phenotypic differences between ATV/r‐treated mice and age‐matched controls are shown. (c–h). p21 Waf1 mRNA levels were measured by qPCR in various tissues. (i). Representative images of p16‐3MR male mice injected with coelenterazine and measured for luminescence using the Xenogen imaging system. (j). Quantification of luminescence presented in I.

Article Snippet: Atazanavir, ritonavir and darunavir were from Medchem Express.

Techniques: Injection, Imaging

Journal: Aging Cell

Article Title: Antiretroviral protease inhibitors induce features of cellular senescence that are reversible upon drug removal

doi: 10.1111/acel.13750

Figure Lengend Snippet: ATV/r‐treated mice accumulate senescent cells at sites of age‐related pathologies. Young adult mice (5 months of age) were treated with 62 mg/kg atazanavir and 21 mg/kg ritonavir in drinking water for 8 weeks. (a). Representative images of SA‐β‐gal staining in subcutaneous fat from vehicle and ATV/r‐treated mice. (b). Quantification of SA‐β‐gal staining presented in a. (c–f). selected SASP factors (MMP3, IL‐1a, GMCSF and IL‐10) and p21 Waf1 mRNA expression levels were measured in subcutaneous fat by qPCR and normalized to Actin. (g). Representative images of sectioned dorsal skin stained for SA‐β‐gal activity. (h). qPCR analysis for senescence marker p16 Ink4a . (i–k). mRNA expression levels of some SASP markers from skin (IL‐10, TIMP1 and MMP3) were measured by qPCR and normalized to Actin. (l–n). cardiac activity was measured by echocardiography.

Article Snippet: Atazanavir, ritonavir and darunavir were from Medchem Express.

Techniques: Staining, Expressing, Activity Assay, Marker

Journal: Aging Cell

Article Title: Antiretroviral protease inhibitors induce features of cellular senescence that are reversible upon drug removal

doi: 10.1111/acel.13750

Figure Lengend Snippet: Removal of ATV/r treatment reverses senescent phenotypes in mice and improves age‐related phenotypes. Young adult mice (5‐months old) were treated with 62 mg/kg atazanavir and 21 mg/kg ritonavir in drinking water for 8 weeks. (a). Schematic of the experimental setup. (b,c). p21 Waf1 and p16 Ink4a mRNA levels in and subcutaneous fat were measured by qPCR. (d). SASP factor MMP3 mRNA levels in the subcutaneous fat tissue were measured by qPCR and normalized to Actin. (e,f). p21 Waf1 and p16 Ink4a mRNA levels in heart tissue were measured by qPCR. (g). SASP factor TIMP1 mRNA levels in the heart were measured by qPCR and normalized to Actin. (h). p16‐3MR male mice were injected with coelentarazine, and luminescence was measured at baseline, after 8 weeks of treatment, and 10 weeks post‐treatment. (i). Schematic of the experimental setup. (j). qPCR analysis for p21 Waf1 mRNA levels in heart tissue. (k). Cardiac activity was measured by echocardiography.

Article Snippet: Atazanavir, ritonavir and darunavir were from Medchem Express.

Techniques: Injection, Activity Assay

Journal: bioRxiv

Article Title: Apixaban, an orally available anticoagulant, inhibits SARS-CoV-2 replication by targeting its major protease in a non-competitive way

doi: 10.1101/2021.09.23.461605

Figure Lengend Snippet: (A) Superposition of the monomeric unit of M pro (in gray, PDB code 7K40) with FXa (on the left in violet, PDB code 2P16) and thrombin (on the right in purple, PDB code 1KTS). The crystallographic structure of apixaban into FXa structure, dabigatran into thrombin structure, and catalytic dyad of M pro (His-41 and Cys-145 residues) are as spheres in pink, cyan, and orange, respectively. For better interpretation the catalytic water (H 2 O cat ) of M pro is not shown. The enzymatic inhibition profile for apixaban, rivaroxaban, and dabigatran (0, 0.08, 0.16, 0.31, 0.63, 1.25, 2.5, 5.0, and 10 mM) into (B) PL pro (8.19 nM) and (C) M pro (88.8 nM) velocity. The positive controls GRL0617 (PL pro ) and GC376 (M pro ) were used under the same condition of anticoagulants. (D) Michaelis-Menten enzymatic mechanism for M pro without and in the presence of a fixed apixaban or atazanavir concentration (2.5 mM) for different substrate concentrations (0, 0.76, 1.56, 3.12, 6.25, 12.5, 25.0, 50.0, and 100 mM). (E) Enzymatic scheme for the experimental mechanism of M pro inhibition by anticoagulants. Best docking pose (ChemPLP function) for the interaction between M pro (F) substrate, and (G) substrate-apixaban into the active site of protease. Best docking pose (ChemPLP function) for the interaction between the dimer interface of M pro (H) apixaban and rivaroxaban, while (I) shows the selected amino acid residues which interact with apixaban. Substrate, rivaroxaban, dabigatran, and apixaban are in stick representation in beige, green, cyan, and pink, respectively, while the catalytic water (H 2 O cat ) is in sphere. Elements’ color: hydrogen, nitrogen, oxygen, sulfur, and chloro are in white, dark blue, red, yellow, and dark green, respectively.

Article Snippet: Nevertheless, apixaban was about 5- and 60-fold less potent in vitro in comparison to the positive control atazanavir and remdesivir, respectively , indicating that apixaban shows an interestingly scaffold for the design of novel compounds to increase its antiviral action.

Techniques: Inhibition, Concentration Assay

Journal: bioRxiv

Article Title: Apixaban, an orally available anticoagulant, inhibits SARS-CoV-2 replication by targeting its major protease in a non-competitive way

doi: 10.1101/2021.09.23.461605

Figure Lengend Snippet: Antiviral activity of anticoagulants, atazanavir, and remdesivir in Calu-3 cells (densities of 2.0 × 10 5 cells/well) infected with SARS-CoV-2 (MOI 0.1) in 96-well plates. The data is presented as (A) virus production (PFU/mL) and (B) percentage of viral replication inhibition. The data represent means ± SEM of three independent experiments.

Article Snippet: Nevertheless, apixaban was about 5- and 60-fold less potent in vitro in comparison to the positive control atazanavir and remdesivir, respectively , indicating that apixaban shows an interestingly scaffold for the design of novel compounds to increase its antiviral action.

Techniques: Activity Assay, Infection, Inhibition

Journal: PLoS ONE

Article Title: Mg-supplementation attenuated lipogenic and oxidative/nitrosative gene expression caused by Combination Antiretroviral Therapy (cART) in HIV-1-transgenic rats

doi: 10.1371/journal.pone.0210107

Figure Lengend Snippet: Control or HIV-Tg rats received cART (atazanavir-ritonavir plus Truvada) for 18 months while on normal or high Mg (6-fold) diets. Real-time quantitative-PCR results were normalized by 18s rRNA, and the values are expressed as means of 4–5± SE; **p<0.01 vs. Ctl, + p<0.05, # p<0.05 vs. Tg alone, ++p<0.01 vs. corresponding normal Mg (N-Mg) groups.

Article Snippet: Therefore, the final dosages of the cART components for both the control or Tg rats on normal or high Mg diets were estimated to be: atazanavir/ritonavir = 16.5/5.5 mg/kg/day; and for Truvada = 16.5 mg TDF plus 11 mg FTC/kg/day.

Techniques: Real-time Polymerase Chain Reaction