Journal: PLOS Pathogens

Article Title: Platelet-like particles released from Ebola virus-infected megakaryocytic cells behave like virus-like particles

doi: 10.1371/journal.ppat.1013985

Figure Lengend Snippet: A. Percentage of GFP-positive MEG-01 VP30 cells. PMA-treated or untreated cells were exposed to EBOVΔVP30-GFP at an MOI of 5 and GFP-positive cells were quantified by flow cytometry 2 days after exposure. Data are representative of two independent experiments. B. EBOVΔVP30 titers from MEG-01 VP30 cells. PMA-treated or untreated cells were exposed to EBOVΔVP30-GFP at an MOI of 1. Supernatants were collected daily, and viral titers were determined using Vero VP30 cells. Data are presented as means ± SD of three independent experiments. The dotted lines indicate the lower limit of detection (20 ffu/ml). Statistical significance was assessed by using the multiple unpaired t-test. * p < 0.05, ** p < 0.01.

Article Snippet: Wild-type MEG-01 cells (ATCC, CRL-2021), MEG-01 VP30 cells (MEG-01 cell line stably expressing EBOV VP30), MEG-01 CD41-mCherry cells (MEG-01 cell line stably expressing CD41 [GenBank accession no. NM_000419.5 ] fused with mCherry at the C-terminus [CD41-mCherry]), and MEG-01 CD41-mCherry/GP cells (MEG-01 cell line stably expressing CD41-mCherry and EBOV GP) were cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS) and antibiotics.

Techniques: Flow Cytometry

Journal: PLOS Pathogens

Article Title: Platelet-like particles released from Ebola virus-infected megakaryocytic cells behave like virus-like particles

doi: 10.1371/journal.ppat.1013985

Figure Lengend Snippet: A. Detection of EBOV proteins by western blot from PLPs released form EBOVΔVP30-exposed MEG-01 VP30 cells. PLPs were collected on day 4 post-exposure from PMA-treated MEG-01 WT or VP30 cells exposed to EBOVΔVP30 at an MOI of 5. The indicated proteins were analyzed by immunoblotting. Data are representative of two independent experiments. B. Relative amount of EBOV gRNA in PLPs released from EBOVΔVP30-exposed MEG-01 VP30 cells. PLPs were prepared as described in (A). EBOV gRNA was quantified by RT-qPCR using the indicated genome-specific primer pairs and normalized to the gRNA in PLPs from MEG-01 WT cells. Data are presented as means ± SD from two independent experiments performed in triplicate. C. Localization of EBOV GP in PLPs released from EBOVΔVP30-exposed MEG-01 VP30 cells. PLPs were prepared as described in (A). The platelet marker CD41 (magenta) and EBOV GP (green) were visualized by immunofluorescence microscopy using specific antibodies, along with the corresponding bright-field image. Scale bars, 5 μm. D. Interaction between EBOV NP/VP35 (top panel) and NP/VP40 (bottom panel) in PLPs released from EBOVΔVP30-exposed MEG-01 VP30 cells. PLPs were prepared as described in (A). NP/VP35 and NP/VP40 complexes (green) were visualized by using a proximity ligation assay (PLA) with specific antibodies and overlaid on the corresponding bright-field image. Scale bars, 5 μm.

Article Snippet: Wild-type MEG-01 cells (ATCC, CRL-2021), MEG-01 VP30 cells (MEG-01 cell line stably expressing EBOV VP30), MEG-01 CD41-mCherry cells (MEG-01 cell line stably expressing CD41 [GenBank accession no. NM_000419.5 ] fused with mCherry at the C-terminus [CD41-mCherry]), and MEG-01 CD41-mCherry/GP cells (MEG-01 cell line stably expressing CD41-mCherry and EBOV GP) were cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS) and antibiotics.

Techniques: Western Blot, Quantitative RT-PCR, Marker, Immunofluorescence, Microscopy, Proximity Ligation Assay

Journal: PLOS Pathogens

Article Title: Platelet-like particles released from Ebola virus-infected megakaryocytic cells behave like virus-like particles

doi: 10.1371/journal.ppat.1013985

Figure Lengend Snippet: Relative expression levels of EBOV mRNA (A) and gRNA (B) in PLPs released from EBOVΔVP30-exposed MEG-01 VP30 cells. PLPs were collected on day 4 post-exposure from PMA-treated MEG-01 VP30 cells exposed to EBOVΔVP30-GFP at an MOI of 5. EBOV mRNA and gRNA were quantified by RT-qPCR using the indicated specific primer pairs and normalized to the amount in the day 0 samples. Data are presented as means ± SD of three independent experiments performed in triplicate.

Article Snippet: Wild-type MEG-01 cells (ATCC, CRL-2021), MEG-01 VP30 cells (MEG-01 cell line stably expressing EBOV VP30), MEG-01 CD41-mCherry cells (MEG-01 cell line stably expressing CD41 [GenBank accession no. NM_000419.5 ] fused with mCherry at the C-terminus [CD41-mCherry]), and MEG-01 CD41-mCherry/GP cells (MEG-01 cell line stably expressing CD41-mCherry and EBOV GP) were cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS) and antibiotics.

Techniques: Expressing, Quantitative RT-PCR

Journal: PLOS Pathogens

Article Title: Platelet-like particles released from Ebola virus-infected megakaryocytic cells behave like virus-like particles

doi: 10.1371/journal.ppat.1013985

Figure Lengend Snippet: A,B. Expression levels of GP and CD41-mCherry in MEG-01 cells stably expressing CD41-mCherry with or without EBOV GP (A), and in PLPs released from these stable cell lines (B). Protein levels were analyzed by immunoblotting using the indicated antibodies. C. Percent of mCherry-positive cells that internalized PLPs containing CD41-mCherry or CD41-mCherry/GP. Huh7 VP30 cells were co-incubated with the indicated PLPs for 1, 3, or 5 h, followed by quantification of mCherry-positive cells by flow cytometry. Data are presented as means ± SD of three independent experiments. Statistical significance was assessed by use of a two-way ANOVA followed by Turkey’s multiple comparisons test. * p < 0.05, **** p < 0.0001.

Article Snippet: Wild-type MEG-01 cells (ATCC, CRL-2021), MEG-01 VP30 cells (MEG-01 cell line stably expressing EBOV VP30), MEG-01 CD41-mCherry cells (MEG-01 cell line stably expressing CD41 [GenBank accession no. NM_000419.5 ] fused with mCherry at the C-terminus [CD41-mCherry]), and MEG-01 CD41-mCherry/GP cells (MEG-01 cell line stably expressing CD41-mCherry and EBOV GP) were cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS) and antibiotics.

Techniques: Expressing, Stable Transfection, Western Blot, Incubation, Flow Cytometry

Journal: PLOS Pathogens

Article Title: Platelet-like particles released from Ebola virus-infected megakaryocytic cells behave like virus-like particles

doi: 10.1371/journal.ppat.1013985

Figure Lengend Snippet: Number of GFP-positive cells following co-incubation with PLPs released from EBOVΔVP30-exposed MEG-01 cells (A). PLPs were collected from PMA-treated MEG-01 WT and VP30 cells exposed to EBOVΔVP30-GFP. Huh7 VP30 cells (2 x 10 5 cells) were co-incubated with the indicated PLPs (2 x 10 6 ) for two days. As a control, cells were cultured in the final wash supernatant (B). GFP-positive cells were quantified by flow cytometry. Data are representative of two independent experiments. C-E. EBOVΔVP30 titers in three different VP30-expressing cell types co-cultured with PLPs released from EBOVΔVP30-exposed MEG-01 VP30 cells. PLPs were prepared as described in (A). Huh7 VP30 cells (2 x 10 5 cells, C), HUVEC VP30 cells (2 x 10 5 cells, D), and PMA-differentiated THP-1 VP30 cells (2 x 10 5 cells, E) were co-cultured with the indicated PLPs (2 x 10 6 ). Virus titers on days 2, 4, and 6 were determined using Vero VP30 cells. Data are presented as means ± SD of three independent experiments. The dotted lines indicate the lower limit of detection (1.3 log10 ffu/ml). Statistical significance was assessed by use of a one-way ANOVA followed by Turkey’s multiple comparisons test. *** p < 0.001, **** p < 0.0001.

Article Snippet: Wild-type MEG-01 cells (ATCC, CRL-2021), MEG-01 VP30 cells (MEG-01 cell line stably expressing EBOV VP30), MEG-01 CD41-mCherry cells (MEG-01 cell line stably expressing CD41 [GenBank accession no. NM_000419.5 ] fused with mCherry at the C-terminus [CD41-mCherry]), and MEG-01 CD41-mCherry/GP cells (MEG-01 cell line stably expressing CD41-mCherry and EBOV GP) were cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS) and antibiotics.

Techniques: Incubation, Control, Cell Culture, Flow Cytometry, Expressing, Virus

Journal: The Journal of Pharmacology and Experimental Therapeutics

Article Title: Aspirin inhibition and recovery of cyclooxygenase activity and thromboxane biosynthesis in human megakaryocytes: a translational surrogate model

doi: 10.1016/j.jpet.2025.103762

Figure Lengend Snippet: Expression of COX-1 and -2 in MEG-01 and CHRF-288-11. (A) Immunoblot analysis of COX-1 and COX-2 expression in CHRF-288-11 cells. (B) Immunoblot analysis of COX-1 and COX-2 expression in MEG-01 cells. Two × 10 6 cells were lysed, and 20 μ g of total proteins were subjected to 9% SDS-polyacrylamide gel. COX-1, COX-2, and β -actin were developed on the same blot ( n = 3). (C) Immunocytochemistry of MEG-01 cells using selective anti-COX-1 and COX-2 antibodies. Representative images of immunostaining of COX-1 or COX-2 are shown; slides were analyzed by light microscopy using the PhenoImager HT 2.0 workstation system (Akoya Biosciences) at main magnification ×20. COX, cyclooxygenase.

Article Snippet: MEG-01 cell line (CRL-2021) was obtained from ATCC and CHRF-288-11 cells kindly provided by Jean-Philippe Rosa (INSERM U1176).

Techniques: Expressing, Western Blot, Immunocytochemistry, Immunostaining, Light Microscopy

Journal: The Journal of Pharmacology and Experimental Therapeutics

Article Title: Aspirin inhibition and recovery of cyclooxygenase activity and thromboxane biosynthesis in human megakaryocytes: a translational surrogate model

doi: 10.1016/j.jpet.2025.103762

Figure Lengend Snippet: Effect of aspirin on TXB 2 biosynthesis and kinetics of COX activity recovery in MEG-01 and CHRF-288-11 cells. (A, B) MK cells were treated with different concentrations of aspirin (0.1, 1, 3, 10, and 30 μ M) for 30 min, followed by TXB 2 measurement. The percentage of TXB 2 in aspirin-treated cells vs vehicle-treated cells as control is plotted. (A) MEG-01 and (B) CHRF-288-11. Results are expressed as a mean ± SEM of experiments performed in triplicates in MEG-01 ( n = 7) and CHRF-288-11 ( n = 3), respectively. IC 50 was determined by GraphPad Prism and calculated as 2.4 ± 0.5 and 2.8 ± 0.6 μ M (mean ± SEM.) for MEG-01 and CHRF-288-11, respectively. (C, D) MK cell lines were treated with 10 μ M aspirin or vehicle (0.1% ethanol, v/v) for 30 min and COX activity was determined. Washed cells were allowed to recover for 24, 48, 72, 96 and 120 hours. TXB 2 levels in (C) MEG-01 and (D) CHRF-288-11 were measured after AA loading and expressed as % TXB 2 of aspirin vs control (vehicle-treated cells). Results are expressed as a mean± SEM. of separate experiments for MEG-01 ( n = 10) and for CHRF-288-11 ( n = 8), performed each in triplicate. AA, arachiconic acid; COX, cyclooxygenase; TX, thromboxane.

Article Snippet: MEG-01 cell line (CRL-2021) was obtained from ATCC and CHRF-288-11 cells kindly provided by Jean-Philippe Rosa (INSERM U1176).

Techniques: Activity Assay, Control

Journal: The Journal of Pharmacology and Experimental Therapeutics

Article Title: Aspirin inhibition and recovery of cyclooxygenase activity and thromboxane biosynthesis in human megakaryocytes: a translational surrogate model

doi: 10.1016/j.jpet.2025.103762

Figure Lengend Snippet: COX isoenzyme contribution in TXB 2 biosynthesis after treatment with 10 μ M aspirin in (A) MEG-01 and (B) CHRF-288-11 cells. All cells were treated with 10 μ M aspirin for 30 min, and basal COX activity was determined (labelled ASA). Cells were next washed and plated for each time point (24, 48, and 72 hours), treated with either DMSO (0.1%; v/ v) (labeled Ctrl.), 10 μ M SC-560 or 1 μ M NS-398 for 30 min, before the addition of 10 μ M AA (see the Methods section). TXB 2 levels were measured as ng/million cells and expressed as % of control. Results are expressed as a mean ± SEM. of three experiments performed in triplicate for both MK cell lines, and statistical analysis was performed using 1-way ANOVA followed by Tukey test, comparing each inhibitor vs control at each time point. ∗∗∗ P < .001; ∗∗∗∗ P < .0001. AA, arachidonic acid; ASA, aspirin; Ctrl., Control; ns, nonsignificant; TX, thromboxane.

Article Snippet: MEG-01 cell line (CRL-2021) was obtained from ATCC and CHRF-288-11 cells kindly provided by Jean-Philippe Rosa (INSERM U1176).

Techniques: Activity Assay, Labeling, Control

Journal: The Journal of Pharmacology and Experimental Therapeutics

Article Title: Aspirin inhibition and recovery of cyclooxygenase activity and thromboxane biosynthesis in human megakaryocytes: a translational surrogate model

doi: 10.1016/j.jpet.2025.103762

Figure Lengend Snippet: Effect of daily treatment with aspirin on TXB 2 biosynthesis in MEG-01. Cells were treated at day 0, washed, and treated daily with 10, 1, or 0.1 μ M aspirin. COX activity was assessed each day by TXB 2 expressed in ng/million cells and represented as a percentage of control (mean ± SEM, n = 3–4, performed in triplicate). Statistical analysis was performed using one-way ANOVA followed by Tukey test, ∗∗∗∗ P < .0001. TX, thromboxane.

Article Snippet: MEG-01 cell line (CRL-2021) was obtained from ATCC and CHRF-288-11 cells kindly provided by Jean-Philippe Rosa (INSERM U1176).

Techniques: Activity Assay, Control