ago2 deletion wild type meg 01 cells  (ATCC)

Journal: Scientific reports

Article Title: Argonaute2 modulates megakaryocyte development and sex-specific control of platelet protein expression and reactivity.

doi: 10.1038/s41598-025-88106-0

Figure Lengend Snippet: Fig. 1. Characterization of Ago2-deleted murine platelets. Whole citrated blood collection from Eif2c2fl/fl/Pf4- Cre (Ago2 KO) or Pf4-Cre mice was subject to complete blood count analysis using a HEMAVET analyzer. (A) Platelet counts. (B) Mean platelet volumes (MPV). (C) Platelet counts by sex, shown as box and whisker distribution plots with max/min. (A, C); no significant difference between groups. (D) MPV by sex. (E) Platelet counts following platelet depletion with anti-Gp1bα antibodies. (F) MPV of platelets from (E). (G) Platelet lifespan monitored by non-depleting X488-Gp1bβ antibodies (X488) as described in Methods. Lines in (A- D) indicate median values. Error bars indicate s.e.m. n.s., no significant difference; other data are n.s. unless indicated otherwise. (A-D), n = 25 each; (E-F), n = 3 each. *, p < 0.05; otherwise, no significant differences between groups.

Article Snippet: MEG-01 cells and AGO2 deletion Wild-type MEG-01 cells (ATCC line CRL-2021) were cultured in RPMI medium (Gibco, Grand Island, NY, USA) with 10% FBS (Corning, Corning, NY, USA), 1% non-essential amino acids (Cytiva, Amersham, United Kingdom), and 1% penicillin/streptomycin (GE HealthCare Life Sciences, Chicago, IL, USA) in a humidified, 5% CO2 atmosphere at 37 C. Cells were cultured in EasYFlasks (Thermo Scientific, Waltham, ME, USA) and treated with 50 ng/mL of phorbol myristate acetate (PMA, Sigma-Aldrich, St. Louis, MO) to induce differentiation and platelet-like particle formation.

Techniques: Whisker Assay

Journal: Scientific reports

Article Title: Argonaute2 modulates megakaryocyte development and sex-specific control of platelet protein expression and reactivity.

doi: 10.1038/s41598-025-88106-0

Figure Lengend Snippet: Fig. 2. Altered megakaryocyte development in Ago2 KO mice. (A) Representative femurs and spleens obtained from Pf4-Cre or Ago2 KO mice, sectioned and stained with hematoxylin and eosin. Bars, 200 μm. (B) Quantification of megakaryocytes from images as in (A). (C) Mean forward scatter (FSC) of femur megakaryocytes identified with Cd42d fluorophore-conjugated antibodies by flow cytometry. (D) Ploidy as a function of FSC in megakaryocytes from (C), identified by propidium iodide staining. (E) Quantification of megakaryocyte area from femoral sections from (A), subject to immunohistochemical staining with Cd42d fluorophore-conjugated antibodies, shown + s.e.m. (F) Percentage of femur megakaryocytes per ploidy class from (D). n = 4; *, p < 0.05; **, p < 0.01; ***, p < 0.001. n.s., no significant differences.

Article Snippet: MEG-01 cells and AGO2 deletion Wild-type MEG-01 cells (ATCC line CRL-2021) were cultured in RPMI medium (Gibco, Grand Island, NY, USA) with 10% FBS (Corning, Corning, NY, USA), 1% non-essential amino acids (Cytiva, Amersham, United Kingdom), and 1% penicillin/streptomycin (GE HealthCare Life Sciences, Chicago, IL, USA) in a humidified, 5% CO2 atmosphere at 37 C. Cells were cultured in EasYFlasks (Thermo Scientific, Waltham, ME, USA) and treated with 50 ng/mL of phorbol myristate acetate (PMA, Sigma-Aldrich, St. Louis, MO) to induce differentiation and platelet-like particle formation.

Techniques: Staining, Flow Cytometry, Immunohistochemical staining

Journal: Scientific reports

Article Title: Argonaute2 modulates megakaryocyte development and sex-specific control of platelet protein expression and reactivity.

doi: 10.1038/s41598-025-88106-0

Figure Lengend Snippet: Fig. 3. Sex-selective increased thromboxane reactivity in Ago2-deleted platelets. Platelets from Pf4-Cre (black lines and circles) or Ago2 KO mice (red lines and circles) were subject to agonist stimulation as indicated in the presence of fluorophore-conjugated antibodies against activated αIIbβ3 integrin (Jon/A) and P-selectin (Cd62p), and measured by flow cytometry. (A) U46619. (B) Thrombin. (C) ADP. (D) Convulxin. (A-D) n = 10 each. (E) Data from (A) separated by sex as indicated. n = 5 each. *, p < 0.05; **, p < 0.01; unlabeled data points showed no significant differences between groups. MFI, mean fluorescence intensities, shown ± s.e.m.

Article Snippet: MEG-01 cells and AGO2 deletion Wild-type MEG-01 cells (ATCC line CRL-2021) were cultured in RPMI medium (Gibco, Grand Island, NY, USA) with 10% FBS (Corning, Corning, NY, USA), 1% non-essential amino acids (Cytiva, Amersham, United Kingdom), and 1% penicillin/streptomycin (GE HealthCare Life Sciences, Chicago, IL, USA) in a humidified, 5% CO2 atmosphere at 37 C. Cells were cultured in EasYFlasks (Thermo Scientific, Waltham, ME, USA) and treated with 50 ng/mL of phorbol myristate acetate (PMA, Sigma-Aldrich, St. Louis, MO) to induce differentiation and platelet-like particle formation.

Techniques: Flow Cytometry, Fluorescence

Journal: Scientific reports

Article Title: Argonaute2 modulates megakaryocyte development and sex-specific control of platelet protein expression and reactivity.

doi: 10.1038/s41598-025-88106-0

Figure Lengend Snippet: Fig. 4. Normal hemostasis and clot dynamics following vascular injury in platelet-specific Ago2-deleted mice. (A) Time to initial stoppage of bleeding after 1-mm diameter tail tip amputation in Pf4-Cre (black lines and circles) or Ago2 KO mice (red lines and circles). (B) Rebleeds following primary hemostasis. The line and error bars indicate median and interquartile ranges. (C) Time to hemostasis after rebleeds. The experiment was stopped at 1200 s. No significant differences between groups were observed. n = 20. (D) Data (A-C) separated by sex as indicated, shown ± s.e.m. (E-I) Hemostatic clot dynamics in cremaster muscle arterioles after laser injury, monitored by confocal intravital fluorescence microscopy. 21 injuries in 3 Pf4-Cre male mice and 20 injuries in 3 Ago2 KO male mice. (A) Platelet accumulation (Cd41 area). (B) Cd62p exposure. (C) Fibrin deposition. (H) Area under curve (AUC) for (E). (I) Peak in curves from (E). The line and error bars indicate median and interquartile ranges.

Article Snippet: MEG-01 cells and AGO2 deletion Wild-type MEG-01 cells (ATCC line CRL-2021) were cultured in RPMI medium (Gibco, Grand Island, NY, USA) with 10% FBS (Corning, Corning, NY, USA), 1% non-essential amino acids (Cytiva, Amersham, United Kingdom), and 1% penicillin/streptomycin (GE HealthCare Life Sciences, Chicago, IL, USA) in a humidified, 5% CO2 atmosphere at 37 C. Cells were cultured in EasYFlasks (Thermo Scientific, Waltham, ME, USA) and treated with 50 ng/mL of phorbol myristate acetate (PMA, Sigma-Aldrich, St. Louis, MO) to induce differentiation and platelet-like particle formation.

Techniques: Fluorescence, Microscopy

Journal: Scientific reports

Article Title: Argonaute2 modulates megakaryocyte development and sex-specific control of platelet protein expression and reactivity.

doi: 10.1038/s41598-025-88106-0

Figure Lengend Snippet: Fig. 5. Induced Ago1 expression in Ago2-deleted platelets. (A) Segment from Supplemental Table 1, showing Argonaute peptides identified in Pf4-Cre (WT) and Ago2 KO (KO) platelets by LC/MS/MS. Red boxes indicate Ago2-specific peptides or Ago1-specific peptides as indicated. (B) Platelet lysates as indicated were separated by SDS-PAGE and subject to immunoblotting with the indicated antibodies. Original blots are presented in Supplemental Fig. 4. (C) Densitometric quantitation of immunoblot results from (B). n = 3 each.

Article Snippet: MEG-01 cells and AGO2 deletion Wild-type MEG-01 cells (ATCC line CRL-2021) were cultured in RPMI medium (Gibco, Grand Island, NY, USA) with 10% FBS (Corning, Corning, NY, USA), 1% non-essential amino acids (Cytiva, Amersham, United Kingdom), and 1% penicillin/streptomycin (GE HealthCare Life Sciences, Chicago, IL, USA) in a humidified, 5% CO2 atmosphere at 37 C. Cells were cultured in EasYFlasks (Thermo Scientific, Waltham, ME, USA) and treated with 50 ng/mL of phorbol myristate acetate (PMA, Sigma-Aldrich, St. Louis, MO) to induce differentiation and platelet-like particle formation.

Techniques: Expressing, Liquid Chromatography with Mass Spectroscopy, SDS Page, Western Blot, Quantitation Assay