Journal: Cell Death & Disease

Article Title: Essential role of zyxin in platelet biogenesis and glycoprotein Ib-IX surface expression

doi: 10.1038/s41419-021-04246-x

Figure Lengend Snippet: A Differential proteomic analysis of proteins expressed in WT and Zyx −/− platelets; n = 3 mice per genotype. B Surface level of GPIb-IX complex on WT and Zyx −/− platelets analyzed by flow cytometry; n = 3 mice per genotype. MFI, mean fluorescence intensity. C Western blot analysis of GPIb-IX in WT and Zyx −/− platelets. Protein concentration has been adjusted to the same level between WT and Zyx −/− platelet lysates. Blots are representative of five independent experiments. D Expression of GPIb-IX complex in WT and Zyx −/− MKs. Left panels, representative confocal images of MKs in WT and Zyx −/− mouse femoral BM sections immunostained with anti-GPIbα, GPIbβ, and GPIX antibodies (original magnification ×200). GPIbα, GPIbβ, and GPIX in MKs are in green, and nuclei are in blue. Scale bar: 100 μm. Right panels, the quantification of MFI of GPIbα, GPIbβ, and GPIX in the left panels analyzed by ImageJ software; n = 10 visual fields from 5 mice per genotype. Data are expressed as means ± SD. E Percentage of GPIbα + cells differentiated from WT and Zyx −/− FL HPCs was analyzed by flow cytometry. F Percentage of GPIbα + platelet-sized particles released from culture-derived MKs in ( E ) was analyzed by flow cytometry. Data are from four independent experiments in ( E ) and ( F ). Means are indicated by horizontal lines in ( A ), ( B ), ( E ), and ( F ). ** P < 0.01, *** P < 0.001, compared with WT mice by unpaired Student’s t -test in ( A ), ( B ), and ( D ), and by two-way ANOVA followed by Bonferroni’s post hoc test in ( E ) and ( F ).

Article Snippet: Antibodies against zyxin (10330-1-AP), GPIX (14-564-1-AP), GFP tag (50430-2-AP), and flag tag (20543-1-AP) were from Proteintech (Wuhan, China).

Techniques: Flow Cytometry, Fluorescence, Western Blot, Protein Concentration, Expressing, Software, Derivative Assay

Journal: Cell Death & Disease

Article Title: Essential role of zyxin in platelet biogenesis and glycoprotein Ib-IX surface expression

doi: 10.1038/s41419-021-04246-x

Figure Lengend Snippet: A mRNA levels of GPIbα, GPIbβ, and GPIX in WT and Zyx −/− platelets. mRNA expression was analyzed by qRT-PCR and determined by a ratio relative to the control GAPDH. The data were expressed as the ratio relative to WT; n = 5 mice per genotype. B Western blot analysis of GPIbα protein in WT and Zyx −/− platelets. Protein concentration was adjusted to the same level between WT and Zyx −/− platelet lysates. Blots are representative of five independent experiments. C, D Dami cells were transfected with siRNAs targeting zyxin (si ZYX -1, and -2) or negative control siRNA (control). The expression of GPIbα and GPIX in Dami cells was analyzed by Western blot; the blots are representative of five independent experiments ( C ). The surface level of GPIbα and GPIX was analyzed by flow cytometry ( D ). E, F Dami cells were treated with or without 10 μg/mL leupeptin plus 10 mM NH 4 Cl (Leu + NH 4 Cl) and MG-132 (100 nM) for 12 h after zyxin siRNA (si ZYX -1) transfection. The total expression of GPIbα was analyzed with Western blot; the blots are representative of five independent experiments ( E ). The surface level of GPIbα was analyzed by flow cytometry ( F ). Data are from five independent experiments in ( C – F ). Means are indicated by horizontal lines in ( A ) and ( C–F ). * P < 0.05, ** P < 0.01, *** P < 0.001, by one-way ANOVA followed by Dunnett’s post hoc test in ( C–F ). NS, not significant.

Article Snippet: Antibodies against zyxin (10330-1-AP), GPIX (14-564-1-AP), GFP tag (50430-2-AP), and flag tag (20543-1-AP) were from Proteintech (Wuhan, China).

Techniques: Expressing, Quantitative RT-PCR, Control, Western Blot, Protein Concentration, Transfection, Negative Control, Flow Cytometry

Journal: Developmental Cell

Article Title: Genome-wide Analysis of Simultaneous GATA1/2, RUNX1, FLI1, and SCL Binding in Megakaryocytes Identifies Hematopoietic Regulators

doi: 10.1016/j.devcel.2011.04.008

Figure Lengend Snippet: GATA1, GATA2, RUNX1, FLI1, and SCL Binding to the Human Megakaryocyte Genome (A–E) Raw ChIP-Seq read data was transformed into density plots and displayed in the UCSC genome browser above the tracks for gene structure and vertebrate homology. The vertical viewing range was set at 0–50. Black vertical bars show the location of the PCR primers used to validate the ChIP material. Shown here are the loci RUNX1 (A), SCL/TAL1 (B), ITGA2B (CD41) (C), GP9 (CD42a) (D), and GP1BB (CD42c) (E). (F) Each peak was allocated to be either within a promoter, intragenic, or intergenic region. The pie chart shows the distribution of the peaks across those three categories (blue, green, and red, respectively). See also Figure S1 .

Article Snippet: Flow cytometry was performed on a CyAn ADP 9 color (Beckman Coulter) as described ( ) using the following antibodies: CD11c PE-Cy5 or V450 (clone B-ly6; Becton Dickinson [BD]), CD14 PB (clone M5E2; BD), CD15 APC (clone HI98; BD) or V450 (clone MMA; BD), CD34 PE (clone 581; Beckman Coulter), CD41 APC (clone HIP8; BD), CD42a F (clone ALMA.16; BD), CD66 PE (clone B6.2; BD), and IgG APC (clone MOPC-21; BD).

Techniques: Binding Assay, ChIP-sequencing, Transformation Assay