Journal: Journal of Extracellular Vesicles

Article Title: Extracellular vesicles are the primary source of blood‐borne tumour‐derived mutant KRAS DNA early in pancreatic cancer

doi: 10.1002/jev2.12142

Figure Lengend Snippet: Differential centrifugation of whole blood efficiently separates different cell and vesicle populations. (a) Schematic of the differential centrifugation protocol used to separate red (RBC) and white blood cells (WBC), platelets (PL), apoptotic bodies (AB), large (LEV) and small extracellular vesicles (SEV), as well as soluble protein (SP) and flow through (FT). (b) Representative transmission electron microscopy images of vesicles enriched in AB, LEV and SEV fractions. Corresponding scale bars are inset. (c) Representative Western blots of blood components for histone H3 (H3), CD42A, cleaved Caspase 9 (CASP9), BAX, CD9 and CD81. A CD9 blotted membrane with vesicular and SP components was additionally overexposed. Molecular weight is indicated beside each blot. (d) Nanoparticle tracking analysis data showing the concentration of particles/ml at sizes ranging from 0–1 μm in LEV, SEV and SP fractions. Inset is a quantification of the concentration of particles from 400–600 nm found in each fraction. (d) Median CD9/63/81 fluorescence for 35 capture antibody coated beads from the MACSplex vesicle surface protein flow cytometry assay in the different blood components. The platelet fraction was diluted 1:10 before analysis

Article Snippet: The antibodies used for this assay were Histone H3 (Santa Cruz FL‐136, 1:250), CD42A (Miltenyi 130‐100‐960, 1:150), cleaved‐CASP9 (Cell Signalling 9505S, 1:1000), BAX (Cell Signalling 2772S, 1:1000), CD9 (Abcam ab92726, 1:2000) and CD81 (Santa Cruz sc‐9158, 1:200).

Techniques: Centrifugation, Transmission Assay, Electron Microscopy, Western Blot, Membrane, Molecular Weight, Concentration Assay, Fluorescence, Flow Cytometry

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