The latter were enriched by flowing blood through a microfluidic channel coated with anti-hCD47 and then staining for the erythroid-specific marker, hGPA (Fig

The latter were enriched by flowing blood through a microfluidic channel coated with anti-hCD47 and then staining for the erythroid-specific marker, hGPA (Fig. inhibition of MII in adherent embryonic stem cells (ESCs) increases survival in culture by preserving intercellular contacts (Chen et al., 2010) inhibition can also lead to multi-nucleated cells (Canman et al., 2003). Actomyosin forces generally stabilize the plasma membrane with an active cortical tension or rigidity (Merkel et al., 2000), but these forces also drive cell rounding in cytokinesis (Sedzinski et al., 2011) and can change dramatically in differentiation (of MSCs) (Engler et al., 2006). Indeed, while it has been known for many years that as granulocytes differentiate they become soft to better traffic from marrow through the endothelial GSK1059615 barrier and into the circulation (Lichtman, 1970), any changes in MII in such cells leaving the marrow or other hematopoietic cells is currently unknown. Mammals express three isoforms of MII: A (sum total intensity (a.u.). (i) Images of co-immunostained MIIA and MIIB (bars = 5 m). (ii) Representative intracellular FACS dot plots show expression GSK1059615 of MIIA, pS1943 and MIIB (Y-axis) across subpopulations (markers indicated in X-axis). GSK1059615 (iii) Mean fluorescent intensity of MII’s for each subpopulation from flow cytometry was normalized to an internal fluorescence control (A549), and B:A was calibrated to an absolute ratio from mass spectrometry analyses of MSCs (B:A = 6:94). The perforated endothelium schematically illustrates the permeable barrier between bone marrow and circulating cells. MKP: MK Progenitor 1 (CD34+CD41+), 2 (CD34-CD41+); ProE: Proerythroblast (CD44+GPA-); EryP: Erythroid Progenitor 1 (CD44+GPA+), 2 (CD44-GPA+); Plt: Platelet; T, B: Lymphoid; Myemid, Myehi: Bone marrow CD33+ myeloid. WBC: Mean result for PB. Mean SEM of n 3, with errors bars omitted if < 5% of mean. (B) Key genes correlated with and ranked by |Pearson correlation| > 0.75 or fit with a power-law. (i) Datasets were derived from RMA summarized microarray analyses of fresh populations of HSC-enriched, MPP, CPP and cultured CD34+-derived cells control or treated with Blebb (see Supplemental Experimental Procedures). Colors in bargraphs and gene symbols respectively represent power law exponents or gene intensities, and they are normalized by minimum levels (Green: 0 or log23) and maximum levels (Red: 3 or log211) of correlated genes using as a reference (Black: 1 or log26). Representative correlation plots between (1/2)11(1/2)5 = 0.000015. This high significance provides a metric of the systematic consistency of our MII measurements. Since MIIB was highest at the protein level in CD34+ subpopulations, microarray profiling of the different stem/progenitor/differentiated cells allowed us to identify genes that correlate with expression of (Fig. 1B, i). correlated strongly with in showing a power law exponent of 1 1.8 (Fig. 1B, ii), whereas the differentiation gene is strongly anti-correlated with a power law of -1.8 exponent (Fig. 1B, iii). shows no correlation with and color-coded for the power law. CCNU Consistent with protein-level analyses, both and transcripts are of similar (mid-range) intensity. About 1% of the microtubule system (marrow elasticity by atomic force microscopy (AFM). Young’s modulus = 2 (2/) ()/(1- v2), where is the indentation, v is the Poisson ration (assumed to be 0.5), and is the half opening angle of the AFM tip (Sneddon, 1965). From 88 measurements done on 4 mouse tibia or GSK1059615 femur samples, cells pulled into micropipettes by aspiration (Ren et al., 2009). Hematopoietic cells were likewise aspirated at low stress (<1 kPa) after transfection of GFP-MIIA or MIIB, and within just 20 min, MIIB polarized by more than 10-fold into the stressed projection (Fig. 2D), while MIIA polarized much less. Importantly, receptors such as integrins do not engage the micropipette wall, and so polarization is independent of adhesion. Partial knockdown of MIIB in CD34+ cells followed by aspiration also showed greater distension of the membrane as well.