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  • In experiments measuring focal adhesion area HUVECs were

    2018-10-25

    In experiments measuring focal adhesion area, HUVECs (4×105) were also settled onto ECM-coated micro-posts as described above. Cells were fixed in 3.7% (w/v) paraformaldehyde in PBS for 10min at R.T. and permeabilized in XMU-MP-1 stabilization buffer (100mM, 300mM sucrose, 3mM MgCl2, 1mM EGTA and 10mM PIPES, pH 6.8) containing 0.3% (v/v) Triton X-100 for 1min at R.T. [4]. Cells were washed once in PBS and incubated in PBS containing 1% (w/v) BSA for 30min at R.T. Cells were then incubated in PBS containing 1% (w/v) BSA and 5µg/ml anti-paxillin (clone 5H11) (Merck) at 4°C overnight followed by Alexa Fluor® 488-conjugated goat anti-mouse IgG secondary antibody (1:600 dilution) (Invitrogen, Carlsbad, CA). Cells were examined under a confocal laser scanning microscope (Zeiss LSM510, Carl Zeiss, Germany) with 63× oil immersion objective lens. Data were analyzed using the Zen 2012 software. To quantify the focal adhesion size, fluorescent images were converted to black-and-white images using the ImageJ software (open source, imagej.net). Background subtraction was performed to reduce noise by setting threshold limit. The number of pixels with values above the threshold which represent the size of focal adhesion was calculated. To quantify the cell spread area, fluorescent images were converted to black-and-white images using the ImageJ software (open source, imagej.net), and Canny edge detection was applied to binarize the images (Mathworks, Novi, MI). Image dilation, erosion, and fill operations were used to fill in the gaps. The number of white pixels was summed to determine the cell spread area. The directionality of contraction was determined by taking the magnitude of the sum of force vectors divided by the sum of their magnitudes [5]. A value of 1.0 suggests parallel forces whereas a value of 0 suggests balanced forces. The correlation value of force vectors of neighboring pillars by taking the cosine of the angle between the two neighboring force vectors. A correlation value of 1.0 is attained when the angle is 0 (i.e. when the two neighboring force vectors are parallel).
    Conflict of interests
    Acknowledgments These experiments were supported by Nanyang Technological University, Singapore, AcRF grant RG39/12 (S.M.T, H.F.T and C.F.) and grant M4081320 (S.M.T.), and Singapore Ministry of Education grants M4011037 and M4080859 (J.J.H. and S.Y.T.). W.K.W. is supported by the program Undergraduate Research Experience on Campus (URECA), Nanyang Technological University, Singapore. J.J.H. and H.F.T contributed equally to the study.
    Data Fig. 1 examines the cell cycle distribution of HeLa cells stably expressing either an empty vector (HeLa EV) or CYB5D2 (HeLa CYB5D2). Fig. 2 shows the status of AKT and ERK1/2 activation in HeLa EV and HeLa CYB5D2 cells. Activation of AKT and ERK1/2 was indirectly determined according to the specific phosphorylation events (see Fig. 2 legend for details). CYB5D2 can be a secretory protein [2,3] that has been indicated to inhibit Neuro2a cell proliferation [2]. The cell cycle distribution of HeLa cells was determined in the presence of either GST or GST-CYB5D2 (Fig. 3). Fig. 4 shows recognition of the CYB5D2 protein in human kidney tissues by the anti-CYB5D2 antibody in the presence of GST or GST-CYB5D2 as a competitor. Table 1 shows the tissues used to examine the CYB5D2 protein levels in normal cervical and cervical cancer tissues.
    Experimental design, materials and methods
    Acknowledgments We like to dedicate this work to a great mother Ms. Guorui Zeng. This work was supported in part by a grant (No. 81302210) from National Natural Science Foundation of China to Y. Xie, and Heart and Stroke Foundation of Canada and CIHR (MOP-84381) to D. Tang.
    Data The calcium-dependent mobility shift of NCS proteins were determined by loading the proteins in native gels and subjecting them to electrophoresis. Apart from the protein and calcium calibration buffer, the loaded sample contained additional components which were tested in this report. They are 20mm Tris pH 7.5 (Chelex100-treated), loading dye and running buffer, and distilled water treated with Chelex-100 (Chelex-water). Samples and standards were diluted in 5% nitric acid in ICP grade water rated at 18.2MΩcm. Data was compiled from at least three independent replicates for each reagent.