Endothelial Glycocalyx-Mediated Nitric Oxide Production in Response to Selective AFM Pulling  Anne Marie W. Bartosch, Rick Mathews, John M. Tarbell  Biophysical Journal  Volume 113, Issue 1, Pages 101-108 (July 2017) DOI: 10.1016/j.bpj.2017.05.033 Copyright © 2017 Biophysical Society Terms and Conditions

Figure 1 AFM pulling and AFM probe functionalization. Cartoons are not drawn to scale. (A) Cell-surface core proteins (syndecan-1, glypican-1, and CD44) and their associated GAGs (HS and HA) were stimulated with corresponding specific antibodies. (B) An AFM scanner stage is implemented to control XYZ-positioning and the deflection of a triangular cantilever is used to monitor indentation depth and pulling-off forces. Glass gridded slides were used to track location of cells probed with AFM for later imaging. Unprobed regions were imaged ∼2 mm away from the probing site. After background correction, percent activation was calculated by comparing the MFI of the probed region to the MFI of the unprobed regions. (C) Silicon nitride cantilevers are functionalized with amine groups, PEG linker, and antibody (Antibodies). Control functionalization schemes include isotype control using normal IgGs (included in Antibodies scheme), PEG linker with varying degrees of active termini (Linker), and bare (None) probes. To see this figure in color, go online. Biophysical Journal 2017 113, 101-108DOI: (10.1016/j.bpj.2017.05.033) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 2 Adhesion forces for functionalized pyramidal AFM probes on RFPEC monolayers. Mean adhesion forces ± SE values are shown for proteoglycans and CD44 in purple, for GAGs in blue, and for control probes in gray; n ≥ 60 indentations, with 20 indentations/cell and three to seven cells probed in each group. The asterisk indicates a significant difference (p < 0.01), using the two-tailed t-test for antibody-functionalized probes versus their isotype controls, HA probes and 1 M EA linker-sham probes versus a bare probe (none). The right panel displays the pulling-off region of a representative anti-glypican-1 force-distance curve during tip retraction (for complete force-distance curves, see Fig. S1 B). The separation curve can include the unbinding of several individual bonds, but adhesion force here measures the maximal detachment force between the cell surface and AFM probe. To see this figure in color, go online. Biophysical Journal 2017 113, 101-108DOI: (10.1016/j.bpj.2017.05.033) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 3 Probing RFPEC with glypican-1 and HS antibodies initiates NO production. Functionalized AFM probes were used to pull on the indicated structures for 10 min. Mean DAF-2 T MFIs from probed regions were normalized by those from unprobed regions on the same slide and converted to activation percentages (mean ± SE), with unstimulated regions indicated by 0%. ∗p < 0.05 compared to paired unstimulated regions. From left to right, n = 28, 20, 20, 22, 21, 16, 13, 11, and 19. The dotted line shows mean activation (n = 12) ± SE for RFPECs exposed to 20 dynes/cm2 shear stress for 10 min, which is significantly different from static regions at 0% activation. Representative DAF-2 T fluorescent images are shown at the left for probed and unprobed regions of the same slide. The scale bar represents 20 μm. To see this figure in color, go online. Biophysical Journal 2017 113, 101-108DOI: (10.1016/j.bpj.2017.05.033) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 4 NO induced by 10 min shear stress is not affected by HA removal in RFPECs. DAF-2 T MFIs normalized by static control MFIs are shown as the mean ± SE (left to right: n = 14, 15, 14, and 18). ∗p < 0.05 compared to paired static control. 20 dynes/cm2 of shear stress was applied to shear samples for 10 min. To see this figure in color, go online. Biophysical Journal 2017 113, 101-108DOI: (10.1016/j.bpj.2017.05.033) Copyright © 2017 Biophysical Society Terms and Conditions