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sc-11-1164_sm_SupplFigure1.tif1462KFig. S1. Analysis of endothelial adhesion molecule expression and MSC senescence (A) Flow cytometric analyses of VCAM-1 and ICAM-1 expression is shown for resting hLMVEC and hCMVEC monolayers and monolayers treated with TNF-α alone, IFN-γ alone, or both TNF-α and IFN-γ. Similarly, FACS analyses of rat VCAM-1 and ICAM-1 expression is shown for GPNT monolayers. (B) The percentage of β-galactosidase positive (i.e. senescent) MSC were quantified for P3, P5 and P7 MSC, and a positive control of P13 MSC treated with etoposide (i). Asterisks indicate statistically significant differences as assessed by a one-way ANOVA test with a Tukey post-hoc test, n=5.Two representative images of (ii) non-senescent P3 MSC and (iii) senescent P13 MSC are shown.
sc-11-1164_sm_SupplFigure2.tif672KFig. S2. Impact of PTX on MSC viability and role of cytokines, VCAM-1 and integrins MSC on adhesion and transmigration (A) Control MSC, MSC treated with 100 ng/ml of PTX or MSC treated with 1mM hydrogen peroxide (H2O2) for 2 h were stained positive for annexin V and propidium iodide (PI) and analyzed by flow cytometry. Percentage of cells in each condition that were non-apototic (annexin V and PI negative), in early apoptosis (annexin V positive, PI negative) and late apoptosis/necrosis (annexin V and PI positive) are shown. Values are mean + s.e.m, n = 3. Asterisks indicate a statistically significant differences as assessed by a one-way ANOVA test with a Tukey post-hoc test. No significant change in the frequency of apoptotic cells was observed in PTX-treated MSC. (B) MSC were incubated on resting, or TNF-α activated and/or IFN-γ activated GPNT for 60 min. Samples were fixed, stained and imaged by fluorescent confocal microscopy. Both the total number of MSC, and the number of MSC in only the transmigrating or basal positions were compared for all conditions. Data were collected from at least 6 microscopic fields for each experimental condition. Values represent mean ± s.e.m. (C) BCECF-loaded MSC were pre-treated with mIgG isotype control or blocking antibody toward α4 integrin (HP2/1) and co-incubated with either resting or TNF-α-activated hCMVEC that were pre-treated with sheep IgG isotype control or blocking antibody toward VCAM-1 for 10 min followed by washing and analysis. Results show the fluorescence signal of adherent MSC (i.e., postwash fluorescence) as a fraction of the total input MSC fluorescence (pre-wash fluorescence; Normalized Fluorescence). Values are mean + s.e.m., n = 5. Asterisks indicate statistically significant differences as assessed by a one-way ANOVA test with a Newman-Keuls post-hoc test. (D) Representative flow cytometric analysis (from three separate stainings) of MSC expression of α4 (7.2R), α4 (HP2/1; blocking antibody), α5, β1, β2 and β5 integrin subunits is shown.
sc-11-1164_sm_SupplFigure3.pdf249KFig. S3. Expression of tight and adherens junctional molecules at site of paracellular migration (A) MSC incubated on hLMVEC for 30 min were fixed and stained for beta-catenin (blue), VEcadherin (red) and CD90 (MSC; green). A representative fluorescent confocal image (single zsection) of an MSC at early-stage of paracellular transmigration is shown. Arrows highlight a discrete endothelial gap in the endothelium and breach of the adherens junction. (B) Samples as in A were stained for CD90 (green), VE-cadherin (red) and the tight junction marker occludin (blue). (i) A representative fluorescent confocal image (single z-section) of an MSC in an early/mid-stage of paracellular diapedesis through an expanded endothelial gap formed discretely at site of MSC diapedesis is shown. (ii) An orthogonal z-stack projection of the same MSC in (i). Arrows highlight a discrete breach of the adherens and tight junctions. (C) Samples as in A were stained for CD90 (green), VCAM-1 (red) and tight junction marker JAM- 1 (red). A (i) confocal projection and (ii) orthogonal z-stack projection of a representative MSC in the process of paracellular transmigration is shown. Note that JAM-1 is note restricted to th junctions as previously described 27 and that it shows co-enrichment with VCAM-1 the transmigratory cup structure (ii, white arrowheads) that surrounds the MSC. Scale bars represent 20 μm.
sc-11-1164_sm_SupplVideo1.mov5549KVideo 1. MSC induce transmigratory cup formation on endothelial surface Video depicts an MSC interacting with TNF-α activated hLMVEC and corresponds to Fig.2B. Samples were stained for CD90 (green, top left panel), VCAM-1 (red, top right panel), and actin (blue, bottom left; merge of all three channels shown in lower right panel), imaged by serial section confocal microscopy and rendered as a series of 3D projections rotated progressively about the y axis for a total of 360°. Note the formation of a cup-like structure formed by VCAM-1 enriched finger-like projections, which extend from the apical surface of the endothelium up the side of, and seemingly embracing, the MSC.
sc-11-1164_sm_SupplVideo2.mov8189KVideo 2. MSC transmigrate across the endothelium in the absence of lateral migration Video depicts dynamic live-cell DIC (right and left panels) and fluorescence (middle and left panels, red) imaging of six MSC on memDsRed-transfected, TNF-α activated hLMVEC and corresponds to Fig.3B. Numbers in first video frame identify 6 separate MSC. MSC #1 and #2 are in the process of paracellular and transcellular diapedesis, respectively. Note that for MSC #2 the transmigration pore gradually expands from ∼1 μm to nearly 20 μm (see red channel) as it progressively spreads its membrane in the subendothelial space in a starburst-like pattern as seen in the DIC channel (see outline in paused frame at 20:31 time point). MSC #3-6 are apically adherent and have not yet initiated transmigration. Notably, these cells do not display any significant spreading, polarization or net lateral migration over a 45 minute duration. However, these do exhibit sporadic ‘jerky’ motions that seem to correlate with bursts of bleb-like protrusions against the endothelial surface (e.g. orange arrow in paused frame 15:31; see also Videos 3-5). Scale bar represents 20 μm.
sc-11-1164_sm_SupplVideo3.mov1439KVideo 3. MSC exhibit multiple actin-negative and actin positive blebs on their surface Movie depicts serial confocal section (played in series) of a MSC initiating transcellular diapedesis across TNF-α activated hLMVEC and corresponds to Fig. 4A. Samples were stained for CD90 (green, top left panel), VCAM-1 (red, top right panel), and actin (blue, bottom left; merge of all three channels shown in lower right panel). Note multiple highly rounded, bleb-like structures (green; one to several mm in diameter) can be seen protruding from the MCS surface, which are both negative and positive for cortical F-actin (blue, see merged panel, bottom, right). Also note that, in addition to blebs on relatively more apical surfaces, blebs can clearly be seen at the MSC-EC interface, including at and just beneath the transcellular pore (green arrows).
sc-11-1164_sm_SupplVideo4.avi6873KVideo 4. MSC exhibit similar blebbing and absence of lateral migration on resting and activated endothelium Left and right panel shows time-lapse DIC imaging of MSC added to resting and TNF-α activated hCMVEC, respectively. No significant difference in blebbing or lateral migration of the MSC is evident. Scale bar represents 50 μm.
sc-11-1164_sm_SupplVideo5.mov23305KVideo 5. MSC blebbing may facilitate transendothelial migration Forceful membrane blebbing is associated with early stages of MSC adherence, transendothelial gap/pore formation, and subendothelial spreading as shown in a mosaic movie composed of 5 example videos representing progressive phases in the transmigration process. Example 1: An MSC apically adherent to hCMVEC imaged by high spatial and temporal (10 frames/minute) DIC imaging. Repetitive cycles of large (1 to nearly 10 mm) blebs formation and retraction over all surfaced of the MSC are seen. Individual blebs protruded rapidly, reaching their maximum diameter within an average of 18 seconds and then somewhat more slowly (average duration of retraction phase of 51s). Example 2: An MSC (DIC, left panel) apically adherent to a memRFP-transfected hCMVEC (red; third panel from left). Note that a large pre-existing paracellular gap is present near, but independent of, the MSC). Interference-contrast reflection microscopy (IRM) is shown in the second panel from the left. IRM reports regions of extremely close contact between cells (i.e., the endothelium) with the underling substrate (i.e., the coverglass) as darkened regions. It was observed that as the MSC formed bleb protrusions against the apical surface of the endothelium dynamic dark spots (with bleb-like spatial and temporal scale) in IRM (e.g., see yellow dashed line in paused frame 1:57). See additional example in Fig.5B. This provides evidence that MSC blebs can exert a force on the endothelium sufficient to locally drive it into closer contact with the underlying basal substrate. Example 3: An MSC (DIC, left panel) apically adherent near the intercellular junction (see faint vertical line of enriched red fluorescence) of two adjacent memRFP-transfected hCMVECs (red). Note that blebbing activity is associated with initial formation of small paracellular gaps. Example 4: An MSC (DIC, left panel) apically adherent near an activated GPNT EC intercellular junction formed between a positive memRFP transfected (red signaling in middle panel) and neighboring non-transfected GPNT ECs (black areas in middle panel) in a confluent monolayer. Note that in this example blebs seem to drive a dramatic expansion of a paracellular gap that becomes further distorted and expanded as the MSC begins to migrate further into the subendothelial space. See also Fig.5C. Example 5: An MSC (DIC, left panel) in late stages of transcellular diapedesis across memYFPtransfected hLMVEC. Over the course of the video MSC progresses from a state of being ∼50% below the endothelium, to being nearly completely spread in the subendothelial space (though the pore has not yet closed over the MSC). Critically, this transition is associated with extensive and dynamic membrane blebbing activity both in the apical and subendothelial portions of the MCS. It is noteworthy, that these blebs clearly exert force against the endothelium as evidenced by the induced distortion of the endothelial membrane (green). Indeed, subendothelial blebs protrusion is seen to give rise to transient bright green rings as the push against the basal surface of the endothelium. During the final ∼10 min of the video, blebbing gradually ceases as the MSC transition to spreading radially in a more lamellipodia-like fashion. Scale bar represents 10m. See also Fig.5D.
sc-11-1164_sm_SupplVideo6.mov7525KVideo 6. Blebbing is specific to MSC interactions with endothelium Movie consists of two distinct segments. In the first segment GFP-actin-transfected MSC were settled on fibronectin-coated glass. DIC is shown in top and bottom panels. GFP (green) is shown in middle and bottom panels. Note that on this substrate MSC display a combination of filopodia and lamellipodia membrane extensions. Although the MSC on the right initially demonstrates some blebbing activity, it quickly switches to the lamellipodial-mediated spreading. In the second segment aortic adventitial fibroblasts have been added to the surface of activated hCMVEC. Note that these cells do not display blebbing activity on endothelium but rather undergo gradual spreading that is coupled to filopodia- or microspike-like membrane protrusions. Scale bars represent 50 μm.
sc-11-1164_sm_SupplVideo7.mov1312KVideo 7. MSC exhibit canonical non-apoptotic migratory blebbing dynamics Movie depicts dynamic live-cell DIC (left and right panels) and fluorescence (green, middle and right panels) imaging of an actin-GFP-transfected (green) MSC transmigrating through TNF-α activated hLMVEC and corresponds to Fig.5G. The shown example is of a relatively late stage diapedesis event in which the MCS is advancing part of its membrane under the endothelium. Note that MSC blebs can be seen (via the DIC imaging) protruding from the MSC that are initially are negative for GFP-actin (green), into which GFP-actin is subsequently recruited followed by the bleb finally retraction in a fashion identical to cycles exhibited by some tumor and embryonic cell undergoing non-apoptotic migratory blebbing 16-18. These cycles of membrane protrusion and retraction seem to be coupled to the overall advancement of the MSC laterally (migrating from bottom to top in the video frame) under the endothelium. Scale bar represents 10 μm.

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