(C,D) Cells under circulation conditions (circulation direction is from left to right). Click here to view.(14M, mov) Supp Movie S2bClick here to view.(13M, mov) Supp Movie S2cClick here to view.(6.0M, mov) Supp Movie S2dClick here to view.(5.1M, mov) Supp Movie S3Supplementary Movie 3. direction of circulation. Scale bar is usually 10 m. NIHMS275527-supplement-Supp_Physique_S1.tif (691K) GUID:?67ACD191-C9F3-4057-89C6-692335C98E1F Supp Physique S2: Supplementary Physique 4. ARH-77 type A and CAG cells show similar pressure response profiles ARH-77 type A and CAG cells were placed inside circulation chambers and subjected to shear pressure of 4 dynes/cm2, which Regorafenib monohydrate was then increased in a stepwise fashion (4 moments per step) to 8, 12, 16, 20, 24, 28 and 32 dynes/cm2. Time-lapse movies were taken throughout the process, and FLIP formation was constantly monitored and Regorafenib monohydrate scored. Offered is the quantity of FLIP-extending cells counted at each time point, calculated as a percentage of the total quantity of FLIP- forming cells in the experiment. As shown, the pressure response profile of CAG cells is very similar to that of ARH-77 type A cells. NIHMS275527-supplement-Supp_Physique_S2.tif (1.4M) GUID:?7D9047F1-DAC2-462C-A2B4-8D10C083A3DE Supp Movie S1: Supplementary Movie 1: ARH-77 type A multiple myeloma cells develop flow-induced protrusions (FLIPs) Cells were seeded and allowed to adhere to fibronectin-coated glass coverslips, placed in a flow chamber, and exposed to shear flow of 20 dynes/cm2 for PDGFC 8 minutes. Numerous FLIPs are obvious. Time was measured from the beginning of circulation. Scale bar indicates 20m. NIHMS275527-supplement-Supp_Movie_S1.avi (5.7M) GUID:?88A943C1-6D70-4F3B-853F-CDE6F11BC793 Supp Movie S2a: Supplementary Movie 2. Three-dimensional reconstruction of the images shown in Physique 4 Fluorescence microscopy images of ARH-77 type A cells. Cells were fixed under circulation and labeled with DAPI, phalloidin-FITC and anti–tubulin. (A, B) Stationary cultures. (C,D) Cells under circulation conditions (circulation direction is usually from left to right). NIHMS275527-supplement-Supp_Movie_S2a.mov (14M) GUID:?44A85CE5-4AF9-4120-87EB-1A26F36B2E36 Supp Movie S2b. NIHMS275527-supplement-Supp_Movie_S2b.mov (13M) GUID:?54A1CEED-5822-4B5D-BD9D-F3666CE207E0 Supp Movie S2c. NIHMS275527-supplement-Supp_Movie_S2c.mov (6.0M) GUID:?74E2FD60-3114-452C-BA7D-DCED480B04A7 Supp Movie S2d. NIHMS275527-supplement-Supp_Movie_S2d.mov (5.1M) GUID:?C80A46AA-9047-4354-BBD9-6A47F6858E22 Supp Movie S3: Supplementary Movie 3. Phase contrast time-lapse movie of ARH-77 type A cells under circulation, showing the same cells as those Regorafenib monohydrate in Physique 5 FLIPs are elongated from their tips, concurrently with a slow backward motion, while concomitantly retracting at their base. Time interval of each frame is usually 5 seconds. Direction of circulation is right to left. NIHMS275527-supplement-Supp_Movie_S3.mov (6.7M) GUID:?A5F5AE4A-4CC6-4400-8102-EA28833E92EC Abstract Exposure of live cells to shear flow induces major changes in cell shape, adhesion to the extracellular matrix, and migration. In the present study, we show that exposure Regorafenib monohydrate of cultured multiple myeloma (MM) cells to shear circulation of 4C36 dynes/cm2 triggers the extension of long tubular protrusions (denoted FLow-Induced Protrusions, or FLIPs) in the direction of the Regorafenib monohydrate circulation. These FLIPs were found to be rich in actin, contain few or no microtubules and, apart from endoplasmic reticulum (ER)-like membranal structures, are devoid of organelles. Studying the dynamics of this process revealed that FLIPs elongate at their suggestions in a shear force-dependent manner, and retract at their bases. Examination of this pressure dependence revealed considerable heterogeneity in the mechanosensitivity of individual cells, most likely reflecting the diversity of the malignant B-cell populace. The mechanisms underlying FLIP formation following mechanical perturbation, and their relevance to the cellular trafficking of MM cells, are discussed. (Sens et al., 2010); extracellular matrix and soluble factors (e.g., EGF) can induce filopodial and lamellipodial protrusions in various cell types (Hu et al., 2010; Mori et al., 2010). While these serve as examples of membrane modification by extracellular biochemical signals, biophysical cues may also trigger the formation of membrane protrusions, including the extension of FLIPs, as explained in the current study. While FLIP formation is usually a novel phenomenon, cellular responses to shear circulation have been documented in diverse cell types. It has been shown that endothelial cells subjected to near-physiological shear undergo uniform alignment (Dewey et al., 1981; Galbraith et al., 1998; Masuda and Fujiwara, 1993), and directional migration and lamellipodial extension in the direction of circulation (Dewey et al., 1981; Wojciak-Stothard and Ridley, 2003; Zaidel-Bar et al., 2005). Hematopoietic cells, which reside, at least transiently, in high-shear vascular environments, respond to circulation in a variety of ways. T cells undergo dynamic shape changes during trans-endothelial migration, including tethering and rolling along the endothelial surfaces, firm attachment to the underlying cells, spreading to them, and trans-migration through the endothelial cell layer (Alon and Dustin, 2007; Dong et al., 1999; Stroka and Aranda-Espinoza, 2010). Platelets also go through several shape changes, including transition from a round morphology, forming multiple elongated extensions during the adhesive process under circulation (Kuwahara et al., 2002). In addition, flow-induced effects were seen in other cell types, such as rolling of human bone-metastatic prostate tumor cells on endothelial cells (Dimitroff et al., 2004); transendothelial migration of melanoma cells (Slattery and Dong, 2003); increased adhesion and distributing in colon cancer cells (Burdick et al., 2003; Kitayama et al., 1999); and.
