Supplementary Materials Supplemental Materials supp_28_23_3156__index
Supplementary Materials Supplemental Materials supp_28_23_3156__index. these forces. These alterations in cell mechanics are abrogated when the actin cytoskeleton is usually dismantled. These data suggest that the tissue-specific variability in DSMCIF network composition provides an opportunity to differentially regulate tissue mechanics by balancing and tuning causes among cytoskeletal systems. INTRODUCTION Cells are integrated into tissues through macromolecular adhesive organelles specialized for anchoring different cytoskeletal components at cellCextracellular matrix and cellCcell adhesion sites. At the cell substrate, actin and intermediate filaments (IFs) are anchored by focal adhesions and hemidesmosomes, respectively, whereas at cellCcell interfaces, actin and IFs are anchored by adherens junctions (AJs) and desmosomes (DSMs) (Simpson = 1. (D) Superresolution micrographs of cells treated with the indicated compounds are shown with actin in white, plakoglobin (PG) in green to indicate the cellCcell junction, and DAPI in blue to show nuclei. Pramipexole dihydrochloride monohyrate (E) The average intercellular pressure and the average traction force per pillar are shown for cells treated with the indicated compounds. Error bars symbolize the standard error of the mean for 7C28 cells from three impartial experiments. *, 0.001; **, = 0.02. Several methods for quantitative assessment of cell causes have been developed, including atomic pressure microscopy (AFM) (Brunner and are the traction force (nN) and lateral deflection of the pillar (m), respectively. Within a cell pair, the net causes are in equilibrium. Therefore the sum of the traction forces in one cell is equivalent and reverse in direction to that of the adjacent cell and vice versa, and thus represents the intercellular pressure (Liu 0.05; N.S., not Pramipexole dihydrochloride monohyrate significant. (D) Representative Western blot indicating knockdown of endogenous DP in cells treated with either nontargeting siRNA (siCtl) or siRNA targeting DP (siDP). Tubulin is usually shown as a loading control. (E) The average traction force per pillar and the average intercellular pressure are shown for cells treated with either nontargeting siRNA (siCtl) or siRNA targeting DP (siDP). Error bars represent the standard error of the mean from at least 30 cells from three impartial experiments. *, 0.0001. To determine whether modulating the DSMCIF linkage influences cell mechanics, we compared cell causes in control and DP variantCexpressing cell pairs. Expression of DPNTP resulted in a decrease in the average intercellular tugging pressure and traction force per pillar compared with uninduced controls (Physique 2C). In addition, small Pramipexole dihydrochloride monohyrate interfering RNA (siRNA) targeting endogenous DP was used to knock down DP expression (Physique 2D). DP knockdown produced effects much like those of DPNTP when compared with nontargeting siRNA controls (Physique 2E). These data suggest that uncoupling the DSMs from your IFs reduces both cellCcell and cellCsubstrate causes. Notably, DP knockdown resulted in a greater decrease in the average traction force per pillar compared with DPNTP. Because DPNTP displaces endogenous DP from cellCcell contacts but does not alter DP protein levels (Physique 2A), Mouse monoclonal to Calreticulin these data raise the possibility that nonjunctional DP impacts cellCsubstrate causes, perhaps indirectly through its ability to interact with other proteins, including kinases (Albrecht 0.0001. (D) Average cell stiffness measurements of individual cells within semiconfluent (80%) cell linens for cells expressing the indicated DP variants are shown (control and induced conditions). Error bars represent the standard error of the mean from at least 91 cells from three impartial experiments. *, 0.0001. (E) Average cell stiffness measurements on single cells, cell pairs, and cell linens for DP knockdown (siDP) and nontargeting siRNA control (siCtl) conditions are shown. Error bars represent the standard error of the mean from at least 55 cells from three impartial experiments. *, 0.0001. (F) Average cell stiffness measurements of calcium-insensitive, confluent cell linens for cells expressing the.