PKCδ Localization at the Membrane Increases Matrix Traction Force Dependent on PLCγ1/EGFR Signaling
Figure 1
Membrane targeted PKCδ increases force of isometric contractions through EGFR/ PLCγ1 signaling.
(Cell Traction Force Microscopy). a) A schematic of PKCδ showing the kras farnesylation motif at the c-terminus of the protein. Membrane targeted PKCδ (PKCδ-CaaX)is represented with the CVIM domain and non-membrane targeted (PKCδ-SaaX) is represented by SVIM. b,c)PKCδ-CaaX cells were placed on (0.5µm red beads) were prepared with 100 µg collagen cross-linked to PAG/beads. b) Cell traction was extrapolated through bead displacement as the cells exerted force. All forces exerted onto the substratum of each cell by bead displacement were computationally measured and analyzed using the software MatLab environment [26–28]. Unconstrained traction is force exerted by the cells in kPa that is derived from bead displacement on 3kPa PAG/bead gel and described previously in (25-27). Traction force output with unconstrained traction and absolute bead displacement from data extrapolation was gathered from all groups for each individual cell. Colorimetric indicators displays red as the most intense in traction force and dark blue displays minimal traction force. Images of cells were taken at 20X objective magnification. c) Boxplot of individual cell constrained force measurements between 25th and 75th percentile. Collective statistical analysis via Student’s T Test was performed between NR6-WT PKCδ-CaaX and NR6-991-PKCδ-Caax after EGF treatment at p = 6.87821e-09). As indicated in results and methods, NR6-WT cell lines contain full length EGFR and NR6-991 cell lines contain truncated EGFR that is deficient in PLCy1 signaling. d) Immunoblot analysis of cells transiently transfected with (PKCδ-C/SaaX) and 50 µM of siRNA of mouse PKCδ siRNA into NR6-WT and NR6-991 fibroblast were then incubated in quiescent media overnight and treated with EGF for 1 hour prior to cell lysis. Western blot analysis of cell lysates was performed. GFP that is expressed with the vector was utilized as control for protein levels. e) Lysates of siRNA knockdown of endogenous PKCδ of fibroblasts is represented in immunoblot. The non-linked GFP on the same vector were utilized for loading control. f) Immunoblot of cell lysates with stably transfected PKCδ-CaaX and PKCδ-SaaX. Non-linked GFP protein levels were utilized for loading control.