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Immunocytochemistry was performed on DRGs that were transfected as described before (Moutal, et al., 2017f ). Briefly, cells were fixed using 4% paraformaldehyde, 4% sucrose for 20 minutes at room temperature. Permeabilization was achieved with a 30-min incubation in phosphate-buffered saline (PBS) containing 0.1 % Triton X-100, following which nonspecific binding sites were saturated by PBS containing 3 % BSA for 30 minutes. Cell staining was performed with primary antibodies in PBS with 3 % BSA overnight at 4°C. Cells were then washed three times in PBS and incubated with PBS containing 3 % BSA and secondary antibodies (Alexa 488 goat anti-mouse and Alexa 594 goat anti-rabbit from Life Technologies) for 1 hour at room temperature. Immunofluorescent micrographs were acquired on a Nikon C1si scanning confocal microscope using CFI Plan APO VC ×60 oil immersion objective with 1.4 numerical aperture. Camera gain and other relevant settings were kept constant. Membrane to cytosol ratio was determined by defining regions of interest on the cytosol and the membrane of each cell using Image J. Total fluorescence intensity was normalized to the area of the region of interest before calculating the membrane to cytosol ratio. Raw images were used for quantification while representative pictures were background subtracted and contrast enhanced for better visualization of the reader.

Images of cells, beads and patterns were acquired with a confocal spinning disk system (Eclipse Ti-E Nikon inverted microscope) equipped with a CSUX1-A1 Yokogawa confocal head, an Evolve EMCCD camera (Roper Scientific, Princeton Instrument) and a Nikon CFI Plan-APO VC ×60–numerical aperture 1.4 oil immersion objective). The system was controlled by MetaMorph software (Universal Imaging).
TFM and image analysis were carried out as described. All processing was carried out using ImageJ (http://rsb.info.nih.gov/ij). Plugins and macros are available at https://sites.google.com/site/qingzongtseng/tfm. First, bead images are aligned to correct experimental drift. Displacement field was calculated using particle image velocimetry on the basis of a normalized cross-correlation algorithm following an iterative scheme. The final grid size for the displacement fields was 0.267 µm × 0.267 µm. Subsequently, the traction-force field was calculated by means of Fourier transform traction cytometry with a regularization parameter set to 2 × 10⁻¹⁰.