Coating for MIET measurements
For MIET measurements glass coverslips (Paul-Marienfeld, Lauda-Königshofen, Germany) were silanized by evaporation with (3-Mercaptopropyl)trimethoxysilane (MPTMS) (Sigma-Aldrich, Darmstadt, Germany) and afterwards coated with 20 nm gold (Kurt-Lesker) as previously described .
To apply fibronectin to the gold surfaces, the gold slides were treated with the crosslinker Dithiobis (succinimidyl propionate) (DSP) (Thermo-Fisher, Waltham, Massachusetts, USA) in Dimethyl sulfoxide (DMSO) (4 mg/mL) for 30 min.
Afterwards coverslips were washed in Phosphate buffered saline (PBS) and coated with 0.025 mg/mL fibronectin (Sigma-Aldrich, Darmstadt, Germany) for 2 h. Fibronectin as an extracellular matrix protein is used to bind fibroblasts to integrins via its RGD sequence.
Cell Culture, drug treatment and immunofluorescence staining
NIH 3T3 mice fibroblasts (ATCC® CRL-1658™, Manassas, Virginia, USA) were cultured in Dulbecco’s Modified Eagle Medium (Gibco by life technologies, Germany) containing 10% Fetal Bovine serum (Bio&Sell, Feucht, Germany) and 1% Antibiotic/Antimycotic solution (GE Healthcare Hyclone, Chicago, Illinois, USA). For MIET measurements cells were trypsinized with Trypsin/EDTA (Biowest, Nuaillé, France) for 3 min and seeded on fibronectin coated gold coverslips for 24 h in the incubator.
The cells were treated with different drugs the next day. Drug treatment was 50 µM Blebbistatin (Sigma-Aldrich, Darmstadt, Germany) for 40 min, 10 µM Y-27632 (Sigma-Aldrich, Darmstadt, Germany) for 1 h and 1 µg/µL Rho-Activator II (Cytoskeleton, Denver, CO, USA) for 3 h, respectively. After each drug treatment cells were briefly rinsed 3x in PBS, which was immediately removed and then fixed in 4% formaldehyde (Polysciences, USA) for 10 min.
For immunofluorescence staining cells were permeabilized in 0.8% Triton X-100 (Sigma-Aldrich, Darmstadt, Germany. For blocking they were incubated in 0.1 M Glycin (Alfa Aesar, Haverhill, Massachusetts, USA) for 30 min and 3% Bovine serum album (Sigma-Aldrich, Darmstadt, Germany) for 30 min. To stain the Golgi apparatus, incubation was performed for 3 h in the primary golgin-97 polyclonal antibody 6.7 µg/mL (Thermo Fisher Scientific, Waltham, Massachusetts, USA) and 3 h in the secondary goat anti-rabbit Alexa fluor 488 antibody 4 µg/mL (Thermo Fisher Scientific, Waltham, Massachusetts, USA). The actin cytoskeleton was stained by incubating 66 µM Alexa Fluor 568 phalloidin (Thermo Fisher Scientific, Waltham, Massachusetts, USA) overnight. The nucleus was stained with DAPI (AppliChem, Darmstadt, Germany) in a concentration of 0.002 µg/mL for 5 min. Except of the overnight staining at 4°C all steps of immunofluorescence staining are performed at room temperature. For imaging PBS was used as mounting medium.
MIET measurements are performed on a fluorescence lifetime imaging microscopy setup MicroTime 200 of PicoQuant (Berlin, Germany). The setup is attached to an inverted microscope Olympus IX73 (Hamburg, Germany). Fluorophores are excited via a pulsed diode laser (LDH-D-TA-560, pulse width ~ 56 ps, repetition rate 40 MHz, wavelength 520 nm), which is coupled into a polarization maintaining single mode fiber. For both focusing excitation light and collecting fluorescence light a high numerical aperture objective (60x1.2 UPlanSApo, Superapochromat, water immersion, WD = 0.28 mm) was used. The collected fluorescence has been splitted by a dichroic beam splitter (zt488/861rpc-UF3, AHF/Chroma, Tübingen, Germany), passing the pinhole and is filtered by an emission short-pass ET750sp-2p8 (AHF/Chroma, Tübingen, Germany) and emission long-pass filter BLP01-594R (AHF/Chroma, Tübingen, Germany) to the hybrid-PMT detector. For data acquisition the multichannel picosecond event timer HydraHarp 400 TCSPC module of PicoQuant (Berlin, Germany) was used. FLIM images of each single cell were recorded with the SymPhoTime 64 software (PicoQuant, Berlin, Germany).
Fluorescence lifetime data evaluation
Fluorescence photons were detected in time-tagged, time-resolved mode, which makes it possible to collect all photons of single pixels and sort them into a histogram according to their arrival time after the last laser pulse. A multi-exponential deconvolution fit with a self-recorded IRF was applied to the data sets in the SymPhoTime 64 software (PicoQuant, Berlin, Germany).
Converting lifetime to height values
The MIET GUI programme of the Enderlein group (University of Göttingen) in MATLAB was used to generate the calibration curve to get the height data from the lifetime data. The theory of how to generate the height data from the lifetimes was described in detail previously [32,33,35,46,47]. The layers of the MIET samples were all of the same composition. On the bottom of the sample was glass (refractive index n = 1.52) and a layer of MPTMS (n = 1,443 ), which was coated with 20 nm of gold. The refractive index of gold is wavelength-dependent and was taken from . Above the gold is the cell (n = 1.36 ) and PBS (n = 1.33 ) as mounting medium. The calibration curve was then used to calculate the height values in a custom-written MATLAB code, which was adapted from the MIET GUI [32,35].
MATLAB height analysis
For further analysis of the calculated heights the MATLAB ver. R2019b software (The MathWorks, Inc, Natick, Massachusetts, USA) was used. In order to determine the edge of the cell, the image was binarized and the largest object was selected. The edge of the largest object corresponds to the actin edge and was calculated with pixel accuracy. To avoid binarization artefacts, the cell was eroded by 2 pixels. Areas of cells larger than the image section were not included in the edge analysis. For the determination of the gradient angle along edge, the cell height along the edge line was derived and the angle was calculated for every edge pixel accordingly. To normalize the delta of the 90th percentile to the 10th percentile of stress fiber height, a parameter we call relative stress fiber rise, the difference was divided by the 90th percentile.
Extract stress fibers
To extract the stress fibers from the intensity weighted height images, these images were analyzed using the TWOMBLI plugin  in Fiji [69,70]. The resulting masks were applied to the existing datasets in MATLAB.
Quantification and statistical analysis
The populations used for the analysis of the entire study are composed of the following number of single cells: Untreated N = 39, Blebbistatin N = 38, Y27632 N = 39, Rho Activator N = 48. Plots in Fig. 2, 3, 6 are generated using MATLAB. Boxplots are generated using PlotsOfData . For statistical analysis Wilcoxon-rank-sum test was performed in MATLAB. ns: p > 0.05, *: p < = 0.05, **: p < = 0.01, ***: p < = 0.001, ****: p < = 0.0001.