frt flanked puromycin resistance cassette  (Addgene inc)

Journal: Communications Biology

Article Title: Linking molecular tension and cellular tractions: a multiscale approach to focal adhesion mechanics

doi: 10.1038/s42003-026-09514-0

Figure Lengend Snippet: a Schematic overview of the experimental procedure: Day 1: preparation of thin (250 μm) and flat PAA gels; Day 2: surface functionalization with Sulfo-SANPAH and collagen coating in acidic solution; Day 3: seeding of cells transiently transfected with VinTS plasmid; Day 4: live cell imaging. b Imaging and data acquisition protocol. Step 1: stressed state bead and cell image acquisition (TFM imaging); Step 2: FLIM data acquisition of donor (Clover) channel (FRET imaging); Step3: intensity data of acceptor (mRuby2) channel; Step 4: relaxed state bead image acquired after the removal of the cells and the relaxation of the gel using a mild SDS solution. c Data processing workflow. Displacement fields generated from the overlay images of stressed and relaxed state bead images are calculated and subsequently converted into tractions. FRET efficiency is derived from the FRET trajectory between τ D and τ BG in the phasor space. FA structural and molecular properties are quantified from the acceptor intensity data after applying a binary FA mask. Illustrations were created using BioRender.com.

Article Snippet: Plasmid DNA expressing free Clover (Addgene #40259) and mRuby2 (Addgene #40260) were gifts from Michael Lin .

Techniques: Transfection, Plasmid Preparation, Live Cell Imaging, Imaging, Generated, Derivative Assay

Journal: Communications Biology

Article Title: Linking molecular tension and cellular tractions: a multiscale approach to focal adhesion mechanics

doi: 10.1038/s42003-026-09514-0

Figure Lengend Snippet: a Traction and FRET overlay images of representative cells on soft (4.5 kPa) and stiff (13 kPa) PAA substrate. Traction arrows are scaled with traction magnitude. b Comparison of cell surface area between soft and stiff PAA substrate. n = 93 and 85 cells. c Comparison of cell-averaged tractions between the soft and stiff substrate ( n = 51 and 38 cells, respectively). Each data point represents a single cell. d Illustration of the structure and working principle of Tension Sensing Module (TSMod) during donor excitation; Clover (C) and mRuby2 (R) FRET pair is separated with a synthetic flexible polypeptide (GGSGGS) 7 ; FRET efficiency remains high due to the module’s inability to support tension. e Illustration of the structure and working principle of Vinculin Tension Sensor (VinTS) used; TSMod is inserted between vinculin head domain (VinHD) and vinculin tail domain (VinTD); FRET efficiency decreases as a result of applied tension. f Averaged focal adhesion FRET efficiencies per cell for VinTS and force-insensitive control TSMod, compared on three different substrates ( n = 20, 93, 29, 85, 22, and 26 cells). Each data point represents a single cell. The middle line of violin plots represents the median, and the upper and lower lines indicate the 75th and 25th percentiles. Statistical analysis between two groups were performed using non-parametric Mann–Whitney U test (Wilcoxon rank-sum test) for ( b , c ), and unpaired two-tailed parametric Student’s t test for ( f ): * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Illustrations ( d , e ) were created using BioRender.com.

Article Snippet: Plasmid DNA expressing free Clover (Addgene #40259) and mRuby2 (Addgene #40260) were gifts from Michael Lin .

Techniques: Comparison, Single Cell, Control, MANN-WHITNEY, Two Tailed Test