Three independent biological replicates (separate passages, performed on separate days), each containing 3 technical replicate coverslips per condition. Conditions: soft gel (0.5 kPa), stiff gel (40 kPa), stiff gel + 50 µM blebbistatin, and glass control. A minimum of 150 cells per condition per biological replicate are imaged across >=10 randomly selected fields to avoid edge artifacts. Field selection is randomized by the DAPI channel only (operator blind to YAP signal) to prevent selection bias. Imaging acquisition settings (laser power, gain, pinhole 1 Airy unit) are fixed across all conditions within a replicate. Fixation is at 24 h post-seeding.
BSL-2 practices for human-derived HaCaT cells; work in a Class II biosafety cabinet. PFA is a fixative and carcinogen — handle in a fume hood with nitrile gloves and dispose as halogenated/aldehyde chemical waste. Triton X-100 and methanol-free PFA are irritants. Blebbistatin is phototoxic and a suspected reproductive hazard; handle with gloves under reduced light. DAPI is a potential mutagen (DNA intercalator). All biological waste is autoclaved or treated with 10% bleach; chemical waste is segregated. Confocal lasers require interlocked enclosure and eye protection during alignment.
Negative control: secondary-antibody-only (no primary) coverslip to define background fluorescence and set the YAP channel threshold. Positive control for nuclear YAP: cells on rigid glass, which reliably drive nuclear YAP. Pharmacological control: blebbistatin (50 µM) to confirm the stiffness response is actomyosin-dependent; its DMSO vehicle (0.1%) is the matched vehicle control. Staining specificity is additionally verified with a YAP siRNA knockdown coverslip showing loss of signal. DAPI alone defines the nuclear mask independent of the YAP channel.
On soft 0.5 kPa gels, the N:C YAP ratio distribution should center near 0.6-0.9 (cytoplasmic YAP, diffuse phalloidin, few stress fibers). On 40 kPa and glass, the distribution should shift to 1.6-2.5 (bright nuclear YAP, prominent stress fibers). Blebbistatin should collapse the stiff-gel ratio back toward 0.8-1.1. A clean dataset shows tight technical-replicate agreement (CV <15% between coverslips) and a clearly bimodal-to-unimodal shift across stiffness, with secondary-only background <5% of specific signal.
To establish a reproducible, quantitative confocal immunofluorescence pipeline that measures the nuclear-to-cytoplasmic (N:C) ratio of endogenous YAP protein in HaCaT keratinocytes as a function of extracellular matrix stiffness. The assay distinguishes mechanically activated (nuclear-enriched YAP) from inactive (cytoplasmic YAP) states and provides a per-cell continuous readout suitable for population distribution analysis rather than binary scoring.
Independent variable: substrate stiffness (0.5 kPa, 40 kPa, glass) and blebbistatin treatment. Dependent variable: per-cell nuclear:cytoplasmic YAP fluorescence intensity ratio (mean nuclear intensity divided by mean perinuclear-cytoplasmic intensity). Controlled variables: cell seeding density, passage number, fixation time, antibody lots and dilutions, all confocal acquisition settings (laser power, gain, pinhole, z-step), collagen coating concentration, and time post-seeding (24 h). Cell crowding is controlled by analyzing only isolated cells with no touching neighbors.
We hypothesize that increasing substrate stiffness from 0.5 kPa (soft) to 40 kPa (stiff) drives YAP nuclear accumulation, increasing the mean single-cell N:C ratio from approximately 0.6-0.9 (cytoplasmic-dominant) on soft gels to approximately 1.6-2.5 (nuclear-dominant) on stiff gels, and that this shift is abolished by the actomyosin inhibitor blebbistatin (50 µM), confirming dependence on actomyosin contractility.
Maximum-intensity projections are generated from z-stacks. Nuclear masks are segmented from the DAPI channel (Otsu or StarDist in Fiji/CellProfiler). The cytoplasmic ring is defined as a 3-pixel-dilated band outside the nuclear mask, excluding overlap with neighbors. Mean fluorescence intensity of the YAP (488) channel is measured within each compartment, background-subtracted using the secondary-only control, and the N:C ratio computed per cell. Segmentation is automated in a CellProfiler pipeline to remove operator bias; all parameters are version-locked and reported.
Per-cell N:C ratios are treated as nested within coverslips within biological replicates; a linear mixed-effects model (condition as fixed effect, biological replicate and coverslip as random effects) is preferred over naive pooling to avoid pseudoreplication. Group means are compared at alpha = 0.05 with Tukey's HSD correction for multiple comparisons across the four conditions. With n = 3 biological replicates and >=150 cells each, power >0.9 to detect a 0.5-unit ratio difference assuming SD ~0.4. Distributions are reported as superplots showing both individual cells and replicate means.