Extended Data Fig. 2: Mechanical signals control YAP activity while osmotic signals control nuclear rheology.
From: Mechano-osmotic signals control chromatin state and fate transitions in pluripotent stem cells
(a) Osmolarity measurements of the various media conditions (mean ± SD; n = 3 independent measurements). (b) Representative images and quantification of p-p38 from hiPSCs 5 min after exchanging pluripotency maintenance medium with pluripotency, basal or basal + FGF2 medium for 5 min. Note moderate but consistent activation of p38 activation in cells in basal but not in basal + FGF2 medium (scale bars 50 µm; n = 5 independent experiments; RM-ANOVA/ Fischer’s). (c) Representative snapshots of live imaging (x/y), optical cross sections (z), and quantifications from LaminB1-RFP-tagged hiPSCs before (Pre) and after (Hyper) hypertonic shock. Note progressive decline in nuclear volume. Line represents median volume and individual dots are average colony volumes at indicated timepoints (scale bars 10 µm; n = 3 independent experiments with 80, 166, 137 nuclei/experiment tracked over the time; Mann-Whitney). (d) Quantification of nuclear envelope fluctuations in cells exposed to hypertonic shock and subsequent washout in pluripotency or basal medium for time points indicated. Note attenuations of fluctuations upon hypertonic shock in both conditions and recovery to more abundant fluctuations in basal medium (n = 428 (Pluri Baseline), 331 (Pluri Hyper 30), 338 (Pluri Rec 0), 400 (Pluri Rec 10), 372 (Pluri Rec 30), 466 (Basal Baseline), 307 (Basal Hyper 30), 392 (Basal Rec 0), 424 (Basal Rec 10), 317 (Basal Rec 30) cells pooled across 3 independent experiments; Kruskal-Wallis/Dunn’s). (e) AFM force indentation experiments of iPS cell nuclei within 20 min of media switch or hypertonic shock. Note data is reproduced from Fig. 1m but with additional condition of hypertonic shock (n = 69 (Pluripotency), 71 (Basal), 76 (Basal+FGF2), 85 (Basal+TGF-β1), 74 (Basal+FGF + TGF-β1), 60 (Hyper Pluripotency) nuclei pooled across 5 independent experiments; ANOVA/Kruskal-Wallis). (f) AFM force indentation experiments of iPS cell nuclei treated with Cytochalasin D in pluripotency or basal medium (n = 14 and 17 nuclei for Pluripotency and Basal conditions, respectively, pooled across 3 independent experiments). (g) Representative snapshots and quantification of live imaging of YAP-Halo-tag hiPSCs during hypertonic shock (scale bars 10 µm; n = 3 independent experiments with 132 total cells tracked across time). (h) Representative snapshots of live imaging and quantification from YAP-Halo-tag hiPSCs during 3 µm compression in pluripotency or basal medium. Note comparable activation of YAP in both conditions (scale bars 30 µm; n = 3 independent experiments representing 255 (Basal), 201 (Pluripotency) cells/condition tracked across time). (i) Representative snapshots of live imaging and quantification from YAP-Halo-tag hiPSCs transfected with NLS-EGFP and compressed to 3 µm height. Note anticorrelated dynamics of YAP and EGFP where YAP nuclear localization is enhanced upon compression whereas EGFP is not (scale bars 50 µm (left panel), 30 µm (right panel); n = 31 cells pooled across 3 independent experiments). All error bars mean±SD. Source numerical data are available in source data.
