Extended Data Fig. 11: Low mechanics disrupt the nuclear envelope and activate cGAS/STING by inhibiting YAP/TAZ.
From: YAP/TAZ activity in stromal cells prevents ageing by controlling cGAS–STING
a, RT-qPCR analysis (left panel) of Lmnb1 and ACTR2 in MAFs and WI-38 cells, respectively, plated on stiff (40 kPa) or soft (0.5 kPa) hydrogels. Lmnb1 and ACTR2 expression on soft substrate is rescued by add-back of constitutively active YAP. Of note, cell cycle inhibition (CDK4/6i treatment, 4 μM Palbociclib for 48 h) in WI-38 cells plated on stiff substrates is inconsequential for LMNB1 and ACTR2 expression levels (right panel). Data (n = 3 independent experiments) are shown as mean ± s.d.; P values are derived from one-way ANOVA with Dunnett’s multiple comparison test (left panel) and from two-sided, unpaired t-test (right panel). b, Superresolution microscopy analysis (left panel) of actin cap integrity, as visualized by F-Actin and Lamin A/C staining, and quantification (right panel) in WI-38 cells plated on adhesive microislands of the indicated sizes. Scale bar, 10 μm. Data (n = 3 independent experiments) are presented as mean ± s.d.; P values are derived from two-sided, unpaired t-test. c, RT-qPCR analysis of YAP/TAZ target genes and SASP marker genes in WI-38 cells plated on stiff (40 kPa) or soft (0.5 kPa) hydrogels in the presence (+YAP) or absence (Control) of constitutively active YAP5SA. Control cells were transduced with empty vector. Data (n = 3 independent experiments) are shown as mean ± s.d.; P values are derived from one-way ANOVA with Dunnett’s multiple comparison test. d, RT-qPCR analysis of YAP/TAZ target genes and SASP marker genes in primary MAFs plated on stiff (40 kPa) or soft (0.5 kPa) hydrogels. MAFs were isolated from R26-rtTAM2; Col-tetO-YAPS127A mice and either left untreated (Control) or treated with doxycycline (+ YAP) ex vivo to sustain YAP activity. Data (n = 3 independent experiments) are shown as mean ± s.d.; P values are derived from one-way ANOVA with Dunnett’s multiple comparison test. e, ChIP-Seq profiles for YAP in human NCI-H2052 cells and human IMR90 fibroblasts showing YAP binding at enhancer elements mapped (red arrow) to the ACTR2 promoter, according to published Hi-C interactome maps. f, Independent repeat experiment of ChIP-qPCR shown in Fig. 5i. Enrichment relative to siY/T: 25.4-fold for TAZ-IP on LMNB1 promoter, 13.8-fold for IP-YAP on LMNB1 promoter; 62.7-fold for IP-TAZ on ACTR2 enhancer, and 99-fold for IP-YAP on ACTR2 enhancer. The panel shows the result of one experiment. g, ChIP-qPCR analysis in human vascular smooth muscle cells (vSMCs) showing that the regulatory elements for LMNB1 and ACTR2 are enriched in YAP- and TAZ-immunoprecipitated chromatin, but not in negative control IP (IgG). Relative DNA binding was calculated as fraction of input and normalized to IgG. The panel shows the result of one experiment, independent repeats for a different cell line (human WI-38) are shown in Fig. 5i and Extended Data Fig. 11f. h, YAP/TAZ-depletion in primary MAFs is inconsequential for Lmna expression levels, as assessed by RNA-Seq. Results of two independent experiments are shown.
