Fig. 6.

Acetylated microtubules are sufficient for dynein-dependent nuclear translocation of YAP upon stimulation with TGF-β1. a Immunolabeling of YAP localization in WT and α-TAT1 KO MEFs treated with EHNA (500 μM) and/or TGF-β1 (2 ng ml⁻¹) and grown on fibronectin-coated soft and stiff matrices. Graphs show the percentage of YAP in the nucleus. Scale bar, 50 μm. b Western blotting analysis of nuclear and cytosolic fractions of WT MEFs treated with EHNA and/or TGF-β1 for 8 h. GAPDH and Lamin A were used as markers for cytosolic and nucleic fractions, respectively. c Microtubule sedimentation assay examining WT and α-TAT1 KO MEFs incubated on a soft matrix. Each fraction was assessed via western blotting to detect the levels of dynein and YAP. S; supernatant (depolymerized tubulin), P; pellet (polymerized tubulin). d Luciferase reporter assay in MEFs treated with TGF-β1 and EHNA and grown on 0.5 kPa PAGs. Data are represented as mean ± S.D. from three independent experiments (n = 6). 8xGTIIC-Luc: one-way ANOVA, F_{3, 28} = 246.9, SBE2-Luc: one-way ANOVA, F_{3, 28} = 19.02. *p < 0.05, **p < 0.05, ***p < 0.005, n.s. not significant. e RT-qPCR analysis of selected genes using the same conditions as those shown in a. Bar graph shows log₂ fold change normalized to WT control. Data are represented as the mean of three independent experiments ± S.D. (n = 6). Statistical significance of the differences between each treatment and control group (baseline; *p < 0.05, **p < 0.05, ***p < 0.005, n.s., not significant) or between TGF-β1 treatment and EHNA + TGF-β1 (^cp < 0.005) was determined by one-way ANOVA with Tukey’s multiple comparison. One-way ANOVA, F_{3, 20} = 201.5 (Acta2), F_{3, 20} = 46.30 (Tagln), F_{3, 20} = 41.05 (Tpm1), F_{3, 20} = 41.29 (Cxcr6), F_{3, 20} = 88.81 (Loxl2), F_{3, 20} = 17.29 (Postn), F_{3, 20} = 177.9 (Cyr61), F_{3, 20} = 143.1 (Ctgf). f WT MEFs were serum starved for 12 h and seeded into the collagen matrix. After 1 h, media containing TGF-β1 and/or EHNA were added into the matrices. Data are represented as the mean of two independent experiments ± S.D. (n = 4). One-way ANOVA, F_{3, 12} = 53.10. ***p < 0.005, n.s. not significant