Fig 1: Tension homoeostasis regulates skin morphogenesis.a Real-time PCR analysis of mechanical stress-related genes (ITGA2, MRTF-A and ACTA2) in HSEs. *P < 0.05, **P < 0.01 and ***P < 0.001, as assessed by Dunnett’s test following two-way analysis of variance (ANOVA; P < 0.001); error bars represent the standard deviation. b Immunohistochemical analyses of mechano-sensitive proteins in HSE. These samples were stained with anti-ITGA2, anti-ITGB1, anti-MRTF-A and anti-aSMA antibodies. Scale bars, 20 µm. c Calculation of the nuclear localization of MRTF-A in cells (b). *P < 0.05, as assessed by Dunnett’s test following two-way analysis of variance (ANOVA; P < 0.001); error bars represent the standard deviation. d Macroscopic images of the THS model (upper) and TRS model without (middle) or with (lower) Y-27632 treatment. Scale bars; 1 mm. e Calculation of the shrinkage rate of (d). *P < 0.001, as assessed by Tukey–Kramer test following two-way analysis of variance (ANOVA; P < 0.001); error bars represent the standard deviation. f Histological analysis of the THS model without (upper columns) or with (middle columns) Y-27632 treatment; the TRS model is also shown (lower columns). H&E staining (left), phalloidin staining (middle) and WGA staining are shown. Scale bars, 20 µm. g Immunohistochemical analyses of focal adhesion proteins and mechano-transducer proteins in the THS model without (upper columns) or with (middle columns) Y-27632 treatment; the TRS model (lower columns) is also shown. Scale bars, 20 µm. h Calculation of the nuclear localization of MRTF-A in cells (g). *P < 0.05, as assessed by Dunnett’s test following two-way analysis of variance (ANOVA; P < 0.001); error bars represent the standard deviation. i Immunohistochemical analyses of collagen fibres in the THS model without (upper) or with (middle) Y-27632 treatment; the TRS model (lower) is also shown. Scale bars, 20 µm. j Real-time PCR analysis of dermal ECM-related genes in HSEs. *P < 0.05, **P < 0.01 and ***P < 0.001, as assessed by Dunnett’s test following two-way analysis of variance (ANOVA; P < 0.001); error bars represent the standard deviation.
Fig 2: Schematic representation of the regulation of skin structure and function by tensional homoeostasis.ROCK signalling maintains cytoskeleton formation and promotes the nuclear localization of MRTF-A, which induces the gene expression of ECM factors and KGF.
Fig 3: Optimized 3D collagen culture conditions promote spheroid formation and the maintenance of hepatic functions over time. (A) TPEF images of Hepoid after 8 days of culture. 3D reconstruction (xyz) of (left) 250 µm and (middle left) 100 µm depth images stack (TPEF stack) at 10× and 60× magnification, respectively; (middle right) and (right) are TPEF images taken at 20× and 60× magnification, respectively. (B) Quantification of the gene expression patterns by RT-qPCR of E-cadherin and N-cadherin in FIH (black), 2D cultures (light grey) and 3D cultured PHH (dark grey), 4 (d4), 15 (d15) and 28 days (d28) after seeding. The results are from at least three independant experiments and are expressed according to the 2D culture level (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, one-way ANOVA test, GraphPad Prism v.7.0). (C) Immunostaining of E-cadherin or N-cadherin (red), Albumin (green) and nuclei (blue) in PHH spheroids at d4, d15 and d28. (D) Immunostaining of MKL1 (pink) and nuclei (blue) in 2D and 3D cultures. Scale bar = 25 µm.
Fig 4: Effects of BAC and IL-10 on the subcellular localization of MRTF-A in HTFs. (a) HTFs were incubated first with or without IL-10 (300 pg/ml) for 24 h and then in the additional absence or presence of BAC (5 × 10-6%) for 6 h, after which the cells were subjected to immunofluorescence staining with antibodies to MRTF-A (green). Nuclei were counterstained with DAPI (blue). Arrows indicate localization of MRTF-A to the nucleus. Scale bars, 20 µm. (b) Cells treated as in (a) with the exception that they were exposed (or not) to BAC for 24 h were subjected to subcellular fractionation, and the nuclear fraction was subjected to immunoblot analysis with antibodies to MRTF-A and to lamin A/C (nuclear marker). Blots for two independent experiments as well as quantitative data (means + s.e.m.) for the relative aSMA/lamin band intensity ratio from four independent experiments are shown. *p < 0.05, n.s. (Holm-Sidak post hoc test). Full-length blots are presented in Supplementary Fig. 4.
Fig 5: Regulation of FHL2. (A) si-RNAs directed against HIF1A and MRTFA inhibit the expression of FHL2 by CoCl2. The bottom panels are immunoblots showing the capacity of the si-RNAs to prevent the induction by CoCl2 of HIF1A and MRTFA. (B) Upregulation of FHL2, ACTG2, SRF and SNAI2 mRNAs in MCF7 cells stably expressing MRTFA-CA. Columns with different superscripts differ significantly (p < .05). (C) Immunodetection of FHL2 in various conditions showing its accumulation in the leading migratory cells in wound assays (top right panel) and its accumulation in the nuclei and the borders of cells either overexpressing MRTFA-CA or treated with CoCl2 (bottom panels). (D) Immunodetection of FHL2 (red) showing its overexpression and accumulation into bundles and focal adhesion points, precisely in the cells with a nuclear enrichment of MRTFA (green).
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