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Fig. 5 | Stem Cell Research & Therapy

Fig. 5

From: Type I collagen deposition via osteoinduction ameliorates YAP/TAZ activity in 3D floating culture clumps of mesenchymal stem cell/extracellular matrix complexes

Fig. 5

OIM-induced YAP/TAZ activity in C-MSCs is due to actin cytoskeletal tension caused by Intβ1-ROCK signaling. aj C-MSCs: C-MSCs cultured with GM for 5 days. OIM-C-MSCs: C-MSCs cultured with OIM for 5 days. ae C-MSCs transfected with negative control or Intβ1 siRNAs were cultured with GM or OIM for 5 days. a Confocal immunofluorescence images of YAP/TAZ (green), F-actin (red), and nuclei (blue) in OIM-C-MSCs. Bar = 20 μm. White boxes show enlarged images. b The graph summarizes the distribution of YAP/TAZ localization patterns. The patterns were classified as mainly nuclear (N > C), diffuse (N = C), and mainly cytoplasmic or undetectable (N < C or undetectable). c Immunoblotting for YAP/TAZ in OIM-C-MSCs. A rabbit anti-YAP/TAZ (D24E4) mAb (Cell Signaling) was used to detect both the YAP and TAZ proteins (middle panel). To show YAP expression more clearly, we also used a rabbit anti-YAP (D8H1X) mAb (upper panel). d Real-time PCR analysis of YAP/TAZ target genes. Data were normalized to the values of control siRNA-transfected C-MSCs. Values represent means ± S.D. of three cultures (**p < 0.01). e RUNX2 expression levels in OIM-C-MSCs were analyzed by real-time PCR. Data were normalized to the values of OIM-C-MSCs transfected with negative control siRNA. Values represent means ± S.D. of three cultures (**p < 0.01). All graphs and images are representative of three independent experiments. fj C-MSCs or OIM-C-MSCs were cultured with or without a ROCK inhibitor (Y27632, 50 μM), the non-muscle myosin inhibitor blebbistatin (Blebbist., 50 μM), or an appropriate concentration of DMSO for 5 days. f Confocal immunofluorescence images of YAP/TAZ (green), F-actin (red), and nuclei (blue) in OIM-C-MSCs. Bar = 20 μm. White boxes show enlarged images. g The graph summarizes the distribution of YAP/TAZ localization patterns. The patterns were classified as mainly nuclear (N > C), diffuse (N = C), and mainly cytoplasmic or undetectable (N < C or undetectable). h Immunoblotting for YAP/TAZ in OIM-C-MSCs. A rabbit anti-YAP/TAZ (D24E4) mAb (Cell Signaling) was used to detect both the YAP and TAZ proteins (middle panel). To show YAP expression more clearly, we also used a rabbit anti-YAP (D8H1X) mAb (upper panel). i Real-time PCR analysis of YAP/TAZ target genes. Data were normalized to the values of C-MSCs treated with DMSO. Values represent means ± S.D. of three cultures (**p < 0.01). j RUNX2 expression levels in OIM-C-MSCs were analyzed by real-time PCR. Data were normalized to the values of OIM-C-MSCs treated with DMSO. Values represent means ± S.D. of three cultures (**p < 0.01). All graphs and images are representative of three independent experiments

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