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Fig. 6 | Stem Cell Research & Therapy

Fig. 6

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

Fig. 6

YAP/TAZ activity is associated with COL1 synthesis and F-actin formation, generating a positive feedback loop in OIM-C-MSCs. (am) C-MSCs: C-MSCs cultured with GM for 5 days. OIM-C-MSCs: OIM-C-MSCs cultured with OIM for 5 days. ae C-MSCs transfected with negative control, YAP and TAZ, or Intβ1 siRNAs were cultured with OIM for 5 days. a, d COL1A1 mRNA expression levels 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). b, e Confocal immunofluorescence images show COL1 (green) and nuclei (blue) in OIM-C-MSCs. Bar = 100 μm. c Confocal immunofluorescence images of YAP/TAZ (green), F-actin (red), and nuclei (blue) in OIM-C-MSCs. Bar = 20 μm. f, g 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 COL1A1 mRNA expression levels were analyzed by real-time PCR. Data were normalized to the values of C-MSCs treated with DMSO. Values represent means ± S.D. of three cultures (**p < 0.01). g Confocal immunofluorescence images show COL1 (green) and nuclei (blue) in C-MSCs. Bar = 100 μm. hl C-MSCs transfected with a constitutively active TAZ mutant (TAZS89A) or control plasmid were maintained in GM for 5 days. h COL1A1 mRNA expression levels were analyzed by real-time PCR. Data were normalized to the values of C-MSCs transfected with control vector (cont). Values represent means ± S.D. of three cultures (**p < 0.01). i Confocal immunofluorescence images show COL1 (green) and nuclei (blue) in C-MSCs. Bar = 100 μm. j Confocal immunofluorescence images of YAP/TAZ/TAZS89A (green), F-actin (red), and nuclei (blue) in C-MSCs. Bar = 20 μm. k Immunoblotting for YAP/TAZ in OIM-C-MSCs. A rabbit anti-YAP/TAZ (D24E4) mAb (Cell Signaling) was used to detect YAP, TAZ, and TAZS89A mutant proteins (middle panel). To show YAP expression more clearly, a rabbit anti-YAP (D8H1X) mAb was also used (upper panel). l Real-time PCR of YAP, TAZ, or YAP/TAZ target genes. Data were normalized to the values of C-MSCs transfected with a control vector (cont). Values represent means ± S.D. of three cultures (**p < 0.01)

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