YAP and TAZ expression at high density and during chondrogenic differentiation of human synovial MSCs. (A) YAP and pYAP expression in human synovial membrane-derived (hSM-)MSCs in monolayer at low (L) and high (H) density detected by immunofluorescence staining, shown without (i) and with sytox green nuclear counterstain (ii). (B) YAP and pYAP expression in hSM-MSCs in monolayer at low (L) and high (H) density detected by western blotting with β-actin as loading control. (C, D) Expression of YAP and TAZ (C) and their target genes CTGF and CYR61 (D) in hSM-MSCs immediately prior to (0 h) or 24 h after plating in micromass culture, determined by quantitative RT-PCR. Data was normalised to GAPDH expression, and is shown as mean ± standard deviation (SD) (three donors) relative to pre-seeding (0 h) control. *
P <0.05; **
P <0.01; ***
P <0.001. (E) Expression of YAP and TAZ in hSM-MSCs after 6 days of treatment with 10 ng/ml TGF-β1 or vehicle only in micromass culture to induce chondrogenic differentiation, determined by quantitative RT-PCR. Data was normalised to GAPDH expression, and is shown as mean ± SD (five donors) relative to vehicle-treated control. *
P <0.05. (F) Detection of YAP by western blotting during chondrogenic differentiation induced by TGF-β1 with detection of β-actin as loading control. MSC, mesenchymal stromal/stem cell; pYAP, phosphorylated YAP; RT-PCR, reverse transcription PCR; TAZ, transcriptional co-activator with PDZ-binding motif; TGF, transforming growth factor; YAP, Yes-associated protein.