Figure 5.

Knockdown of peripheral benzodiazepine receptor (PBR) by siRNA rescues Midazolam‐induced suppression of chondrogenesis. The specific small interference RNA against human PBR (siPBR) was transfected to knockdown PBR expression in primary mesenchymal stem cells (MSCs). A scrambled siRNA (siN) served as a negative control. (A) The protein level of PBRs in transfected cells was accessed using Western blot analysis. Cells were then treated without or with chondrogenic induction medium in the absence or presence of Midazolam (10 μmol/L, MDZ, CHON + MDZ, respectively) for 7 d. Cells were then fixed and stained with (B, C) Alcian blue or (D) immunostained with type II collagen. The nuclei were stained with DAPI. C, Quantification of Alcian blue and these quantification results are normalized with untreated control. (E‐G) Primary MSCs, transfected with either siN or siPBR, were seeded at high cell density and then treated with or without chondrogenic induction medium or co‐treated with or without Midazolam (10 μmol/L) for 7 d. Protein levels of SOX5, SOX6, SOX9 and type II collagen were analysed using Western blot analysis. Levels of vinculin served as an internal control (upper panel). Quantification results of (F) SOX9 and (G) type II collagen are shown. Quantification results are presented as mean ± SEM of three independent experiments (*P ≦ .05, ***P ≦ .005)