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Fig. 2 | Stem Cell Research & Therapy

Fig. 2

From: Protein deubiquitinase USP7 is required for osteogenic differentiation of human adipose-derived stem cells

Fig. 2

Knockdown of USP7 inhibits osteogenic differentiation of hASCs in vitro. a Left panel: Microscopic images of GFP-positive hASCs under ordinary and fluorescent light. Scale bars =100 μm. Middle and right panel: Validation of USP7 knockdown effect by qRT-PCR and Western blotting, respectively. b Left panel: Images of ALP staining on day 7, and AZR staining on day 14 in shNC, shUSP7-1, and shUSP7-2 groups treated with proliferation or osteogenic media. Right panel: Histograms show ALP activity and quantification of AZR staining by spectrophotometry. c Relative mRNA expression of RUNX2, ALP, OC, and OSX measured by qRT-PCR on day 14 of osteogenic induction. GAPDH was used for normalization. d Confocal microscopy of OC with DAPI counterstaining in shNC, shUSP7-1, and shUSP7-2 groups on day 14 of osteogenic induction. Scale bars = 50 μm. e Western blotting of the protein expression of RUNX2, OSX, and the internal control GAPDH on day 14 of osteogenic induction with antibodies as indicated. Results are presented as the mean ± SD, n = 3. *P < 0.05, **P < 0.01. ALP alkaline phosphatase, AZR alizarin red S, GFP green fluorescent protein, hASC human adipose-derived stem cell, NC negative control, OC osteocalcin, OM osteogenic media, OSX osterix, PM proliferation media, RUNX2 runt-related transcription factor 2, USP7 ubiquitin specific protease 7

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