Fig. 3

YY1 knockdown inhibits the osteogenic differentiation of hBMSCs. a Relative mRNA expression of YY1 during the osteoinduction of hBMSCs (n = 3). GAPDH was used for normalization relative to the day 0 group. b Protein expression of YY1 and the internal control β-actin during osteoinduction of hBMSCs (n = 3). Histograms show the quantification of band intensities. β-actin was used for normalization relative to the day 0 group. c Images of ALP staining after 7 days of osteogenic differentiation, and ARS staining after 14 days of osteogenic differentiation in the si-NC, si-YY1-2, and si-YY1-3 groups (n = 3). Histograms show ALP activity and ARS staining quantification by spectrophotometry. d Relative mRNA expression of ALP, RUNX2, and OCN measured via qRT-PCR in PM and OM on day 7 (n = 3). GAPDH was used for normalization. e Confocal microscopy of RUNX2 with DAPI counterstaining of the si-NC and si-YY1 groups after osteogenic induction for 7 days (n = 3). Scale bars: 20 μm. f Western blotting analyses of the protein expression of RUNX2, OCN, and β-actin in the si-NC, si-YY1-2, and si-YY1-3 groups after osteogenic induction for 7 days (n = 3). Histograms show the quantification of band intensities. β-actin was used for normalization relative to the si-NC group (*p < 0.05; **p < 0.01; ***p < 0.001). ALP, alkaline phosphatase; ARS, alizarin red S; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; hBMSCs, human bone marrow mesenchymal stem cells; PM, proliferation medium; OCN, osteocalcin; OM, osteogenic medium; qRT-PCR, quantitative reverse transcription-polymerase chain reaction; RUNX2, runt-related transcription factor 2; YY1, Yin Yang 1