Fig. 6

GDF5 knockdown inhibits osteogenic differentiation of hBMSCs. a Relative mRNA expression of GDF5 during osteoinduction of hBMSCs (n = 3). GAPDH was used for normalization relative to the day 0 group. b Protein expression of GDF5 and 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 The efficiency of transient transfection of si-GDF5-1 and si-GDF5-2 by qRT-PCR. GAPDH was used for normalization relative to the si-NC group (n = 3). d The efficiency of transient transfection of si-GDF5-1 and si-GDF5-2 by western blotting analyses. Histograms show the quantification of band intensities (n = 3). β-actin was used for normalization relative to the si-NC group. e Western blotting analyses of the protein expression of RUNX2, OCN, and β-actin in the si-NC, si-GDF5-1, and si-GDF5-2 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. f Images of ALP staining after 7 days of osteogenic differentiation, and ARS staining after 14 days of osteogenic differentiation in the si-NC, si-GDF5-1, and si-GDF5-2 groups (n = 3). Histograms show ALP activity and AZR staining quantification by spectrophotometry. g Confocal microscopy of RUNX2 with DAPI counterstaining in the si-NC and si-GDF5 groups after osteogenic induction for 7 days (n = 3). Scale bars: 20 μm. h 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. (**p < 0.01; ***p < 0.001). ALP, alkaline phosphatase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GDF5, growth differentiation factor 5; 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