Figure 3

Osteogenic and adipogenic differentiation of human PSC with WISP-1 knockdown. WISP-1 siRNA or scramble siRNA treated PSC were evaluated for osteogenic and adipogenic differentiation. (A) Knockdown efficiency of WISP-1 in human PSC, assessed by qRT-PCR. (B–D) Osteogenic differentiation of human PSC with or without WISP-1 knockdown. (B) Expression levels of osteogenic gene markers by qRT-PCR at 3 days of osteogenic differentiation, including RUNX2 (Runt-related transcription factor 2), ALP (Alkaline Phosphatase), COL1A1 (Type I Collagen), and OCN (Osteocalcin). (C) Alkaline phosphatase (ALP) staining and photometric quantification after 12 days of osteogenic differentiation with or without WISP-1 knockdown. (D) Bone nodule formation examined by Alizarin red (AR) staining after 12 days of osteogenic differentiation with or without WISP-1 knockdown. (E) Expression levels of markers of BMP and Wnt signaling activity by qRT-PCR at 3 days of osteogenic differentiation with or without WISP-1 knockdown, including ID1 (Inhibitor of DNA binding 1) and AXIN2 (Axis Inhibition Protein 2). (F) PSC were treated with rBMP2 (50 ng/mL), with or without WISP-1 knockdown. Expression levels of osteogenic gene markers by qRT-PCR at 3 days of osteogenic differentiation, including RUNX2, ALP, and OCN. (G) Adipogenic differentiation of human PSC with or without WISP-1 knockdown. Adipocytic gene markers assessed by quantitative RT-PCR at 3 days of differentiation, including PPARγ (Peroxisome proliferator-activated receptor gamma), CEBPα (CCAAT/enhancer-binding protein alpha), FABP4 (Fatty acid binding protein 4), and LPL (Lipoprotein lipase). *P < 0.05; **P < 0.01. Scale bars (part C) 10 mm; scale bars (part D) 200 µm.