Fig. 4

Effect of Wnt–β-catenin signaling on CCN2 mRNA and protein expression in rat nucleus pulposus (NP) cells. a mRNA expression of CCN members (CCN1, CCN2, CCN3, CCN4, CCN5, and CCN6) after exposure of NP cells to 6-bromoindirubin-3′-oxime (BIO) (1.0 μM) for 24 h assessed using real-time PCR. Results are presented as mean and 95% CI (n = 12 for each group). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an endogenous control. The unpaired Student’s t test was used. b Western blot analysis of CCN2 protein after treatment of NP cells with BIO (1.0 μM). Western blot analysis showed there was a decrease in the levels of CCN2 protein after BIO treatment. β-actin was used as a loading control. Immunoblots shown are representative of experiments with similar results (n = 8). The paired Student’s t test was used. c Detection of CCN2 protein expression by immunofluorescence microscopy. NP cells were cultured with or without 1.0 μM BIO for 24 h, fixed, and stained with antibody against CCN2. CCN2 protein is decreased with treatment compared with untreated control. Left: cells stained with antibody to CCN2; middle: cells stained with 4′,6-diamidino-2-phenylindole (DAPI) to identify healthy nuclei; right: cells stained with antibody to CCN2 and DAPI. Scale bar = 100 μm (× 20 original magnification)