A The effectiveness of BMP4 blocking antibody on the inhibition of BMP4 was demonstrated by measurements of BMP4 protein in the spent medium by ELISA (p<0.001; one-way-ANOVA with Tukey-Kramer post-test; n=3).
B. Addition of BMP4 blocking antibody to the cultured NCM460 cells progressively reduced the mRNA expression of CHST11 (p<0.01, p<0.001; one-way ANOVA with Tukey-Kramer post-test; n=3).
C. In the HT-29 and NCM460 cells, exogenous recombinant human BMP4 increased CHST11 expression significantly, compared to untreated control cell preparations (p<0.001; one-way ANOVA with Tukey-Kramer post-test; n=6).
D. Both exogenous BMP4 and ARSB silencing significantly increased the ratio of chondroitin 4-sulfate to chondroitin 6-sulfate in the NCM460 cells (p<0.05, p<0.001, respectively; one-way ANOVA with Tukey-Kramer post-test; n=3).
E. In contrast to effects on C4S, neither ARSB siRNA nor exogenous BMP4 affected the C6S content in the NCM460 cells (n=3).
F. Exogenous BMP4 (20 ng/ml × 24 h) had no impact on ARSB activity in the NCM460 or HT-29 cells (n=5).
[ARSB=arylsulfatase B; BMP=bone morphogenetic protein; C4S=chondroitin 4-sulfate; C6S=chondroitin 6-sulfate; CHST11=carbohydrate (chondroitin 4) sulfotransferase 11; N.D.=no difference; O.D.=optical density]