Fig. 6: PI-88 inhibits sulfatases leading to increased HSPG sulfation, and inhibition of WNT and BMP signaling in OPCs.

a, b Flow cytometry of HS sulfation using RB4CD12 antibody on rat CG-4 (a) and human primary (b) OPCs. No antibody control is shown (grey). PI-88 treatment (100 μg/mL) resulted in a clear increase in OPC sulfation in a time-dependent manner. c, d Western blots for active β-catenin (αABC) and phosphorylated Smad 1/5 (p-Smad) following treatment of rat CG-4 OPCs treated with BMP7, WNT3a (both 50 ng/mL), and/or PI-88 (100 μg/mL). e, f Dose–response curves for WNT and BMP reporter activity in the presence/absence of PI-88 (20 h following treatment, n = 3, one-way ANOVA). ** and *** indicate significant effect of PI-88 by Dunnett’s post-test p < 0.01 and <0.001, respectively. g human primary OPCs were infected with lentiviral TCF reporter and treated with PI-88 (2 µg/mL) and/or WNT3a (50 ng/mL) (n = 3 fetal human samples, normalized mean ± SEM). h similarly, hOPCs were infected with BMP response element-dependent reporter and treated with PI-88 and/or BMP7 (n = 3). g, h Two-way ANOVA revealed significant effects of WNT/BMP and PI-88 treatment, * and **** indicates Sidak’s post-test p < 0.05 and <0.001, respectively. i–n The effect of PI-88 treatment on hOPC differentiation was assessed in the context of inhibitory BMP treatment. hOPC were treated with BMP and/or PI-88 and O4⁺ oligodendrocyte (red) and GFAP⁺ astrocyte (green) differentiation assessed after removal of mitogens. Quantification of O4% (m, n = 5 fetal samples) and GFAP% (n, n = 3 fetal samples). Two-way RM ANOVA revealed significant effects of BMP and PI-88 on O4⁺ differentiation (main effect p < 0.05). Mean ± SEM is shown. Scale: 50 µm.