Figure 1: BM-MSC-derived VEGF restores SphK activity in NP–C mice PNs.
From: Pathological roles of the VEGF/SphK pathway in Niemann–Pick type C neurons
(a) SphK activities between NP–C and control were analysed in human fibroblast (n=7 per group), mouse cerebellum tissue (n=7 per group) and primary mouse PN samples (n=9 per group). SphK activity did not show passage differences between NP–C and normal fibroblasts. (b) Three days after cocultures, we measured SphK activities in PNs derived from WT and NP–C mice (n=8 per group). (c) VEGF levels were measured in CM derived from PNs with or without BM-MSCs by ELISA (n=7 per group). (d) Primary cultures of NP–C PNs were immunostained with anti-calbindin and anti-VEGF (scale bar, 50 μm). Arrowheads indicate VEGF expression by PNs. Values represent normalized fluorescence intensities of VEGF in PNs (WT PN, n=8; and NP–C PN, n=9). (e) SphK activities were measured in NP–C PNs alone (n=7) and NP–C PNs cocultured with BM-MSCs, VEGF siRNA BM-MSCs and VEGFtg BM-MSCs (n=8 per group). (f) Effect of the PTK787 on BM-MSCs mediated SphK activation. NP–C PNs were pretreated with PTK787 at 10 μM for 1 day and cocultured for 3 days with BM-MSCs, and then SphK activity was assayed (n=7 per group). (g) Representative images of PNs stained with anti-calbindin (scale bar, 100 μm). The mean number of PNs per squared millimetre was counted (n=8 per group). (h) Effect of VEGF knockdown on SphK activity in PNs (control, n=6; and VEGF siRNA, n=8 per group). (i) Representative images and quantification of neuronal survival in normal and VEGF-knockdown PNs (scale bar, 50 μm; n=8 per group). a,h,i, Student’s t-test. b–g, one-way analysis of variance, Tukey’s post hoc test. *P<0.05, **P<0.01, ***P<0.005. All error bars indicate s.e.m.
