Figure 7. PDGF-BB upregulates phosphate transporters PiT1 (Slc20a1) in astrocytes.

(A) qRT-PCR analysis of Slc20a1 mRNA expression in isolated mouse brain microvessels with different dosages of PDGFBB treatment for 16 hours. n = 4. (B) Western blot analysis of PiT1 protein expression in isolated mouse brain microvessels with different dosages of PDGF-BB treatment for 16 hours. n = 3. (C) The relative density of PiT1 was calculated using ImageJ. n = 3. (D) Double-immunofluorescence staining of frozen brain tissue sections from 3- and 22-month-old male mice using antibodies against CD13 and PiT1. n = 3. (E) Double-immunofluorescence staining of frozen brain tissue sections from 3- and 22-month-old male mice using antibodies against CD31 and PiT1. n = 3. (F and G) qRT-PCR analysis of Slc20a1 mRNA expression in primary brain astrocytes treated with PDGF-BB at different dosages (F) and for different durations (G). n = 3. (H) Western blot analysis of PiT1 protein expression in brain astrocytes treated with PDGF-BB at different dosages. (I) Western blot analysis of PiT1 protein expression in brain PCs treated with 50 ng/mL PDGF-BB at different time points. (J) Isolated brain microvessels were incubated under different culture conditions indicated for 16 hours with 2.6 mM Pi, 2.7 mM Ca, and 20 ng/mL PDGF-BB. The concentrations of the Pi transporter inhibitor PFA were 1 mM and 3 mM. Calcification was detected by Alizarin red staining. (K) Double-immunofluorescence staining of frozen brain tissue sections from 3- and 22-month-old male mice using antibodies against GFAP and OPN. n = 3. (L) Double-immunofluorescence staining of frozen brain tissue sections from WT and Pdgfb^cTG mice using antibodies against GFAP and PiT1. n = 3. Scale bars: 100 μm (J–L); 200 μm (D and E). Relative fold-change results are shown as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, by ordinary 1-way ANOVA for multiple-group comparisons (A, C, F and G). Magnification value is ×10 for D, E, and K.