Supplementary Information s29

SUPPLEMENTARY INFORMATION

Materials and Methods

Reagents

Recombinant rat FGF2 was purchased from PeproTech (Rocky Hill, NJ).

Western blot

The following antibodies were used for the supplemental data: anti-phospho-p38 MAPK

(Thr180/Tyr182) antibody, anti-p38 MAPK antibody, anti-phospho-SAPK/JNK

(Thr183/Tyr185) antibody, anti-SAPK/JNK antibody, anti-phospho-Akt (Ser473) antibody, and anti-Akt antibody (Cell Signaling Inc., Beverly, MA).

Real-time PCR

Real-time PCR was carried out using TaqMan Gene Expression Assays Rn00579811_m1 for

PIT-1, Rn00568130_ml for PIT-2, and Rn00667869_m1 for β-actin.

1 Supplementary Figure Legends

Supplementary Figure 1. (a) Vascular calcification was increased by FGF23. Rat aortic rings were cultured for 6 days in control medium or high-phosphate (HP) medium (4 mM) in the absence or presence of recombinant FGF23 (10 ng/ml). Results are expressed as mean ±

SEM. *P<0.05 versus control, **P<0.05 versus HP medium without FGF23. (b) Aortic expression of Klotho in rat model of CKD. Aorta lysates from rats that underwent 5/6 nephrectomy (Nx) or sham treatment (control) were analyzed by western blotting with antibodies against Klotho.

Supplementary Figure 2. (a, b) Responsiveness of rat vascular smooth muscle cells to

FGF23. (a) Vascular smooth muscle cells (VSMCs) were serum starved for 24 h and then treated with FGF23 (5 ng/ml) for the indicated time or with 10 ng/ml FGF2 for 15 min as a positive control. Cell lysates were analyzed by western blotting with antibodies against phospho-ERK1/2 (p-ERK) and total ERK1/2 (t-ERK). (b) VSMCs were serum starved for 24 h and then treated with FGF23 (0–10 ng/ml) for 30 min or with 10 ng/ml FGF2 for 15 min. Cell lysates were analyzed by western blotting with antibodies against p-ERK and t-ERK.

(c) Effect of FGF23 on signaling pathways other than ERK1/2 in Klotho-overexpressing

VSMCs. Cell lysates from Figure 3c were analyzed by western blotting with antibodies against

2 phospho-p38 (p-p38), total p38 (t-p38), phospho-JNK (p-JNK), total JNK (t-JNK), phospho-

AKT (p-AKT), and total AKT (t-AKT).

Supplementary Figure 3. Effect of FGFR and MEK inhibitors on FGF23-induced ERK1/2 phosphorylation.

Klotho-overexpressing VSMCs were serum starved for 24 h and pretreated with (a)

FGFR1 inhibitor (SU5402, 0–10 μM) or with (b) MEK inhibitor (U0126, 0–10 μM) or inactive compound U0124 (5 µM) for 60 min, followed by FGF23 (5 ng/ml) stimulation for 30 min.

Cell lysates were analyzed by western blotting with antibodies against p-ERK and t-ERK.

Results are expressed as mean ± SEM. *P<0.05 versus control, **P<0.05 versus FGF23 alone.

Supplementary Figure 4. (a) Effect of FGF23 on vascular smooth muscle cell calcification.

Klotho-deficient vascular smooth muscle cells (VSMCs) were cultured for 6 days in control medium or high-phosphate (HP) medium (5 mM) in the absence or presence of recombinant

FGF23 (2–10 ng/ml). Results of calcium deposition quantification are expressed as mean ±

SEM. FGF23 had no effect on vascular calcification in Klotho-deficient VSMCs (b) Klotho expression after treatment with phosphate and FGF23. Klotho-overexpressing VSMCs were cultured in control medium or HP medium (5 mM) in the absence or presence of recombinant

3 FGF23 (5 ng/ml) for 24 h. Cell lysates were analyzed by western blotting with antibodies against Klotho and β-actin. Real-time RT-PCR analysis of sodium-dependent phosphate transporters PIT-1 (c) and PIT-2 (d). Klotho-overexpressing VSMCs were incubated in control medium or high-phosphate (HP) medium for 48 h in the absence or presence of FGF23 (5 ng/ml). Results are expressed as mean ± SEM. N.S. indicates that group differences were not significant.