<p>SUPPLEMENTARY INFORMATION</p><p>Materials and Methods</p><p>Reagents</p><p>Recombinant rat FGF2 was purchased from PeproTech (Rocky Hill, NJ). </p><p>Western blot</p><p>The following antibodies were used for the supplemental data: anti-phospho-p38 MAPK</p><p>(Thr180/Tyr182) antibody, anti-p38 MAPK antibody, anti-phospho-SAPK/JNK</p><p>(Thr183/Tyr185) antibody, anti-SAPK/JNK antibody, anti-phospho-Akt (Ser473) antibody, and anti-Akt antibody (Cell Signaling Inc., Beverly, MA). </p><p>Real-time PCR</p><p>Real-time PCR was carried out using TaqMan Gene Expression Assays Rn00579811_m1 for</p><p>PIT-1, Rn00568130_ml for PIT-2, and Rn00667869_m1 for β-actin.</p><p>1 Supplementary Figure Legends</p><p>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 ±</p><p>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.</p><p>Supplementary Figure 2. (a, b) Responsiveness of rat vascular smooth muscle cells to</p><p>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. </p><p>(c) Effect of FGF23 on signaling pathways other than ERK1/2 in Klotho-overexpressing</p><p>VSMCs. Cell lysates from Figure 3c were analyzed by western blotting with antibodies against</p><p>2 phospho-p38 (p-p38), total p38 (t-p38), phospho-JNK (p-JNK), total JNK (t-JNK), phospho-</p><p>AKT (p-AKT), and total AKT (t-AKT). </p><p>Supplementary Figure 3. Effect of FGFR and MEK inhibitors on FGF23-induced ERK1/2 phosphorylation. </p><p>Klotho-overexpressing VSMCs were serum starved for 24 h and pretreated with (a)</p><p>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.</p><p>Cell lysates were analyzed by western blotting with antibodies against p-ERK and t-ERK.</p><p>Results are expressed as mean ± SEM. *P<0.05 versus control, **P<0.05 versus FGF23 alone.</p><p>Supplementary Figure 4. (a) Effect of FGF23 on vascular smooth muscle cell calcification.</p><p>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</p><p>FGF23 (2–10 ng/ml). Results of calcium deposition quantification are expressed as mean ±</p><p>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</p><p>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.</p><p>4</p>