Fgf23

Before analysis, with the lids tightly secured, the tubes were placed in a water bath and heated at 15–20°C. After thawing, samples were centrifuged for 15 min at 1000 g (15°C) to remove precipitates if any formed. After that, the supernatant was diluted 1 : 1 by transferring 35 μl of serum into a new tube containing 35 μl of analyzed protein-deprived serum-like buffer. As a quality control, lyophilized human serum was used. Before analysis, lyophilisates and calibration standards were dissolved in distilled water. Sample analysis was performed at 22°C.
Analysis of osteogenesis/osteolysis factor serum concentrations was carried out using commercially available, high-sensitive Luminex kits: (1) DKK-1, TNF-α, OPG, OCN, OPN, and FGF-23—EMD Millipore Corporation, and (2) NT-proANP, PCSK9, TRAIL, sRANKL, TSP-2—R&D Systems. The analyses were carried out according to the manufacturer's instructions in duplicates.

FGF23, DEX, LY294002, Trizol reagents, polybrene and puromycin were obtained from Sigma-Aldrich Chemicals Co. (St Louis, MO, USA). The cell culture reagents were provided by Gibco Co. (Shanghai, China). FGFR1 antibody (#3472) and all other antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).

Hek293-Klotho cells were seeded at density of 200 000 cells/well on 12-well tissue culture plates and transfected with ARHGDIA Ambion™ Silencer™ Select Pre-Designed siRNA (4427038 Ambion, distributed through Thermo Fisher Scientific, Reinach, Switzerland) or with RhoGDI1 (ARHGDIA) Human siRNA Oligo Duplex (Locus ID 396) (SR300287 OriGene Technologies Inc., Rockville MD, USA) using Lipofectamine 3000 Transfection Reagent (L3000015 Invitrogen, distributed through Thermo Fisher Scientific, Reinach, Switzerland) following the manufacturer’s protocol. 72 h after transfection with Rho-GDI1 or negative control siRNAs, cells were treated with vehicle (0.1% BSA) or with 100 ng/ml FGF23 (SRP3039 Sigma-Aldrich, Buchs, Switzerland) for 5 or 15 min. Following treatment, cellular protein was extracted in RIPA (R0278 Sigma-Aldrich) by 1 h incubation on a shaker, followed by centrifugation at 10000 g.

HEK293 cells were obtained from ATCC and were tested negative for mycoplasma using LookOut® PCR-based kit (MP0035-1KT Sigma-Aldrich). HEK293-Klotho cells stably expressing Klotho were a gift from Dr. Bettina Lorenz-Depiereux, HelmholtzZentrum München and were described in Diener et al. (2015 (link)). Cells were transfected with Myc-DDK-tagged Rho-GDI1 (ARHGDIA) (MR202112 OriGene) using Lipofectamine 3000 (L3000015 Invitrogen, distributed through Thermo Fisher Scientific, Reinach, Switzerland). HEK293-Klotho cells were seeded at density of 500 000 cells/well on 6-well tissue culture plates and transfected with RhoGDI1 (ARHGDIA) (NM_004309) Human Tagged ORF Clone (RG200902 OriGene) using Lipofectamine 3000 Transfection Reagent (L3000015 Invitrogen, distributed through Thermo Fisher Scientific, Reinach, Switzerland) following the manufacturer’s protocol. 72 h after transfection, cells were treated with vehicle (0.1% BSA) or with 100 ng/ml FGF23 (SRP3039 Sigma-Aldrich, Buchs, Switzerland) for 5 or 15 min. Following treatment, cellular protein was extracted in RIPA (R0278 Sigma-Aldrich) by 1 h incubation on a shaker, followed by centrifugation at 10000 g.