Fgfr3

Cells were lysed in TNE buffer (10 mM Tris [pH 7.5], 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid [EDTA]) supplemented with 1% NP-40, protease inhibitor cocktail, and phosphatase inhibitor. Samples were separated using a NuPAGE system (Thermo Fisher Scientific) on 4–12% Bis-Tris gels in MOPS-SDS buffer, and then transferred to a polyvinylidene difluoride membrane. Antibodies were purchased from the indicated suppliers: FGFR3 (sc-13121, Santa Cruz; dilution ratio 1:200), TACC3 (sc-22773, Santa Cruz; dilution ratio 1:200), phosphorylated ERK (#4370, Cell Signaling Technology; dilution ratio 1:1000), ERK (#4695, Cell Signaling Technology; dilution ratio 1:1000), phosphorylated-AKT (#4060, Cell Signaling Technology; dilution ratio 1:1000), AKT (#4691, Cell Signaling Technology; dilution ratio 1:1000), phosphorylated-STAT3 (#9145, Cell Signaling Technology; dilution ratio, 1:1000), STAT3 (#9139, Cell Signaling Technology; dilution ratio 1:1000), and actin (MAB1501R, Merck Millipore Headquarters; dilution ratio 1:1000). Blots were then incubated with either anti-mouse or anti-rabbit horseradish peroxidase-conjugated secondary antibodies (#115-035-166 and #111-035-144, Jackson Immuno Research; dilution ratio 1:10,000), and visualized by chemiluminescence. Images have been cropped for presentation. Full size images are presented in Supplementary Figures 8–14.

Decalcified bone sections were deparaffinized with xylene, and endogenous peroxidase activity was quenched by treatment with 3% H₂O₂ for 15 min, followed by antigen retrieval by trypsinization for 10 min. Sections were then blocked with normal goat serum for 30 min and incubated at 4 °C overnight with primary antibody followed by the appropriate biotinylated secondary antibody and horseradish peroxidase-conjugated streptavidin-biotin staining. Immunoreactivity was visualized with a 3,3′-diaminobenzidine tetrahydrochloride kit (ZSGB-BIO, Beijing, China) followed by counterstaining with Methyl green. Primary antibodies against the following proteins were used: collagen II (1:400; Chondrex, Redmond, WA, USA), FGFR3 (1:200; Santa Cruz Biotechnology, Dallas, Texas, USA), Osteocalcin (1:200; Santa Cruz Biotechnology), PCNA (1:200; Epitomics, Burlingame, CA, USA), Collagen X (1:200; Abcam, Cambridge, MA, USA), Aggrecan (1:200; Millipore, Billerica, MA, USA), MMP13 (1:200; Proteintech, Chicago, IL, USA), ADANTS5(1:200; Abcam), lubricin (1:600; Abcam), IHH (1:100; Abcam) and RUNX2 (1:200; Santa Cruz Biotechnology). The number of immunoreactive cells in three central regions of condylar cartilage section was counted by using Image-Pro Plus 5.1.

Protein extraction was performed by lysing the cells in RIPA (radio-immunoprecipitation assay) buffer. The protein concentration was determined using the DC protein assay, and equal amounts of protein were loaded onto SDS-PAGE gels. After electrophoresis, the proteins were transferred onto PVDF membranes and blocked with 5% nonfat milk in TBST for 1 h. The primary antibodies were added and incubated overnight at 4 °C with shaking. The membranes were then washed and incubated with secondary antibodies for 1 h at room temperature. The primary antibodies of TTl1, FGFR3, α-actin, p53, p21, ATF3, FAS, TOM20, mtTFA, and PGC1α were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA); TELO2, Cyclin D1, and γ-H2A.x were obtained from Abcam (Cambridge, UK); and p-mTOR, mTOR, p-ATM, ATM, p-p53 (ser15), MDM2, DEC1, p-c-JUN, c-JUN, p-Akt, Akt, p-p38, p38, p-PI3K, PI3K, p-ERK, ERK, p-Chk2, Chk2, p-AMPK, AMPK, p-eIF2α eIF2α, p-P70S6K, P70S6K, p-TSC2, TSC2, and 4EBP1 were obtained from Cell Signaling (Danvers, MA, USA).

Immunostaining was performed using an automated stainer (Ventana Medical Systems, Tucson, AZ, USA), according to the manufacturer’s protocol. The electrocharged slides were stained with CK20 (M7019, Dako Cytomation, Glostrup, Denmark), p16 (06594441001, CINtech, Roche, Manheim, Germany) p21 (556431, BD Pharmigen, San Jose, CA, USA), p53 (DO-7, Novocastra, Leica Biosystems, Wetzlar, Germany), MIB-1 (M7240, Dako Cytomation, Glostrup, Denmark), p-HH3 (117C826, IMGENEX, San Diego, CA, USA) and FGFR3 (sc-13121, Santa Cruz Biotechnology, Heidelberg, Germany). Immunohistochemistry was quantified using the histoscore, as described below. The staining was scored according to both intensity and the cell percentage. Intensity was assessed in the membrane and cytoplasm and scored as 0: negative, +: weak, ++: moderate, +++: strong. Histoscore was then calculated by multiplying the intensity of the stains by the percentage of positively stained cells. In addition, p-HH3 immunohistochemistry was used to identify cells undergoing mitosis. Staining was assessed by counting the number of stained cells in 2 mm², approximately 10 consecutive high-power fields [HPF] of upper layers of the urothelium with a Zeiss Axioskop microscope (Zeiss, Oberkochen, Deutschland).

Protein lysates were prepared as previously described in RIPA [65 (link)]. Western blot analysis was performed with antibodies specific for beta-actin, Flag (Sigma Aldrich), GAPDH (Life technologies), CR-1, E-cadherin (Epitomics), vimentin (Dako, Trappes, France), p-SRC, SRC, p-SMAD2, p-ERK1/2, ERK1/2, p-FGFR1-4, p-AKT, AKT (Cell Signaling), FGFR3, FGFR1, SMAD2 (Santa Cruz, CA, USA).

Western blot technique was used for protein analysis of cell lysates as previously described [12 (link)]. Briefly, sample media was mixed with 4X loading (sample) buffer (Sigma, St Louis, MO) and Radio-Immuno Precipitation Assay buffer, pH 7.4 (Cat No. BP-115, Boston BioProducts, Ashland, MA). 15–50 μg of sample was subjected to Western blot depending on target proteins. The same amount of protein was used within a Western blot and target proteins were then visualized with an enhanced chemiluminescence detection system. Antibodies used in this study were for endothelial nitric oxide synthase (eNOS) (Cat No. 9572; Cell signaling technology, Danvers, MA) 1:1000; phospho-eNOS (Ser1177) (p-eNOS) (Cat No. 9571; Cell signaling technology, Danvers, MA) 1:1000; α-Klotho (Cat No. Ab75023; Abcam, Cambridge, MA) 1:1000; phospho-FGFR (Tyr653/654) (p-FGFR) (Cat No.3471; Cell signaling technology, Danvers, MA) 1:1000; FGFR1 (Cat No. SC-121; Santa Cruz Biotechnology, Santa Cruz, CA) 1:1000; FGFR3 (Cat No. SC-123; Santa Cruz Biotechnology, Santa Cruz, CA) 1:1000; FGFR4 (Cat No. Ab5481; Abcam, Cambridge, MA) 1:1000 and Actin (Cat No. MAB1501; Millipore, Billerica, MA) 1:1000.