Supplementary table 1. Inhibitor used to study the FGF18 signalling pathways. *IC50 from 1;
†IC50 from 2; ‡from 3; §Kd values from 4; for p38δ from 3.
Name Targets Provider Concentrations
PD173074* FGFR1 3.6 nM FGFR2 3.3 nM FGFR3 5.3 nM FGFR4 5-10 µM
Sigma- Aldrich
1, 10, 100 nM
SR3306† JNK1 67 nM JNK2 283 nM JNK3 159 nM
Merck Millipore
0.1, 0.3, 1 µM
PD0325901‡ ERK1/2 full inhibition at 100 nM
MKK1 1 µM
Merck Millipore
0.1, 0.3, 1 µM
SB203580§ P38α 17 nM P38β 250 nM P38γ 1700 nM P38δ none
Merck Millipore
0.1, 0.3, 1 µM 1
2
3
4
Supplementary table 2. PCR primers.
Gene Primers
Porcine type II collagen
Forward: 5’- GGATGGGCAGAGGTATAATG -3’
Reverse: 5’- TCTCCAGGTTCTCCTTTCTG -3’
Porcine type I collagen
Forward: 5’- CCCCAGAAGAACTGGTACAT -3’
Reverse: 5’- CCTACAGGTACCCTGTGTCC -3’
Porcine SOX9
Forward: 5’- CAGAACTCCGGCTCCTACTA -3’
Reverse: 5’- GGTCTGGTGAGCTGTGTGTA -3’
Porcine GAPDH
Forward: 5’- TCAAGAAGGTGGTGAAGCAG -3’
Reverse: 5’- TGTCGTACGAGGAAATGAGC -3’
Porcine FGFR3
Forward: 5’- TGGAGCCTGGTCATGGA -3’
Reverse: 5’- TGCTGGATGCTGCCAAA -3’
Porcine RLP13A
Forward: 5’- TACGTTCTTTTCCGCCTGCT-3’
Reverse: 5’- TCAAGGTGGTGCGTCTGAAG-3’
Human type II collagen
Forward: 5’- CCTGAGTGGAAGAGTGGAGA -3’
Reverse: 5’- TCCATAGCTGAAATGGAAGC -3’
Human type I collagen
Forward: 5’- AAAGGATCTCCTGGTGAAGC -3’
Reverse: 5’- CACCTTTAGGTCCAGGGAAT -3’
Human SOX9
Forward : 5’- CCGCTCACAGTACGACTACA -3’
Reverse: 5’- GTGTGTAGACGGGTTGTTCC -3’
Human FGFR3
Forward : 5’- ACCTGGTGTCCTGTGCCTAC -3’
Reverse; 5’- GCCGTTGGTTGTCTTCTTGT -3’
Human EF1α
Forward: 5’- CCTTGTGGAAATTTGAGACC -3’
Reverse: 5’- CCATTTTGTTAACACCGACA -3’
5
6
Supplementary Methods Biochemical measurements
A dimethylmethylene blue (DMMB) assay was used to quantify GAG in the papain lysate of the 3D cell constructs or the culture medium of the monolayer culture. The absorbance at 525 nM of the samples was compared to that of chondroitin sulphate C standards (Sigma- Aldrich, C4384 diluted in medium or in PBS).
DNA was measured with the Quant-iT PicoGreen dsDNA assay kit (Life Technologies).
Fluorescence in the samples was compared to that of a DNA standard from 31.25 to 1,000 ng/mL diluted from the DNA stock solution provided with the PicoGreen kit.
From the literature it is known that a chondrocyte contains 7.7 ± 0.5 pg DNA5. Consequently, the number of cells/construct was calculated using the formula:
Million
cell/ construct = DNA (ng / construct ) 7 .7 x 1000
HPro measurement was performed using a high performance liquid chromatography-mass spectrometry/ mass spectrometry (HPLC-MS/MS) assay, using 4-Hydroxyproline (VWR International) 0.1 to 50.0 µg/mL for calibration standards. 5 µL samples were mixed with 10 µL of internal standard (1.2 µg/mL 4-Hydroxyproline [2H3] from C/D/N-Isotopes) and 200 µL of hydrochloric acid 25% (v/v) and hydrolysed overnight at approximately 110°C. After
centrifugation, samples were evaporated at 55°C/10 Torr, re-suspended in 1 mL water and 7
8 9 10 11 12
13 14 15
16 17
18
19 20 21 22 23 24
Actin staining
Cells were fixed with 4% paraformaldehyde, permeabilised with 0.2% Triton X100 and blocked with 2% (w/v) bovine serum albumin (BSA) and 0.1% Tween 20 in phosphate buffered saline (PBS). Fixed cells were stained with Alexa 488 Phalloidin (Life Technologies, Cat. No. A12379) 1/1,000 in BSA 2% (w/v), 0.1% Tween 20 in PBS (30 min at room
temperature). Images were acquired with an inverted microscope (Zeiss, Axio Observer) using a filter set for green fluorescence.
32
33 34 35 36 37 38
39
40
Supplementary figure 1: (A) FGFR3 expression in monolayer. For porcine chondrocytes:
cells were isolated and cultured as already described (see Method in the main manuscript).
The culture medium was DMEMHG with 10% FBS (Biochrom), 0.4 mM Proline and 50 µg/mL ascorbate-2-phosphate (both from Sigma). For the human OA chondrocytes in monolayer:
human material from a patient who underwent total knee replacement was provided by the Uniklinik Mainz (ethical approval No. 837.339.10 (7348)). Cells were isolated and cultured in 24-well plates with sprifermin 0 – 1,000ng/mL in Chondrocyte Growth Medium (Cell
Application). After seven days, gene expression analysis was performed by real-time PCR (n
= 4). Individual data values (●) are shown. P-values correspond to comparison with the control (0 ng/mL Sprifermin). (B) FGFR3 gene expression in human OA chondrocytes in 3D from the Grade II-2 culture (see Method in the main manuscript). Individual data values (■) as well as the median values (-) are shown. P-values correspond to comparison with the control (CTR).
Supplementary figure 2. Human OA chondrocytes in monolayer. Cells were cultured in 24-
well plates with sprifermin 100 ng/mL in HAM’s F12 with 10% FCS and 50 µg/mL ascorbate- 2-phosphate. After seven days, the cells were detached and counted with a Vi-CELL
Analyzer counter (Beckman Coulter Inc.). Gene expression analysis was performed by real- time PCR (n = 4). Data were normalised to the control (no sprifermin). Each data point represents the median obtained from each donor (A; n = 4). In parallel, cells were cultured in 41
42 43 44 45 46 47 48 49 50 51 52 53
54 55 56 57 58 59
to the control. The fold increase (x) and decrease (/) over control are indicated directly on the graphs. CTR = control; perm = permanent; 1w = 1 week; 1d/w = 1 day per week for 3 weeks.
67 68
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Small Molecule c-jun-N-terminal Kinase (JNK) Inhibitors Protect Dopaminergic Neurons in a Model of Parkinson's Disease. ACS Chem Neurosci 2011; 2: 198-206.
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