Supporting Information

Hammitzsch et al. 10.1073/pnas.1501956112 SI Materials and Methods scaling was performed with AIMLESS (45). Initial phases were Thermal Shift Assay. Thermal melting experiments were carried out calculated by molecular replacement with PHASER using an en- using an Mx3005p Real Time PCR machine (Stratagene) as de- semble of known bromodomain models (PDB ID codes 2OSS, scribed (43). were buffered in 10 mM Hepes, pH 7.5, 2OUO, 2GRC, 2OO1, 3DAI, 3D7C, 3DWY, and 3G0L). Unique 500 mM NaCl and assayed in a 96-well plate at a final concentration and initial solutions were improved in a total of 50 cycles of au- of 2 μMin20-μL volume. Compounds were added at a final con- tomated chain tracing, starting from existing model and centration of 10 μM. SYPRO Orange (Molecular Probes) was computed using ARP/wARP (46). Further manual building was added as a fluorescence probe at a dilution of 1:1,000. Excitation performed with COOT (47), and refinement against maximum and emission filters for the SYPRO-Orange dye were set to 465 nm likelihood target using REFMAC5 (48). Thermal motions were and 590 nm, respectively. The temperature was raised with a step of analyzed using TLSMD (translation/libration/screw motion de- 3 °C per minute from 25 °C to 96 °C, and fluorescence readings termination), and hydrogen atoms were included in late refinement weretakenateachinterval. cycles. PRODRG (49) was used to generate the CBP30 inhibitor coordinates and .cif files. All model validations were carried out Protein Expression and Purification. cDNA encoding human bro- using MolProbity (50). Data collection and refinement statistics are modomains was cloned, expressed, and purified as previously de- compiled in Table S3. The models and structure factors have PDB scribed (10). For purification of in vivo biotinylated protein expression, ID codes 5BT3 (EP300), 5BT4 [BRD4(1)], and 5BT5 [BRD2(2)]. the same construct boundaries (e.g., CBP residues R1081-G1198) were subcloned into pNIC-BIO1 vector, a derivative from pNIC28- FRAP. FRAP studies were performed using a recently published Bsa4 vector (GenBank EF198106), containing a 10 His-tag and TEV protocol (25). In brief, U2OS cells were transfected (Lipofect- protease cleavage site at the N terminus and an in-frame biotinylation amine 2000 transfection reagent; Life Technologies), with sequence inserted at the C terminus. The constructs were transformed mammalian overexpression constructs encoding an N-terminal into the BL21 (DE3)-R3-BirA cell line (BL21 derivative coexpressing GFP and an NLS, followed by three tandem repeats of the ex- BirA using a pACYC coexpression vector). Cells were grown over- tended CBP bromodomain (amino acids 868–1,341). The FRAP nightat37°Cin10mLofLuria–Bertani medium with 50 μg/mL and imaging system consisted of a Zeiss LSM 710 laser-scanning kanamycin and 34 μg/mL chloramphenicol (start-up culture). The and control system coupled to an inverted Zeiss Axio Observer. start-up culture was diluted 1:1,000 in fresh medium, and cell growth Z1 microscope equipped with a high-numerical-aperture (N.A. × was allowed at 37 °C to an optical density of about ∼1.0 (OD600) 1.3) 40 oil immersion objective (Zeiss). Samples were placed in before the temperature was decreased to 25 °C. d-Biotine was dis- an incubator chamber capable of maintaining temperature and solved into 10 mM bicine, pH 8.3, and added to the culture at humidity. FRAP and GFP fluorescence imaging were carried out 500 μM final concentration. The protein expression was induced for with an argon-ion laser (488 nm) and with a PMT (photomultiplier) 8 h at 25 °C with 50 μM isopropyl-β-D-thiogalactopyranoside (IPTG). detector set to detect fluorescence between 500 and 550 nm. Once Proteins were purified using Ni-affinity chromatography and size an initial scan had been taken, a region of interest corresponding to exclusion chromatography. ∼50% of the entire GFP-positive nucleus was empirically selected for bleaching. A time-lapse series was then taken to record GFP Crystallization, Data Collection, and Structure Solution. EP300 con- recovery using 1% of the power used for bleaching. The image struct (Uniprot identifier as EP300_HUMAN Q09472-1 fragment datasets and fluorescence recovery data were exported from ZEN 1048–1161), BRD4 construct (Uniprot identifier as BRD4_HUMAN 2009, the microscope control software. On average, 10–20 cells O60885-1 fragment 44–168), and BRD2 construct (Uniprot were imaged per group. One-way ANOVA with Tukey–Kramer identifier as BRD2_HUMAN P25440-1 fragment 344–455) were correction for multiple comparisons was used to detect significant used for crystallographic studies. Aliquots of the purified proteins differences (P < 0.05) between treatment groups. were set up for crystallization using a mosquito crystallization robot (TTP Labtech). Coarse screens were typically setup onto Greiner ChIP-q-PCR. ChIP was performed using the Low Cell# ChIP kit + three-well plates using three different drop ratios of precipitant to (Diagenode). CD4 T cells were negatively selected from leu- protein per condition (200 + 100 nL, 150 + 150 nL, and 100 + 200 nL). kocyte cones and cultured under Th17-promoting conditions as All crystallizations were carried out using the sitting drop vapor described in Cell Purification and Cell Culture. After 6 d of cul- diffusion method at 4 °C. EP300 rod crystals with CBP30 (2.8 mM ture, 3 × 105 Th17 cells were purified with an IL-17 Secretion final concentration) were obtained by mixing 100 nL of the protein Assay (Miltenyi Biotec) for each ChIP and incubated with 2 μM (13mg/mL)and200nLofcrystallization buffer [0.1 M trisodium CBP30 or DMSO for 24 h. Cells were cross-linked with 1% citrate dehydrate, pH 5.6, 20% (vol/vol) iso-propanol, 20% (wt/vol) formaldehyde for 10 min, and 1.25 M Glycine was added for 5 min. PEG 4000]. Bar-shaped crystals of BRD4(1) with CBP30 (5.54 mM Sonication was performed by three runs of 10 cycles (30 s “ON,” final concentration) were obtained by mixing 200 nL of the protein 30 s “OFF”) at high power setting (BioruptorPico; Diagenode). Cell (17.8 mg/mL) and 100 nL of crystallization buffer [0.1 M succinic lysates were immunoprecipitated with anti-p300 (C-20; Santa Cruz) acid, 20% (wt/vol) PEG 3350]. Plate crystals of BRD2(2) with and anti-H3K56Ac (C15410213; Diagenode), and DNA isolation CBP30 (8 mM final concentration) were grown by mixing equal was performed with DNA isolation buffer kit (Diagenode) ac- volumes of 150 nL of protein (7 mg/mL) and crystallization buffer cording to the manufacturer’s instructions. q-PCR was performed [0.2 M sodium/potassium tartrate, 20% (wt/vol) PEG 3350, 10% with SYBR Green (Qiagen) on ViiA7 PCR instruments (Life (vol/vol) ethylene glycol]. Technologies), using primer pairs in the human IL 17A (−43, Crystals were cryoprotected using the well solution supplemented forward 5′-AGGCACAAACTCATCCATCC-3′, reverse 5′-GTC- with additional 20% (vol/vol) ethylene glycol and were flash frozen AGAACCCAGCGTTTCAT-3′; +94, forward 5′-ATGAAACGC- in liquid nitrogen. Data were collected at Diamond Light Source TGGGTTCTGAC-3′, reverse 5′-CGGCTTTCAAAATCTCAAG- beamlines I02 and I03 at a wavelength of 0.9795 Å and 0.9763 Å. G-3′; +4,000, forward 5′-ACCACACAACCCAGAAAGGA-3′,re- Indexing and integration were carried out using XDS (44), and verse 5′-GCAGGATGGAGTGAAGAGGA-3′)andhumanIL 17F

Hammitzsch et al. www.pnas.org/cgi/content/short/1501956112 1of8 locus (+29, forward 5′-GAAATCCTAGGCATGACAGTCC-3′,re- subjected to LUMINEX analysis with a custom-made premixed verse 5′-AACACAGGCATACACAGGAAGA-3′). All values Multiplex Screening Assay for IL-10, IL-22, TNFα, and GM-CSF obtained were normalized to input. The relative enrichment was (R&D Systems) following the manufacturers instructions. Sam- expressed as mean ± SEM. ples were read on a Bio-Plex 200 System (Bio-Rad) using Bio- Plex Manager software version 5.0. Cell Purification and Cell Culture. Mononuclear cells from peripheral + blood (PBMCs) or synovial fluid (SFMCs) were isolated by Ficoll Transcriptional Profiling. CD4 Tcells(1× 106)wereculturedin48- + + density-gradient centrifugation (Histopaque; Sigma-Aldrich). CD4 well plates as described above for 4 d. CD4 T cells were subjected T cells were negatively selected from PBMCs or SFMCs with a + to total RNA isolation with the RNeasy Mini kit (Qiagen) on day human CD4 T-cell Isolation Kit (Miltenyi Biotec). The purity of 0 or day 4, including on-column digestion of DNA with RNase-Free ≥ + this population was 89.1% by flow cytometry. CD4 T cells were DNase Set I (Qiagen). RNA quality was checked using a 2100 cultured in conditions that promoted Th17 numbers and preserved Bioanalyzer and a Eukaryote Total RNA Nano Assay (Agilent Th1, Th2, and Treg numbers as follows: RPMI-1640 (Sigma-Aldrich) Technologies). Microarray analysis was performed by the Cam- supplemented with penicillin-streptomycin (50,000 units, 50 mg) bridge Genomic Service using Illumina beadchip array HT12v4.1. (Sigma-Aldrich), L-glutamine (2 mM) (Gibco/Life Technologies), and 10% (vol/vol) FBS (Sigma-Aldrich) at 37 °C and 5% CO in a RMA (robust multiarray average) expression values were normal- 2 ized by quantiles, using R (Bioconductor). differentially ex- humidified atmosphere. We used recombinant human IL-2 (100 P IU/mL), IL-1β (20 ng/mL), IL-6 (20 ng/mL), and IL-23 (20 ng/mL) pressed were selected using ANOVA, with a value, based on < < (all Peprotech), together with anti-CD2/3/28 beads (Miltenyi Bio- permutations, 0.01 and a false discovery rate 0.05, using Multiple tec) at a ratio of one bead per 20 cells in 96-well plates (5 × 104 cells Experiment Viewer (MEV) (Dana-Farber Cancer Institute). Genes per well), for 3 d for cytokine analysis and cell viability analysis, and werefilteredforalogratio≥ 1 in at least one condition. The 6 d for proliferation analysis. For transcriptional analysis, a ratio of transcripts were further organized with K-means clustering after one bead per 10 cells in 48-well plates (1 × 106 cells per well) for median centering using MEV. Pathways and ontology over- 4dwasused. representation were assessed using Ingenuity Pathways Analysis

+ (Ingenuity Systems) (51). Data (compliant for Minimum In- Cell Proliferation and Viability, ELISA, and LUMINEX Analysis. CD4 formation About a Microarray Experiment) for the datasets used in T cells were labeled with 5 μM CFSE (carboxyfluorescein this study have been deposited in the Gene Expression Omnibus succinimidyl ester) (Molecular Probes/Invitrogen/Life Technolo- (GEO) profiles database, www.ncbi.nlm.nih.gov/geo/ (accession no.  gies) according to the manufacturers instructions and cultured as GSE71231). above. Before flow cytometry, cells were additionally stained for For quantitative PCR, RNA was reverse transcribed with the High surface markers (anti-CD4-APC and anti-CD8a-BV510; Bio- Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Legend) and viability (fixable viability dye eFluor780; ebioscience). TaqMan Fast Universal PCR Master Mix and TaqMan probes for Samples were analyzed on an LRSFortessa (BD Bioscience), and + transcripts encoding IL 17A (Assay ID Hs00174383_m1), IL 17F 2 × 104 live CD4 T cells were recorded per sample. Data analysis (Hs00369400_m1), IL 21 (Hs00222327_m1), IL 22 (Hs01574154_ was performed using FlowJo software (version X 10.0.7). m1), CSF2 (Hs00929873_m1), IL 10 (Hs00961622_m1), For viability assays, cells were washed twice in cold PBS and IFNG (Hs00989291_m1), IL 23R (Hs00332759_m1), RORC stained with anti-Annexin V-FITC and 7-AAD (both BD Bio- TBX21 HIST1H2BK science) in Annexin V binding buffer (10 mM Hepes, pH 7.4, (Hs01076122_m1), (Hs00203436_m1), CCL17 GZMB 140 mM NaCl, and 2.5 mM CaCl ) for 15 min at room temperature (Hs00955067_g1), (Hs00171074_m1), (Hs01554355_ 2 TIMD4 NOTCH1 Hs01062014_m1 in the dark. Flow cytometry analysis was performed within 1 h of m1), (Hs00293316_m1), and ( ) staining in Annexin V binding buffer. were used for q-PCR on a ViiA 7 Real-Time PCR System (Life + CD4 T cells (5 × 104 per well) were cultured in 96-well plates Technologies). Assays were performed in triplicate, and gene ex- as described above for 3 d. Inhibitors were added at a final pression levels were normalized to ACTB (β-actin) (Hs01060665_g1) concentration of 1:1,000 on day 0. Supernatants were analyzed or RPL13A (Hs04194366_g1), and internal controls (no cDNA) with with an IL-17A, IL-17F, and IFNγ ELISA kit (ebioscience) and ΔΔCt calculations.

Hammitzsch et al. www.pnas.org/cgi/content/short/1501956112 2of8 Fig. S1. Comparison of the binding modes of CBP30 for p300 with the BRD4(1) and BRD2(2) bromodomains. (A) Comparison of the CBP30 binding modes in the p300 and BRD4(1) bromodomain complexes reveals a 170° rotation around the isoxazole–benzimidazole bond. CBP30 and key interacting residues are shown in ball and stick representation. Carbon atoms of CBP30 in the p300 complex are highlighted in yellow and in the BRD4(1) complex in cyan, respectively. (B) Alternative view of the two binding modes of CBP30 in BRD4(1) and p300 binding mode. The binding pocket of p300 is shown as a solid surface. The Inset shows a 2FoFc electron density map of the CBP30/p300 complex contoured at 2σ around CBP30. (C) Comparison of the CBP30 binding modes in BRD4(1) and BRD2(2) reveals a similar binding mode. The electron density for both benzimidazole inhibitor decorations was diffuse suggesting disorder. However, the central core of the inhibitor was well defined by electron density, allowing assignment of the binding pose. The tryptophan side chains (W81 and W370) assume a different orientation in both structures. This orientation positions the 3-chloro-4-methoxyphenol ring system next to W81 in BRD4. The aromatic interaction with the ring is probably more favorable than interactions with the morpholine moiety, which would also be sterically excluded by the bulky tryptophan side chain conserved in BET bromodomains, where the tryptophan side chain (W370) assumes a different conformation that does not allow ar- omatic stacking with the CBP30 3-chloro-4-methoxyphenol ring.

Fig. S2. Time dependence of fluorescence recovery for the BRD4 and 3xCBP fluorescence recovery after photobleaching (FRAP). Curves represent the means in the bleached area at each time point of at least 15 cells in each group, and SE bars are shown for each data point. The curves are colored as indicated in the figure.

Hammitzsch et al. www.pnas.org/cgi/content/short/1501956112 3of8 amtshe al. et Hammitzsch www.pnas.org/cgi/content/short/1501956112

4of8 Fig. S3. Full BioMAP profile showing effects of CBP30 on primary hematopoietic cells. Response of CBP30 has been studied using four different concentrations as indicated in the figure. Monitored marker proteins are shown on the x axis, and relevant proteins have been highlighted in the figure. Historic variations of DMSO-treated cells of this platform are indicated by the gray shaded area. Antiproliferative effects are indicated by gray arrows. For details on studied cell types, see www.discoverx.com/services/drug-discovery-development-services/primary-cell-phenotypic-profiling/diversity-plus. Fig. S4. BioMAP comparison of the effects of CBP30 and JQ1 on primary hematopoietic cells. The effect of CBP30 was studied at 1.1 and 10 μM. Monitored marker proteins are shown on the x axis, and relevant proteins have been highlighted in the figure. Historic variations of DMSO-treated cells of this platform are indicated by the gray shaded area. Antiproliferative effects are indicated by gray arrows (JQ1). Studied cell systems were as follows: peripheral blood mononuclear cells plus venular endothelial cells stimulated with LPS; peripheral blood mononuclear cells plus venular endothelial cells (SAg); B cells plus peripheral blood mononuclear cells (BT); macrophages plus venular endothelial cells (IMphg).

Fig. S5. Effects of CBP30 and JQ1 on viability and proliferation of human T cells. Toxic effects of the compounds were assessed using Annexin V/7-AAD staining by flow cytometry on day 3 of culture with CBP30 or JQ1. CFSE dilution was used to evaluate % divided cells on day 6 of culture. Means ± SD are shown. Representative of n = 4 experiments.

Hammitzsch et al. www.pnas.org/cgi/content/short/1501956112 5of8 Table S1. Melting temperatures and temperature shifts with CBP30 on bromodomain family members

BRD target DMSO Tm,°C ΔTm 10 μM CBP30, °C

ASH1L 53.8 ± 0.2 −0.4 ± 0.3 ATAD2 45.8 ± 0.7 −0.7 ± 1.3 BAZ1A 50.8 ± 0.3 0.2 ± 1.0 BAZ2A 44.7 ± 0.4 0.3 ± 0.7 BAZ2B 53.8 ± 0.0 0.2 ± 0.1 BRD1 49.1 ± 0.2 0.1 ± 0.1 BRD2(1) 52.5 ± 0.1 0.9 ± 0.4 BRD2(2) 49.5 ± 0.1 1.4 ± 0.0 BRD3 (1) 48.3 ± 0.1 1.9 ± 0.1 BRD3 (2) 49.5 ± 0.2 1.2 ± 0.3 BRD4(1) 49.0 ± 0.4 2.0 ± 0.4 BRD4(2) 50.6 ± 0.1 1.0 ± 0.4 BRDT (1) 48.5 ± 0.5 0.0 ± 0.4 BRDT (2) 51.9 ± 0.5 0.5 ± 0.1 BRD9 42.4 ± 0.2 0.3 ± 0.2 BRPF1A 42.2 ± 0.4 0.3 ± 0.1 BRPF3 46.6 ± 0.4 0.3 ± 0.2 BRWD3 (2) 53.3 ± 1.0 0.1 ± 0.7 CECR2 50.9 ± 0.2 0.1 ± 0.1 CBP 45.8 ± 0.2 9.6 ± 0.1 p300 41.2 ± 0.1 10.4 ± 0.4 FALZ 52.6 ± 0.2 0.4 ± 0.4 GCN5L2 55.4 ± 0.1 0.2 ± 0.2 KIAA1240 53.2 ± 0.1 0.3 ± 0.1 LOC93349* 56.9 ± 0.2 0.1 ± 0.1 PB1 (1) 58.5 ± 0.7 0.6 ± 0.7 PB1 (2) 53.8 ± 0.2 0.3 ± 0.2 PB1 (3) 45.1 ± 0.1 0.2 ± 0.2 PB1 (4) 46.4 ± 0.1 0.0 ± 0.2 PB1 (5) 48.3 ± 0.2 0.2 ± 0.2 PB1 (6) 61.0 ± 0.2 −0.1 ± 0.2 PCAF 52.7 ± 0.2 0.3 ± 0.3 PHIP(2) 46.9 ± 0.2 0.2 ± 0.1 SMARCA2A 44.4 ± 0.0 0.1 ± 0.1 SMARCA4 53.2 ± 0.3 0.2 ± 0.3 SP140 58.2 ± 0.6 0.4 ± 0.8 TAF1(1) 46.3 ± 0.0 0.1 ± 0.0 TAF1(2) 48.1 ± 0.1 −0.3 ± 0.1 TAF1L(1) 44.4 ± 0.1 0.0 ± 0.2 TAF1L(2) 46.3 ± 0.3 0.2 ± 0.8 TIF1alpha 39.9 ± 0.3 0.5 ± 0.6 TIF1alpha* 39.6 ± 0.1 0.6 ± 0.0 TRIM28* 59.4 ± 0.2 0.2 ± 0.3 TRIM66* 32.4 ± 0.9 0.3 ± 0.4 WDR9(2) 48.2 ± 0.4 −0.1 ± 0.2

Significant changes are in bold. Mean ± SEM, n = 3. *Constructs containing the PHD-bromodomain.

Hammitzsch et al. www.pnas.org/cgi/content/short/1501956112 6of8 Table S2. Molecular selectivity of CBP30 for CBP and p300 compared with the BET family Selectivity, fold 6 −1 obs Protein KA*, 10 M N KD,nM ΔH , cal/mol TΔS, kcal/mol ΔG, kcal/mol relative to CBP

CBP† 47.4 ± 4.4‡ 0.98 ± 0.02 21 −10,910 ± 48 −0.789 −10.1 n/a CBP 38.5 ± 2.6 0.94 ± 0.02 26 −10,570 ± 36 −0.564 −10.0 p300 31.4 ± 2.49 0.97 ± 0.03 32 −10,500 ± 57 −0.616 −9.88 n/a BRD4(1)† 1.17 ± 0.18 1.01 ± 0.03 854 −8,470 ± 160 −0.472 −7.99 40.6 BRD4(1) 1.13 ± 0.07 0.9 ± 0.02 885 −9,561 ± 107 −1.42 −8.14 34.0 BRD4(2) 0.205 ± 0.01 0.81 ± 0.04 4,878 −3,439 ± 80 3.629 −7.07 232 BRD3(1) 0.92 ± 0.03 0.93 ± 0.08 1,086 −9,332 ± 85 −1.232 −8.1 52 BRD3(2) 0.456 ± 0.02 0.962 ± 0.02 2,193 −10,630 ± 214 3.082 −7.63 104 BRD2(1) 0.992 ± 0.03 0.92 ± 0.005 1,008 −9,877 ± 65 −1.801 −8.07 48 BRD2(2) 0.567 ± 0.03 1.02 ± 0.01 1,763 −4,579 ± 56 3.082 −7.66 84

*All experiments were carried out at 15 °C. †Data published in ref. 22 for compound 59. ‡Errors shown are errors of the nonlinear least square fits. n/a, not applicable.

Table S3. Crystallographic data collection and refinement statistics Protein ID

EP300 BRD BRD4(1) BRD BRD2(2) BRD

Ligand CBP30 CBP30 CBP30 Data collection

Space group P 61 P212121 P21212 Cell dimensions a,b,c, Å 53.43 53.43 77.00 41.92 91.80 113.76 52.76 71.81 32.00 α, β, γ, ° 90.00 90.0 120.00 90.00 90.0 90.00 90.00 90.00 90.00 Resolution, Å* 29.60 (1.05) a 29.87 (1.50) 29.68 (1.40) Unique observations* 56,052 (1671) 71,462 (3204) 24,507 (3365) Completeness (%)* 95.9 (58.0) 99.6 (92.8) 99.3 (95.9) Redundancy* 10.8 (2.8) 9.7 (8.7) 9.2 (7.3)

Rsym or Rmerge* 0.066 (0.349) 0.098 (0.426) 0.079 (0.165) I/σI* 22.2 (3.1) 14.4 (4.8) 22.3 (11.6) Wavelength 0.9795 0.9763 0.9763 Phasing MR MR MR Refinement

Rwork/Rfree, % 16.50/17.80 19.12 (21.42) 16.44 (18.53) No. of atoms Protein/other/solvent 976/40/189 3,155/116/346 905/30/118 B-Factors, Å2 Protein/other/solvent 8.56/6.78/17.49 16.55/24.19/24.68 9.63/21.80/22.23 rmsd bond, Å 0.009 0.009 0.010 rmsd angle, o 1.384 1.422 1.405 Ramachandran statistics Favored,% 100.00 100.00 100.00 Outliers, % 0.00 0.00 0.00 PDB ID code 5BT3 5BT4 5BT5

MR, molecular replacement. *Highest resolution shell (in Å) shown in parentheses.

Hammitzsch et al. www.pnas.org/cgi/content/short/1501956112 7of8 Table S4. Patient characteristics for samples used in ELISA/LUMINEX assays and for q-PCR ELISA/LUMINEX q-PCR

HC (n = 5) AS (n = 11) PSA (n = 6) HC (n = 4) AS (n = 3)

Age, y (SD) 52.27 (11.20) 51.92 (10.91) 40.0 (7.48) 54.00 (7.00) 44.00 (15.85) Male/female, no. 4/1 9/2 2/4 4/0 0/3

HLA-B27 positive, % na 100 na na 100 BASDAI* (SD) na 3.38 (2.53) na na 5.07 (3.55) † CRP, mg/L (SD) na 6.47 (5.12) 21.04 (13.99) na 16.53 (12.31) Anti-TNF treatment, no. na 4/11 2/6 na 0/3 ‡ DMARD treatment, no. na 2/11 5/6 na 0/3 Steroid treatment, no. na 1/11 1/6 na 0/3

Synovial fluid mononuclear cells used in Fig. 5H were from an additional AS patient with the following characteristics: 58 y, female, BASDAI 9.9, CRP 27.0 mg/L, on anti-TNF (adalimumab) and DMARD (methotrexate) treatment, no steroid treatment. *Bath ankylosing spondylitis disease activity index. † C-reactive protein. ‡ Disease-modifying antirheumatic drug. na, not applicable. Mean and SD are shown.

Other Supporting Information Files

Dataset S1 (XLS)

Hammitzsch et al. www.pnas.org/cgi/content/short/1501956112 8of8