SUPPLEMENTARY DATA

Pleiotropic roles of metallothioneins as regulators of chondrocyte apoptosis and

catabolic and anabolic pathways during osteoarthritis pathogenesis

Yoonkyung Won, Youngnim Shin, Churl-Hong Chun, Yongsik Cho, Chul-Won Ha, Jin-Hong Kim, and Jang-Soo Chun

Supplementary Materials and Methods Supplementary Tables 17 Supplementary Figures 17

Supplementary Materials and Methods

Human OA cartilage and experimental OA in mice OA cartilage was sourced from individuals undergoing arthroplasty.1819 The Institutional Review Board of Wonkwang University Hospital approved the use of these materials, and all participants provided written informed consent before the operative procedure. Male WT mice of the 129S1/SvImJ strain and Mt1/Mt2 double-KO mice (129S7/SvEvBrd-Mt1tm1BriMt2tm1Bri/J; The Jackson Laboratory) were used for experimental OA studies. The C57BL/6 strain (Zip8fl/fl and Mtf1fl/fl) and cartilage-specific conditional knockout (cKO) mice for Zip8 (Zip8fl/fl;Col2a1-Cre) and Mtf1 (Mtf1 fl/fl;Col2a1-Cre) were also used for experimental OA studies.7 All experiments were approved by the Gwangju Institute of Science and Technology Animal Care and Use Committee. Experimental OA in 12 week- old male mice was induced by destabilization of the medial meniscus (DMM) surgery2022 or by intra-articular (IA) injection (once weekly for 3 weeks) of adenovirus (1 × 109 plaque forming units [PFUs] in a total volume of 10 l) expressing MT1 (Ad-Mt1), MT2 (Ad-Mt2), ZIP8 (Ad-Zip8), MTF1 (Ad-Mtf1), HIF-2α (Ad-Epas1), or NAMPT (Ad-Nampt).1822 Mice were sacrificed 6 or 8 weeks after DMM and 3 or 8 weeks after the first IA injection for histological and biochemical analyses. Human OA cartilage sections were stained with alcian blue. Cartilage destruction in mice was examined using safranin-O staining and scored using the OARSI grading system.40

Histology and immunohistochemistry Human OA cartilage was frozen, sectioned into 10-μm-thick slices, and fixed in paraformaldehyde. Sulfate was detected with alcian blue staining. Cartilage destruction in mice was examined by safranin-O and hematoxylin staining.7,41,42 Synovial inflammation, osteophyte formation, and subchondral bone sclerosis were determined as described previously.7,42 Briefly, knee joints were fixed in 4% paraformaldehyde, decalcified in 0.5 M EDTA, and embedded in paraffin. Paraffin blocks were sectioned at a thickness of 5 μm; serial sections were obtained from the entire joint at 40-μm intervals. Sections were deparaffinized in xylene, hydrated with graded ethanol, and stained with safranin-O. Cartilage destruction was scored by two blinded observers using the OARSI grading system.40 Cartilage sections were immunostained for ZIP8 (sc-133415; Santa Cruz Biotechnology, Santa Cruz, CA, USA), MTF1 (NBPI-86380; Novus Biologicals, Littleton, CO, USA), MT1/MT2 (NBP1- 97493; Novus Biologicals), MMP3 (ab52915; Abcam, Cambridge, UK), MMP13 (ab51072; Abcam), SOX9 (EPR14335-78; Abcam)

Primary culture of articular chondrocytes and adenovirus infection Chondrocytes were isolated from femoral condyles and tibial plateaus of mice, as described previously.7 Chondrocytes were maintained as a monolayer in Dulbecco’s Modified Eagle’s Medium supplemented with 10% fetal bovine serum and antibiotics, and cells at culture day 2 were treated as indicated in each experiment. Ad-Mt1, Ad-Mt2, Ad-Zip8, and Ad-Mtf1 were purchased from Vector Biolabs (Philadelphia, PA, USA). Ad-Epas1 was purchased from Newgex Inc. (Seoul, Korea). Ad-Nampt was produced by Seoulin Bioscience using the pAdEasy System (QBiogene). Mouse articular chondrocytes were cultured for 2 days, infected with adenovirus for 2 hours at the indicated multiplicity of infection (MOI), and cultured for 24 hours, followed by further analyses.

Apoptosis of chondrocytes Chondrocyte apoptosis in articular cartilage was determined by TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labeling) assay using a kit from Roche Diagnostics (Mannheim, Germany) and then counterstained with 4',6-diamidino-2- phenylindole (DAPI) to label nuclei.21 Specimens were visualized under a fluorescence microscope (Zeiss, Oberkochen, Germany). The number of apoptotic chondrocytes in relation to the total number of cells was quantified in superficial/middle zones cartilage and calcified cartilage. Multiple field images from least three sections were used for each experimental group.

Determination of intracellular ROS For intracellular ROS analysis, primary cultured chondrocytes were loaded with 20 μM DCF- DA (Invitrogen Corp.), incubated at 37 °C for 30 min. Cells were immediately examined by flow cytometry using a FACSCanto instrument (BD) and analyzed with FlowJo software (Tree Star). cDNA microarray Microarrays were used to analyze the transcriptome of chondrocytes deficient Mt1 and Mt2 or overexpressing MT1, MT2, MTF1, or ZIP8. Briefly, total RNA was isolated from mouse articular chondrocytes from WT or Mt1/Mt2 double KO mice or chondrocytes infected with Ad-Mt1, Ad-Mt2, Ad-Mtf1, or Ad-Zip8 for 24 hours, and subjected to a microarray analysis using Affymetrix GeneChip Mouse 2.0 ST Array (Affymetrix, Santa Clara, CA, USA), as described by the manufacturer.

Transcription factor arrays Transcription factor array analyses were performed using a Cignal 45-Pathway Reporter Array (Qiagen, Hilden, Germany). Briefly, articular chondrocytes plated on 96-well plates were transfected with reporter constructs reconstituted from the kit using Attractene (Qiagen). Transfected cells were infected with either Ad-C or Ad-Mt2 for 2 hours. After incubating cells for an additional 36 hours, firefly luciferase and Renilla luciferase activities were measured using a Dual-Glo Luciferase Assay System (Promega, Madison, WI, USA). Firefly luciferase activity was expressed relative to that of Renilla luciferase to normalize for transfection efficiency.

Reporter gene assay The previously described SOX9-dependent Col2a1 enhancer elements reporter construct was a kind gift from Veronique Lefebvre of the Cleveland Clinic, Cleveland.42,43 Chondrocytes were treated with hyaluronidase type I-S (4 units/ml) for 3 hours under serum-free conditions, and transfected with both a reporter vector (1.0 μg) and a constitutive Renilla luciferase vector (0.1 μg) using lipofectamine 2000 (Invitrogen Corp., Carlsbad, CA, USA). Transfected cells were cultured for 36 hours prior to assaying luciferase activity. Reporter gene activity was measured using a Dual Luciferase Assay Kit (Promega).

RT-PCR, qRT-PCR, and siRNA Total RNA was extracted from primary cultured mouse chondrocytes using the TRI reagent (Molecular Research Center Inc., Cincinnati, OH, USA). The RNA was reverse transcribed, and the resulting cDNA was amplified by PCR. PCR primers are summarized in Table S1. Transcript levels were quantified by qRT-PCR. siRNAs targeting Mt1, Mt2, Zip8, Mtf1, Epas1, and Nampt were obtained from GE Dharmacon (Lafayette, CO, USA); nontargeting (scrambled) siRNA was used as a negative control. Chondrocytes were transfected by incubating for 6 hours with siRNA and Lipofectamine 2000 (Invitrogen Corp.), as described by the manufacturer, and infected with adenoviruses as described above.

Western blotting Total cell lysates were prepared in lysis buffer (150 mM NaCl, 1% NP-40, 50 mM Tris, 0.2% sodium dodecyl sulfate [SDS], 5 mM NaF) and used to detect MT1/MT2, MTF1, SOX9, COL2A1 ( type II alpha 1), aggrecan, and ERK1 (extracellular signal-regulated kinase- 1). Secreted proteins (MMP3, MMP13, and ADAMTS5) were detected after trichloroacetic acid (TCA) precipitation from 900 µl of serum-free conditioned medium. For the detection of ZIP8, the cell pellet was resuspended using loading buffer containing the anionic denaturing detergent SDS. All lysis buffers contained a cocktail of protease inhibitors and phosphatase inhibitors (Roche). The following antibodies were used for Western blotting: ZIP8 (sc-133415; Santa Cruz Biotechnology), MTF1 (NBP-86380; Novus Biologicals), MT1 and MT2 (NBP1- 97493; Novus Biologicals), MMP3 (ab52915; Abcam), MMP13 (ab51072; Abcam), ADMATS5 (PA5-14350; Thermo Fisher Scientific Inc., MA, USA), SOX9 (sc-20095; Santa Cruz Biotechnology), COL2A1 (MAB8887; Millipore, MA, USA), aggrecan (AB1031; Millipore), and ERK1 (610408; BD Biosciences, NJ, USA).

Statistical analysis For cell-based in vitro studies, each independent experiment was conducted using primary cultured articular chondrocytes derived from a set of littermates. Normal distribution of acquired data was confirmed using the Shapiro–Wilk test. A two-tailed Student’s t-test with unequal sample sizes and variances and two-way analysis of variance (ANOVA) with post-hoc tests (LSD) were used for pair-wise comparisons and multi-comparisons, respectively. Significance was accepted at the 0.05 level of probability (P < 0.05). For animal-based in vivo experiments, each independent trial was conducted using an individual mouse. For each animal, at least three slides spanning the medial tibial plateaus in knee joints were used for histological analysis. The most advanced lesion in these serial sections of medial tibial plateaus was selected for further data quantification. Cartilage destruction and synovitis were scored based on OARSI grade, a discrete grading system. Data collected from laboratory experiments on mice were analyzed using the non-parametric Mann-Whitney U test.

Supplemental references 40. Glasson SS, Chambers MG, Van Den Berg WB, et al. The OARSI histopathology initiative - recommendations for histological assessments of osteoarthritis in the mouse. Osteoarthritis Cartilage 2010;Suppl 3:S1723. 41. Ryu JH, Chae CS, Kwak JS, et al. Hypoxia-inducible factor-2α is an essential catabolic regulator of inflammatory rheumatoid arthritis. PLoS Biol 2014:12:e1001881. 42. Kim JH, Lee G, Won Y, et al. Matrix cross-linking-mediated mechanotransduction promotes posttraumatic osteoarthritis. Proc Natl Acad Sci U S A 2015: 112:94249. 43. Murakami S, Lefebvre V, de Crombrugghe B. Potent inhibition of the master chondrogenic factor Sox9 gene by interleukin-1 and tumor necrosis factor-alpha. J Biol Chem 2000: 275:368792.

Supplementary Table S1. PCR primers and conditions

Size AT Strand Primer sequences Origin (bp) (°C) S 5´-GCCATTGTAATAACCCTGCACC-3´ Adamts5 292 58 Mouse AS 5´-TCAGTCCCATCCGTAACCTTTG-3´ S 5´-CTGTCTTTGTCACCCACACATG-3´ Acan 581 55 Mouse AS 5´-GAAGACGACATCACCATCCAG-3´ S 5´-ATATCGCTGCGCTGGTCGTC-3´ Actin 517 58 Mouse AS 5´-AGGATGGCGTGAGGGAGAGC-3´ S 5´-ATGGCAGTGGATTGGTTAGG-3´ Atf2 252 64 Mouse AS 5´-TCTCTAAGGACTGAACCCACA-3´ S 5´-CACACTGGTAAGTGGGGCAAGACCG-3´ Col2a1 173 57 Mouse AS 5´-GGATTGTGTTGTTTCAGGGTTCGGG-3´ S 5´-CGAGAAGAACGACGTGGTGTTC-3´ Epas1 333 64 Mouse AS 5´-GTGAAGGCTGGCAGGCTCC-3´ S 5´-TCACTGCCACCCAGAAGAC-3´ Gapdh 450 58 Mouse AS 5´-TGTAGGCCATGAGGTCCAC-3´ S 5´-AAAGAGAGAAAGTCCAAGTCCAGCC-3´ Irf1 344 61 Mouse AS 5´-GCTATCTTCCCTTCCTCATCCTCG -3´ S 5´-AGGGATGATGATGCTGGTATGG-3´ Mmp3 434 58 Mouse AS 5´-CCATGTTCTCCAACTGCAAAGG-3´ S 5´-TGCACTGGGCTTAGATCATTCC-3´ Mmp9 450 58 Mouse AS 5´-CGGTCCTTGAAGAAATGCAGAG-3´ S 5´-CCCAGAGGTCAAGATGGATG-3´ Mmp12 482 60 Mouse AS 5´-GGCTCCATAGAGGGACTGAA-3´ S 5´-TGATGGACCTTCTGGTCTTCTGG-3´ Mmp13 473 58 Mouse AS 5´-CATCCACATGGTTGGGAAGTTCT-3´ S 5´-CAACTGCTCCTGCTCCACCG-3´ Mt1 132 60 Mouse AS 5´-CCCTGGGCACATTTGGAGCAG-3´ S 5´-AGCCCTGGGAGCACTTCGCAC-3´ Mt2 138 58 Mouse AS 5´-ACCCCAACTGCTCCTGTGCCTC-3´ S 5´-ACAGATACTGTGGCGGGATTG-3´ Nampt 424 58 Mouse AS 5´-TGATATCCACGCCATCTCCTTG-3´ S 5´-GTTTTAATGGTGATGCAGAGTCCGTC-3´ Mtf1 461 64 Mouse AS 5´-GGGATTATTAGTTAGGACAGAGTTGGC-3´ S 5´-CACTGGCAGTTACGGCATCAG-3´ Sox9 457 61 Mouse AS 5´-CATGTAAGTGAAGGTGGAGTAGAGC-3´ S 5´-CCTTCTGGCCTTGGATTGACACC-3´ Stat1 412 60 Mouse AS 5´-GGTTCTGGTGCTTCCTTTGGTCTG-3´ S 5´-GAACAATTGCCTGGATGATCACGC-3´ Zip8 430 58 Mouse AS 5´-AAGCCGGTTAACATCCCTGCATTC-3´

AT, annealing temperature; S, sense; AS, antisense.

Supplementary Table S2. List of upregulated (A; fold change ≥ 2; P < 0.05) and downregulated (B; fold change ≤ 0.5; P < 0.05) genes in chondrocytes infected with Ad-Mt2 (800 MOI)

A. Upregulated genes

Fold change P value Gene accession Gene symbol (Ad-Mt2) (Ad-Mt2) BC034858 Saa2 96.60043 0.00012 AK144158 Cxcl3 87.32795 0.00000 AK148467 Mmp3 78.21605 0.00003 AK150430 Hp 68.07137 0.00000 BC052495 Saa1 53.60302 0.00000 BC019212 Saa4 35.64665 0.00000 AK003101 Ccl5 34.08537 0.00001 AK143562 Lbp 24.57527 0.00000 AK003905 Steap4 22.33843 0.00065 BC029976 C3 22.25393 0.00031 AK150728 Mmp13 22.18268 0.00051 AK010170 Slpi 20.56522 0.00000 AK014895 Slc39a8 20.44150 0.00000 BC055885 Saa3 19.62024 0.00098 AK132590 Ccl2 19.52451 0.00000 AK149531 Cfb 17.42540 0.00000 AJ132103 Vnn3 15.51554 0.00000 AK002773 Vnn1 15.45479 0.00051 AK140312 Cxcl1 13.94237 0.00001 ENSMUST00000174425 Gm20419 13.66814 0.00002 AK003143 Fabp4 12.95102 0.00104 BC010546 Ifi204 10.88619 0.00005 AK008517 Xdh 10.44445 0.00002 AK003407 Ifitm3 9.20096 0.00003 AK051475 Chi3l1 9.14633 0.00061 AK050355 Slfn9 8.90524 0.00000 AF065919 Nos2 8.52496 0.00069 AK078827 Ccl7 8.25900 0.00025 AK041059 Bst1 8.24926 0.00010 AF272948 Gpr84 8.14085 0.00001 AK089780 Il6 8.06560 0.00007 AK080147 Il13ra1 7.47355 0.00000 AK036666 Gbp7 7.26828 0.00218 AF140672 Ifi202b 7.04348 0.00011 AK007477 Rtp4 7.00411 0.00762 AK140360 Ch25h 6.93595 0.00001 AK139833 A4galt 6.90749 0.00001 BC031424 Isg15 6.84992 0.00377 BC117110 Hist1h2ab 6.80773 0.00006 EF660528 AW112010 6.49338 0.00017 XR_105655 Gm3601 6.40435 0.00013 XR_105655 Gm3601 6.39086 0.00015 AK053190 Cebpd 6.23583 0.00006 AK087960 Srgn 6.17798 0.00020 BC065803 Hist1h2ag 6.00027 0.00004 AK009161 Angptl4 5.93530 0.00275 AK033934 Fgf7 5.74869 0.00002 BC013651 Serpina3n 5.67286 0.00001 AK075570 Sod3 5.67089 0.00014 AK151131 Ccl9 5.64740 0.00005 AK004651 Mmp9 5.61343 0.00017 BC033044 Nfkbiz 5.54251 0.00002 AK018602 Ifih1 5.47260 0.00570 AK149774 Lcn2 5.42727 0.00187 AK018830 Fam111a 5.42641 0.00003 AK155016 Bcl3 5.31606 0.00000 AK088907 Rrm2 5.23038 0.00001 AK010768 Glipr1 5.09979 0.00012 AK077326 Tm4sf1 4.95872 0.00005 BC027838 Ereg 4.93278 0.00009 AK017282 Prr16 4.83648 0.00137 AK002830 Irf7 4.75271 0.00474 XM_917927 LOC640793 4.74884 0.00035 AK133465 Ampd3 4.71625 0.00001 AK018777 Kitl 4.52868 0.00000 AK019924 Gcnt2 4.49955 0.00003 AK085754 Trf 4.46036 0.00027 AK014783 Irak3 4.40092 0.00000 AK132597 Prg4 4.39378 0.00037 AK044924 Ccna2 4.35629 0.00005 AK137203 Avpr1a 4.33824 0.00114 AK089687 Il13ra2 4.33497 0.00009 AK011243 Chaf1b 4.23636 0.00000 AK010166 Car13 4.17462 0.00005 AK035320 Gna14 4.16962 0.00000 AK020117 Hist1h1b 4.15125 0.00009 AK041985 Tmem255a 4.13853 0.00050 AY462056 Kng2 4.12892 0.00005 BC003748 Sntb1 4.12677 0.00028 AK013034 Cyp7b1 4.10821 0.00000 AK020144 Hmmr 4.09955 0.00000 BC062809 Figf 4.09024 0.00040 AK033321 Top2a 4.06593 0.00000 BC066148 4632434I11Rik 4.06530 0.00006 AK034466 Il4ra 4.01897 0.00001 AK077435 Stom 4.01260 0.00004 AK002590 Fas 3.93614 0.00001 AY773477 Cpa6 3.91195 0.00039 AK034534 Ckap2 3.91078 0.00006 AK050778 Kif11 3.90322 0.00001 AK150138 Gbp2 3.89400 0.00434 AK010929 Ccne2 3.88605 0.00009 AK136911 Epas1 3.88227 0.00000 AK044764 Gpnmb 3.88155 0.00569 BC005501 Cd302 3.87893 0.00104 BC046640 Tmem173 3.86508 0.00016 BC053042 Fabp5 3.85299 0.00000 AK010121 Hist1h3f 3.84826 0.00223 AK028919 Ncaph 3.84551 0.00005 AK040174 Ect2 3.80737 0.00013 BC023918 Prr11 3.80613 0.00002 BC109166 Npas2 3.80383 0.00000 AK141381 Slc16a3 3.75865 0.00010 BC060216 Chl1 3.75455 0.00069 AK018553 Slc6a14 3.68827 0.00008 AK027921 Epb4.1l3 3.66783 0.00001 BC147143 Hist1h2bk 3.62179 0.00070 AK018587 Batf 3.55203 0.00001 AK152518 Zc3h12a 3.54805 0.00035 AK002843 Igtp 3.53340 0.00778 AK051873 Nrp2 3.51950 0.00001 AK085188 Tk1 3.51316 0.00004 AK052772 Mki67 3.47948 0.00000 AK002451 Cks2 3.42033 0.00016 ENSMUST00000119358 Gm12891 3.42033 0.00016 AK049601 Rrm1 3.41915 0.00001 AK035110 Robo2 3.41722 0.00010 BC057985 Orm2 3.40201 0.00319 BC053042 Fabp5 3.37863 0.00382 AK044746 Adamts5 3.37517 0.00034 AK021258 Bub1 3.36567 0.00005 AK033196 Mcm5 3.35162 0.00000 AK144181 Adm 3.35107 0.00092 AK134045 Plk1 3.34755 0.00000 AK158142 Ska3 3.34679 0.00005 AK052980 Ltbp2 3.33913 0.00024 AK014207 Ly6e 3.32779 0.00110 AK083728 Esco2 3.32193 0.00115 AK005415 Stat2 3.30566 0.00300 AK014340 Moxd1 3.30345 0.00003 BC125011 Hist1h2af 3.27506 0.00004 AY455829 Ncapg2 3.27390 0.00001 AK137073 Diap3 3.26907 0.00060 AK050456 Depdc1a 3.24783 0.00002 BC023743 Olfml2b 3.19989 0.00079 AK002451 Cks2 3.19796 0.00000 AK003838 Pbk 3.19638 0.00014 AK012870 Gtse1 3.19362 0.00001 XR_141327 Gm10309 3.18763 0.00056 AK030231 Cdk1 3.17969 0.00035 AK019471 Stil 3.17539 0.00088 BC060304 Hist1h2bg 3.16744 0.00262 NM_144938 C1s 3.15582 0.00045 AK048550 Il1r1 3.15468 0.00002 AK005646 Asf1b 3.15459 0.00002 AK077513 Aurka 3.14201 0.00003 AK042697 Nusap1 3.13352 0.00244 BC116724 Serpina3c 3.13200 0.00049 AK135524 Pdzrn4 3.11279 0.00064 BC028559 Kif23 3.10070 0.00005 AK053906 Lig1 3.09703 0.00011 AK132030 Clu 3.09280 0.00000 AK004231 Tcf19 3.08905 0.00053 AK012040 Ckap2l 3.06630 0.00018 ENSMUST00000102979 Hist1h4m 3.05083 0.00568 BC019757 Hist1h4i 3.04152 0.00091 AK036316 Lrrn3 3.04149 0.00003 BC090671 Hells 3.03072 0.00006 BC120800 Hist1h3c 3.01920 0.00012 AK141392 Ifitm2 3.01700 0.00001 AK129034 Arhgap11a 3.00535 0.00006 AK132236 Sdc1 2.99118 0.00005 AK008638 Mad2l1 2.98417 0.00000 AK004692 Sord 2.96249 0.00011 AK143672 Atp2b4 2.94606 0.00008 AK019885 Tnfrsf9 2.94151 0.00008 BC024848 Cenpi 2.93857 0.00005 AK134319 Atad2 2.93195 0.00020 AK033205 Brip1 2.93156 0.00402 AK079749 Ifi44 2.93021 0.02994 AK007221 Herc6 2.92416 0.00665 AK040324 B4galnt1 2.92283 0.00002 AK049856 Slc11a2 2.91767 0.00000 AK089950 Ccne1 2.91619 0.00001 AK009085 Anxa8 2.90339 0.00053 AK011276 Ppap2b 2.90083 0.00007 AK039737 Pcdh7 2.87775 0.00004 AK002545 Irgm1 2.87276 0.01085 BC099406 Hist1h2ai 2.87158 0.00229 AK045067 Kif20b 2.85365 0.00028 AK012192 Cdc6 2.85037 0.00068 AK041487 Ttk 2.84679 0.00001 AK003065 S100a4 2.84318 0.00055 AK144785 Fosl1 2.84250 0.00177 AK011311 Tpx2 2.83919 0.00008 BC068159 Iqgap3 2.83706 0.00005 AK011502 Egln3 2.83247 0.00012 AK013312 Ccnb2 2.82627 0.00004 AK047165 Socs3 2.82118 0.00001 AK086930 Layn 2.82103 0.00041 AK043023 Prnd 2.80280 0.00139 BC021501 Lurap1l 2.79790 0.00001 AK036390 Wdhd1 2.79170 0.00000 AK030851 Alcam 2.78902 0.00019 AK020421 Hist1h3d 2.78867 0.00631 XM_001005050 Gm5593 2.78491 0.00000 AK075871 2310008H04Rik 2.77016 0.00000 AK028728 Exo1 2.74748 0.00004 AK005501 Plac1 2.73245 0.00271 BC021433 Dram1 2.73186 0.00007 AK150795 Niacr1 2.72256 0.00038 BC023850 Tlr3 2.71614 0.01205 AK133234 Tgfbr3 2.71294 0.00033 AK075951 Aurkb 2.69755 0.00008 AK141509 Sncg 2.68851 0.00150 AK007822 Aldh1l1 2.68725 0.00080 AK079862 Ccl20 2.68443 0.00081 AF529169 AF529169 2.68297 0.00001 AK088142 Mcm3 2.67712 0.00000 BC034832 Apon 2.67572 0.00034 AK045076 Clspn 2.66981 0.00000 BC021874 C330027C09Rik 2.66711 0.00003 AK032722 Rnf150 2.65292 0.00000 AK164473 Mis18bp1 2.64606 0.00041 AK011820 Fbxo5 2.64483 0.00432 AK079915 Lgals3bp 2.63853 0.00048 AK076451 Nsg1 2.63832 0.00000 AK014612 Fbxo32 2.63373 0.00008 AK088502 Bub1b 2.63198 0.00037 BC003281 Parp9 2.63151 0.00259 AK004753 Ggta1 2.62425 0.00023 AK008124 H2afx 2.61852 0.00006 AK041009 Map3k8 2.61656 0.00004 AK005043 Tlr2 2.60727 0.00028 AK139985 Tnfaip6 2.59900 0.00013 AK149744 Cd14 2.59606 0.00019 AK051920 Abca1 2.59442 0.00003 AK143374 Cebpb 2.59341 0.00000 BC022176 Ptx3 2.59331 0.00092 AK036384 Dlgap5 2.56690 0.00017 AK011090 2810417H13Rik 2.55548 0.00138 AK021066 Ankrd55 2.55491 0.00002 AK004702 Lrdd 2.55078 0.00207 AK079915 Lgals3bp 2.54730 0.00331 AK012027 Angpt1 2.54521 0.00015 AK020346 Scel 2.54234 0.00054 AK134420 Cdc45 2.53912 0.00002 AK046595 Fam102b 2.53555 0.00076 AK154786 Tnfrsf23 2.52129 0.00039 AK052863 Atp1a2 2.51843 0.00061 NM_029617 Casc5 2.51758 0.00025 AK051201 Kif20a 2.51363 0.00001 AK150580 Mastl 2.50980 0.00002 AK143383 Smim3 2.50838 0.00005 AK133445 Cenpe 2.49728 0.00003 AK157802 Ticrr 2.49186 0.00002 AK043725 Dnajc9 2.48960 0.00000 AK040615 Fam83d 2.48864 0.00011 AK050821 Kif15 2.48213 0.00007 AK035309 Parp10 2.47485 0.00255 AK034839 Chaf1a 2.46985 0.00144 AK019136 Fancd2 2.44898 0.00008 BC021847 Smc2 2.43620 0.00001 AK011131 Cdc20 2.43077 0.00005 AK016699 Cyp1b1 2.42419 0.00843 AK028996 Man1a 2.42308 0.00011 AK002546 Tmem176a 2.42250 0.00007 AK030970 Parpbp 2.42140 0.00004 AK003466 Ier3 2.41516 0.00033 AK046691 Car5b 2.41461 0.00002 AK084837 Kif4 2.41343 0.00009 AK151469 B4galt5 2.41283 0.00053 AK012614 Pcna 2.41170 0.00000 AK003690 St8sia4 2.40496 0.00666 AK041638 Foxm1 2.40364 0.00017 BC025635 Cd44 2.39868 0.00027 BC113158 4833403I15Rik 2.39645 0.00405 AK015393 Artn 2.39149 0.00074 AK089215 Nfkbia 2.38470 0.00005 NM_178195 Hist1h2bf 2.37986 0.00000 BC103533 Mybl1 2.36748 0.00005 AK079570 Emb 2.36529 0.00198 AK033516 Slc16a1 2.35341 0.00022 AK133985 H2-T22 2.35181 0.00232 AK003408 Spc25 2.34590 0.00001 AK075819 Uhrf1 2.33997 0.00001 AK149748 Icam1 2.33298 0.00003 AK005062 Dusp6 2.33199 0.00146 AK002219 Gpx3 2.33173 0.00216 AK019283 Ly96 2.33138 0.00021 AK182234 Ptges2 2.32633 0.00052 AK002516 Arhgdib 2.31508 0.00571 AK156348 Prss23 2.31478 0.00001 AK047965 Otor 2.31414 0.00111 NM_178199 Hist1h2bl 2.30391 0.00022 AK028271 Cdh11 2.29902 0.00459 AK031556 Traf3ip2 2.29393 0.00008 AK014528 Ptpn2 2.29097 0.00000 NM_178184 Hist1h2an 2.28703 0.00007 AK034764 Incenp 2.28670 0.00009 AK049923 Ptgs2 2.28523 0.00014 AK011710 Casp6 2.28329 0.00182 AK089921 Bckdhb 2.28003 0.00000 AK006208 Cenph 2.27869 0.00009 BC018793 Fam46c 2.27731 0.00017 XM_001005050 Gm5593 2.27704 0.00100 AK004883 Egfr 2.27650 0.00026 AK087931 Mndal 2.27150 0.02274 AK009958 Tubb6 2.26501 0.00003 AK034348 Ptger4 2.25798 0.00002 BC025462 Fanci 2.24759 0.00076 AB112350 Napepld 2.24641 0.00002 AK009690 Stx6 2.23443 0.00000 BC006072 Ucp2 2.23171 0.00497 AK002443 Dgat2 2.22460 0.00036 XM_003945997 LOC101056305 2.22170 0.00002 AK006328 Knstrn 2.21993 0.00668 AK004925 Nek6 2.21773 0.00000 AK157231 Mocs1 2.21764 0.00218 AK154578 Acvr1b 2.21666 0.00004 AK002454 Ctsc 2.21603 0.00123 AK028384 Rassf2 2.21381 0.00000 AK010821 Fam49a 2.21358 0.00320 AK010885 Cenpm 2.20298 0.00029 AK087680 Tnfaip2 2.20029 0.00016 AK033257 Dock11 2.19596 0.00007 AK009000 Klk10 2.19119 0.00026 AK076282 Tacc3 2.19037 0.00003 AK010426 Cdkn3 2.19016 0.00061 AK147751 Lonrf3 2.18914 0.00015 AK011435 Tyms 2.17890 0.00183 AK016909 Atoh8 2.17550 0.00013 AK011197 Psrc1 2.17518 0.00833 AK040220 Cx3cl1 2.16581 0.00284 AK045954 Serpine2 2.16145 0.00157 AK003328 Csrp2 2.15511 0.00020 ENSMUST00000083837 n-R5s54 2.15391 0.00460 BC112328 Gbp10 2.14819 0.01266 AK036743 Slc16a10 2.14710 0.00206 AK086962 Slc43a3 2.14655 0.00000 AK014566 Tapbp 2.14637 0.00012 AK014357 Sgol1 2.14352 0.00012 AK137142 Arg2 2.13924 0.00026 AK028497 Mybl2 2.13426 0.00036 AK040020 Osmr 2.13070 0.00000 AK087541 Nfil3 2.13069 0.00005 AK003773 Hmgb2 2.12953 0.00001 AK031241 Dhfr 2.12622 0.00139 AK005029 Isyna1 2.12305 0.00008 AK007408 Tmem176b 2.12115 0.00001 AK018527 Clmp 2.11932 0.00003 AK186355 Fam63b 2.11788 0.00050 AF459018 C1rb 2.11337 0.00006 AK031083 Cdca2 2.11334 0.00005 AK151086 Micall2 2.11096 0.00001 AK172247 Ccdc3 2.10761 0.00010 AF065903 Fgf2 2.10641 0.00019 AK011242 Rad51 2.10230 0.00010 NM_001042421 Kntc1 2.09794 0.00044 BC076592 Tnfrsf22 2.09338 0.00027 AK139711 Fmnl3 2.09226 0.00063 AK002567 Mt2 2.09118 0.00764 AK012919 Dtl 2.08911 0.00048 BC080752 Hrct1 2.08467 0.00002 AK006216 Cdhr4 2.08452 0.00004 AK033288 Zfp367 2.08382 0.00002 BC120684 Bdkrb1 2.08340 0.00080 BC003276 St6galnac2 2.08232 0.00018 AK019687 C1ra 2.07476 0.00903 BC025900 Spdl1 2.07083 0.00075 AK199612 Cdca5 2.06634 0.00012 BC020489 Apol9a 2.06352 0.00232 AK010622 Scarb1 2.06333 0.00018 AK133962 Plk2 2.06200 0.00004 AK086173 Brca1 2.05943 0.00061 AK052998 Macrod1 2.05702 0.00006 AK005353 Evi2a 2.05680 0.00001 AK030685 Slc39a10 2.05625 0.00454 AK009581 Hfe 2.05303 0.00163 AK037836 Pola1 2.04982 0.00052 AK156273 Cables1 2.04539 0.00033 AK032066 Hspa4l 2.03965 0.00112 AK029474 B4galt1 2.03551 0.00000 AK011489 Rfc5 2.03467 0.00027 AK012177 Ripk3 2.03152 0.00516 AK021408 Troap 2.02858 0.00013 AK038940 Enpp2 2.02716 0.00079 BC138877 Lrrc8b 2.02616 0.00014 AK010888 Ptgr1 2.01291 0.00483 NM_197997 4930422G04Rik 2.00965 0.00028 AK159139 Hspa1b 2.00787 0.00089 AK011352 Ncapd2 2.00505 0.00015 BC023391 D930015E06Rik 2.00501 0.00099 AK042942 Fen1 2.00295 0.00003 AK039481 Nov 2.00063 0.00445

B. Downregulated genes

Fold change P value Gene accession Gene symbol (Ad-Mt2) (Ad-Mt2) AK010786 Tubb2b 0.49913 0.00598 BC031853 Slc38a7 0.49886 0.00007 AK122559 Rab11fip4 0.49530 0.00005 AK012888 Angptl1 0.49018 0.00036 BC089158 Epb4.1l4a 0.48968 0.00016 AJ001692 Glp1r 0.48907 0.00009 AK049685 Wscd2 0.48509 0.00006 AK028833 Cobl 0.48488 0.00102 BC132471 Gm129 0.48391 0.00019 AK035548 Cnn1 0.48341 0.00133 AK079196 Npr3 0.48327 0.00428 AK018106 6330403K07Rik 0.48210 0.00054 AF241833 Scamp5 0.48150 0.00008 AK019881 Rps6ka2 0.48065 0.00018 AK137667 Mdga2 0.47748 0.00016 AK122564 Gm22 0.47651 0.00087 AK002880 Tagln 0.47516 0.00008 AK139185 Pycr1 0.47431 0.00024 NR_045177 2900055J20Rik 0.46896 0.00037 BC022696 Dusp12 0.46785 0.00075 AK040564 Bcam 0.46482 0.00492 AK085667 Tnc 0.46272 0.00007 AK077393 Rarres1 0.46100 0.00087 BC021324 Slc38a3 0.45959 0.00013 AK048770 Kif21b 0.45817 0.00004 AK140217 Fgfr3 0.45662 0.00016 BC009815 Mia 0.45624 0.00001 AK006589 Aaed1 0.45396 0.00014 AK132611 Cpxm1 0.45060 0.00002 AK048456 Matn3 0.44920 0.00114 AK028770 Boc 0.44825 0.00024 AK147397 Slc22a23 0.44620 0.00503 AK029323 Galnt5 0.44215 0.00003 AY572454 Trim66 0.43751 0.00013 AK004279 Sync 0.43694 0.00020 NM_001081027 Kcnt2 0.43441 0.00103 AK050297 Omd 0.43227 0.00006 AK007574 Fgf21 0.42349 0.00425 AK134583 Pth1r 0.41758 0.00003 AK044928 Thbd 0.41561 0.00167 AK003959 Cdkn1c 0.41551 0.00003 AK008175 Tspan13 0.41480 0.00061 NM_009925 Col10a1 0.40881 0.00040 AK029720 Slc2a5 0.40793 0.00067 AK008849 Cybrd1 0.40757 0.00054 BC064033 Fam180a 0.40578 0.00322 AK142805 Chrdl1 0.40518 0.00011 AK154923 H2-DMb2 0.39843 0.00388 BC010816 Lims2 0.39522 0.00003 AK003888 Fxyd6 0.38883 0.00007 AK085272 Cyb5r2 0.38640 0.00006 AK029068 Itgbl1 0.38550 0.00034 AK052838 Colgalt2 0.38435 0.00153 AK004477 Tmem8c 0.35648 0.00099 AK037740 Satb1 0.35581 0.00001 AK044636 Slc8a3 0.34971 0.00028 AK014342 Matn1 0.33893 0.00156 AK014504 Aspn 0.31529 0.02984 AK019476 Smpd3 0.29480 0.00005 AK003423 Slc16a14 0.28666 0.00002 AK016571 Scube2 0.22055 0.00009 AK083768 Slc35f1 0.17983 0.00013 AK029352 C1qtnf3 0.17908 0.00303 AK003506 Il17b 0.13505 0.00005 AF117759 Sfrp5 0.10571 0.00001

Supplementary Table S3. List of genes upregulated in common by Ad-Mt2 and Ad-Mtf1 infection in chondrocytes (fold change ≥ 2; P < 0.05)

Fold change Fold change P value P value Gene accession Gene symbol (Ad-Mt2) (Ad-Mtf1) (Ad-Mt2) (Ad-Mtf1) BC034858 Saa2 96.60043 24.2647 0.00012 0.00008 AK144158 Cxcl3 87.32795 12.05285 0.00000 0.00006 AK148467 Mmp3 78.21605 20.27975 0.00003 0.00003 AK150430 Hp 68.07137 25.51925 0.00000 0.00000 BC052495 Saa1 53.60302 13.97818 0.00000 0.00010 BC019212 Saa4 35.64665 12.46459 0.00000 0.00001 AK003101 Ccl5 34.08537 8.899312 0.00001 0.00015 AK143562 Lbp 24.57527 12.28706 0.00000 0.00001 AK003905 Steap4 22.33843 12.52124 0.00065 0.00072 BC029976 C3 22.25393 12.40415 0.00031 0.00029 AK150728 Mmp13 22.18268 13.30671 0.00051 0.00036 AK010170 Slpi 20.56522 9.436339 0.00000 0.00006 AK014895 Slc39a8 20.4415 7.184332 0.00000 0.00006 BC055885 Saa3 19.62024 14.39575 0.00098 0.00093 AK132590 Ccl2 19.52451 4.855447 0.00000 0.00028 AK149531 Cfb 17.4254 6.563163 0.00000 0.00001 AJ132103 Vnn3 15.51554 4.869322 0.00000 0.00004 AK002773 Vnn1 15.45479 9.015094 0.00051 0.00021 AK140312 Cxcl1 13.94237 3.593797 0.00001 0.00029 ENSMUST00000174425 Gm20419 13.66814 6.629757 0.00002 0.00009 AK003143 Fabp4 12.95102 4.462663 0.00104 0.00822 BC010546 Ifi204 10.88619 3.72209 0.00005 0.00034 AK008517 Xdh 10.44445 4.756782 0.00002 0.00001 AK003407 Ifitm3 9.200959 3.954263 0.00003 0.00035 AK051475 Chi3l1 9.146325 5.927864 0.00061 0.00064 AK050355 Slfn9 8.905243 4.476949 0.00000 0.00004 AF065919 Nos2 8.52496 2.268262 0.00069 0.00094 AK078827 Ccl7 8.259001 2.758231 0.00025 0.00423 AK041059 Bst1 8.249263 2.303911 0.00010 0.00250 AF272948 Gpr84 8.140849 3.775507 0.00001 0.00002 AK080147 Il13ra1 7.473545 2.854076 0.00000 0.00001 AK036666 Gbp7 7.268283 2.815275 0.00218 0.00033 AF140672 Ifi202b 7.043477 2.711639 0.00011 0.00637 AK007477 Rtp4 7.00411 3.278154 0.00762 0.00611 AK140360 Ch25h 6.935945 2.228542 0.00001 0.00231 AK139833 A4galt 6.907491 2.297404 0.00001 0.00153 BC031424 Isg15 6.849921 4.340083 0.00377 0.00136 BC117110 Hist1h2ab 6.807732 4.058616 0.00006 0.00023 EF660528 AW112010 6.493383 3.058779 0.00017 0.00261 XR_105655 Gm3601 6.404353 3.72299 0.00013 0.00014 XR_105655 Gm3601 6.390864 3.774777 0.00015 0.00011 AK053190 Cebpd 6.235834 3.822443 0.00006 0.00003 AK087960 Srgn 6.177983 3.054898 0.00020 0.00043 BC065803 Hist1h2ag 6.00027 3.995715 0.00004 0.00016 AK009161 Angptl4 5.9353 2.345213 0.00275 0.01985 AK033934 Fgf7 5.748685 2.090315 0.00002 0.00202 BC013651 Serpina3n 5.672865 4.238974 0.00001 0.00001 AK075570 Sod3 5.670892 3.132767 0.00014 0.00057 AK151131 Ccl9 5.647404 4.182777 0.00005 0.00002 AK004651 Mmp9 5.61343 2.287531 0.00017 0.00671 BC033044 Nfkbiz 5.542515 2.906147 0.00002 0.00010 AK018602 Ifih1 5.472604 2.199517 0.00570 0.00027 AK149774 Lcn2 5.427273 4.188388 0.00187 0.00247 AK018830 Fam111a 5.426406 3.307999 0.00003 0.00014 AK155016 Bcl3 5.316064 3.085611 0.00000 0.00001 AK088907 Rrm2 5.230384 3.17838 0.00001 0.00007 AK010768 Glipr1 5.099789 3.589245 0.00012 0.00055 AK077326 Tm4sf1 4.958723 2.152427 0.00005 0.00263 AK017282 Prr16 4.836482 3.009399 0.00137 0.00593 AK002830 Irf7 4.752715 2.198749 0.00474 0.00011 XM_917927 LOC640793 4.748835 2.48035 0.00035 0.00002 AK133465 Ampd3 4.716253 2.04609 0.00001 0.00087 AK018777 Kitl 4.52868 3.026796 0.00000 0.00021 AK019924 Gcnt2 4.499549 2.170602 0.00003 0.00109 AK085754 Trf 4.460362 2.618377 0.00027 0.00103 AK014783 Irak3 4.400925 2.162217 0.00000 0.00032 AK132597 Prg4 4.393782 2.528544 0.00037 0.00349 AK044924 Ccna2 4.356286 2.983986 0.00005 0.00015 AK137203 Avpr1a 4.338242 2.083971 0.00114 0.00759 AK011243 Chaf1b 4.236358 3.357223 0.00000 0.00009 AK010166 Car13 4.174618 2.304785 0.00005 0.00090 AK020117 Hist1h1b 4.15125 3.174702 0.00009 0.00019 AK041985 Tmem255a 4.138532 2.75318 0.00050 0.00003 AY462056 Kng2 4.128916 2.072079 0.00005 0.00460 AK013034 Cyp7b1 4.108213 2.264848 0.00000 0.00003 AK020144 Hmmr 4.099548 2.599013 0.00000 0.00008 BC062809 Figf 4.090241 2.202339 0.00040 0.00796 AK033321 Top2a 4.06593 2.946578 0.00000 0.00000 BC066148 4632434I11Rik 4.065296 2.768075 0.00006 0.00040 AK002590 Fas 3.936136 2.614915 0.00001 0.00007 AK034534 Ckap2 3.910782 2.71332 0.00006 0.00034 AK050778 Kif11 3.903225 2.983621 0.00001 0.00001 AK010929 Ccne2 3.88605 3.655501 0.00009 0.00032 AK136911 Epas1 3.882268 2.533606 0.00000 0.00001 AK044764 Gpnmb 3.881549 2.833224 0.00569 0.01892 BC005501 Cd302 3.878927 2.13382 0.00104 0.00448 BC046640 Tmem173 3.86508 2.004502 0.00016 0.00158 BC053042 Fabp5 3.852989 2.421103 0.00000 0.00002 AK010121 Hist1h3f 3.848258 2.754168 0.00223 0.00753 AK028919 Ncaph 3.845509 2.839674 0.00005 0.00009 AK040174 Ect2 3.807371 2.55311 0.00013 0.00018 BC023918 Prr11 3.806128 2.243597 0.00002 0.00033 AK027921 Epb4.1l3 3.667832 3.134705 0.00001 0.00001 BC147143 Hist1h2bk 3.62179 2.868462 0.00070 0.00082 AK002843 Igtp 3.533397 2.109183 0.00778 0.00089 AK085188 Tk1 3.513158 2.398965 0.00004 0.00008 AK052772 Mki67 3.479479 3.036617 0.00000 0.00000 AK049601 Rrm1 3.419151 2.471324 0.00001 0.00002 AK035110 Robo2 3.417219 2.040298 0.00010 0.00011 BC053042 Fabp5 3.378628 2.170133 0.00382 0.01862 AK021258 Bub1 3.365665 2.413469 0.00005 0.00030 AK033196 Mcm5 3.351623 2.438272 0.00000 0.00005 AK134045 Plk1 3.347549 2.580292 0.00000 0.00017 AK158142 Ska3 3.346786 2.438884 0.00005 0.00027 AK014207 Ly6e 3.327792 3.211022 0.00110 0.00046 AK083728 Esco2 3.321932 2.59992 0.00115 0.00232 AK014340 Moxd1 3.303451 2.457193 0.00003 0.00005 BC125011 Hist1h2af 3.27506 2.428823 0.00004 0.00094 AY455829 Ncapg2 3.273902 2.484263 0.00001 0.00019 AK137073 Diap3 3.269065 3.183627 0.00060 0.00019 AK050456 Depdc1a 3.247834 2.568832 0.00002 0.00027 BC023743 Olfml2b 3.199891 2.944911 0.00079 0.00084 AK002451 Cks2 3.197963 2.046897 0.00000 0.00006 AK003838 Pbk 3.196375 2.680772 0.00014 0.00029 AK012870 Gtse1 3.193619 2.498412 0.00001 0.00011 XR_141327 Gm10309 3.187629 2.113415 0.00056 0.00117 AK030231 Cdk1 3.179691 2.193025 0.00035 0.00000 AK019471 Stil 3.17539 2.180444 0.00088 0.00292 BC060304 Hist1h2bg 3.167436 2.648032 0.00262 0.00543 AK005646 Asf1b 3.154585 2.131435 0.00002 0.00009 AK077513 Aurka 3.142007 2.389108 0.00003 0.00014 AK042697 Nusap1 3.133519 2.136376 0.00244 0.00792 BC116724 Serpina3c 3.131995 2.162867 0.00049 0.00328 BC028559 Kif23 3.100701 2.633978 0.00005 0.00007 AK053906 Lig1 3.097029 2.365902 0.00011 0.00000 AK132030 Clu 3.092802 3.025618 0.00000 0.00000 AK004231 Tcf19 3.089049 2.355349 0.00053 0.00065 AK012040 Ckap2l 3.066297 2.316945 0.00018 0.00006 ENSMUST00000102979 Hist1h4m 3.050828 2.515077 0.00568 0.00082 BC019757 Hist1h4i 3.041524 2.037779 0.00091 0.01108 AK036316 Lrrn3 3.041489 2.006738 0.00003 0.00084 BC090671 Hells 3.030724 2.768108 0.00006 0.00003 BC120800 Hist1h3c 3.019196 2.780675 0.00012 0.00026 AK129034 Arhgap11a 3.005345 2.160411 0.00006 0.00094 AK008638 Mad2l1 2.984169 2.049789 0.00000 0.00156 AK019885 Tnfrsf9 2.941514 2.451291 0.00008 0.00015 BC024848 Cenpi 2.938571 2.376988 0.00005 0.00012 AK134319 Atad2 2.931953 2.056589 0.00020 0.00024 AK033205 Brip1 2.931556 2.71277 0.00402 0.00221 AK049856 Slc11a2 2.917666 2.072238 0.00000 0.00000 AK089950 Ccne1 2.916194 3.459355 0.00001 0.00008 AK039737 Pcdh7 2.877747 2.284305 0.00004 0.00078 AK012192 Cdc6 2.850372 2.711029 0.00068 0.00001 AK041487 Ttk 2.846785 2.204085 0.00001 0.00005 AK144785 Fosl1 2.842498 2.526481 0.00177 0.00144 AK011311 Tpx2 2.839186 2.258164 0.00008 0.00005 BC068159 Iqgap3 2.837056 2.074824 0.00005 0.00151 AK047165 Socs3 2.821183 2.10329 0.00001 0.00081 AK043023 Prnd 2.802805 2.341532 0.00139 0.00167 AK036390 Wdhd1 2.791696 2.836485 0.00000 0.00000 AK020421 Hist1h3d 2.788665 2.618837 0.00631 0.01012 XM_001005050 Gm5593 2.784909 2.006236 0.00000 0.00003 AK075951 Aurkb 2.697555 2.360374 0.00008 0.00038 AK088142 Mcm3 2.677121 2.091109 0.00000 0.00002 AK045076 Clspn 2.669812 2.237832 0.00000 0.00002 AK164473 Mis18bp1 2.646057 2.07785 0.00041 0.00079 AK011820 Fbxo5 2.644834 2.099524 0.00432 0.00788 AK088502 Bub1b 2.631984 2.007698 0.00037 0.00131 AK008124 H2afx 2.618525 2.481843 0.00006 0.00002 AK020346 Scel 2.542338 6.514211 0.00054 0.00000 AK154786 Tnfrsf23 2.521287 2.965352 0.00039 0.00012 NM_029617 Casc5 2.517576 2.039557 0.00025 0.00056 AK040615 Fam83d 2.488645 2.025487 0.00011 0.00003 AK003466 Ier3 2.415157 2.117775 0.00033 0.00093 AK012614 Pcna 2.411695 2.111827 0.00000 0.00002 BC103533 Mybl1 2.367484 2.003214 0.00005 0.00016 AK075819 Uhrf1 2.339968 2.282986 0.00001 0.00004 AK149748 Icam1 2.332983 2.079322 0.00003 0.00031 AK009958 Tubb6 2.265015 2.027912 0.00003 0.00086 BC006072 Ucp2 2.231713 2.053606 0.00497 0.00032 AK028497 Mybl2 2.134263 2.288545 0.00036 0.00065 AK011242 Rad51 2.102299 3.094121 0.00010 0.00010 BC076592 Tnfrsf22 2.093382 2.697632 0.00027 0.00001 AK002567 Mt2 2.091184 2.242072 0.00764 0.00680 BC138877 Lrrc8b 2.026157 2.206725 0.00014 0.00004

Supplementary Table S4. List of genes upregulated in common by Ad-Mt2 and Ad-Zip8 infection in chondrocytes (fold change ≥ 2; P < 0.05)

Fold change Fold change P value P value Gene accession Gene symbol (Ad-Mt2) (Ad-Zip8) (Ad-Mt2) (Ad-Zip8) BC034858 Saa2 96.60043 55.08124 0.00012 0.00006 AK144158 Cxcl3 87.32795 56.65844 0.00000 0.00000 AK148467 Mmp3 78.21605 63.6611 0.00003 0.00001 AK150430 Hp 68.07137 42.06763 0.00000 0.00000 BC052495 Saa1 53.60302 26.95952 0.00000 0.00003 BC019212 Saa4 35.64665 12.76148 0.00000 0.00000 AK003101 Ccl5 34.08537 13.56748 0.00001 0.00008 AK143562 Lbp 24.57527 21.84639 0.00000 0.00001 AK003905 Steap4 22.33843 21.59643 0.00065 0.00063 BC029976 C3 22.25393 21.14663 0.00031 0.00033 AK150728 Mmp13 22.18268 21.26615 0.00051 0.00052 AK010170 Slpi 20.56522 15.56371 0.00000 0.00000 AK014895 Slc39a8 20.4415 30.42593 0.00000 0.00000 BC055885 Saa3 19.62024 19.19776 0.00098 0.00099 AK132590 Ccl2 19.52451 16.15223 0.00000 0.00003 AK149531 Cfb 17.4254 13.12618 0.00000 0.00000 AJ132103 Vnn3 15.51554 14.56365 0.00000 0.00000 AK002773 Vnn1 15.45479 14.63372 0.00051 0.00060 AK140312 Cxcl1 13.94237 14.04267 0.00001 0.00001 ENSMUST00000174425 Gm20419 13.66814 8.713394 0.00002 0.00002 AK003143 Fabp4 12.95102 6.870779 0.00104 0.00360 BC010546 Ifi204 10.88619 6.310496 0.00005 0.00004 AK008517 Xdh 10.44445 10.05781 0.00002 0.00013 AK003407 Ifitm3 9.200959 5.771282 0.00003 0.00010 AK051475 Chi3l1 9.146325 8.597485 0.00061 0.00071 AK050355 Slfn9 8.905243 3.389684 0.00000 0.00013 AF065919 Nos2 8.52496 6.776468 0.00069 0.00072 AK078827 Ccl7 8.259001 8.610187 0.00025 0.00073 AK041059 Bst1 8.249263 8.110897 0.00010 0.00040 AF272948 Gpr84 8.140849 9.013507 0.00001 0.00001 AK089780 Il6 8.065599 3.781924 0.00007 0.00148 AK080147 Il13ra1 7.473545 5.319193 0.00000 0.00010 AF140672 Ifi202b 7.043477 4.652537 0.00011 0.00039 AK140360 Ch25h 6.935945 3.911698 0.00001 0.00053 AK139833 A4galt 6.907491 5.022715 0.00001 0.00003 BC117110 Hist1h2ab 6.807732 3.312814 0.00006 0.00203 EF660528 AW112010 6.493383 6.519243 0.00017 0.00034 XR_105655 Gm3601 6.404353 4.199841 0.00013 0.00006 XR_105655 Gm3601 6.390864 4.064342 0.00015 0.00015 AK053190 Cebpd 6.235834 5.932958 0.00006 0.00002 AK087960 Srgn 6.177983 5.226686 0.00020 0.00012 BC065803 Hist1h2ag 6.00027 3.037499 0.00004 0.00162 AK009161 Angptl4 5.9353 5.672122 0.00275 0.00273 AK033934 Fgf7 5.748685 5.521054 0.00002 0.00002 BC013651 Serpina3n 5.672865 4.712867 0.00001 0.00001 AK075570 Sod3 5.670892 5.376531 0.00014 0.00009 AK151131 Ccl9 5.647404 4.05048 0.00005 0.00019 AK004651 Mmp9 5.61343 4.604836 0.00017 0.00070 BC033044 Nfkbiz 5.542515 5.11664 0.00002 0.00000 AK149774 Lcn2 5.427273 5.166223 0.00187 0.00187 AK018830 Fam111a 5.426406 3.349733 0.00003 0.00009 AK155016 Bcl3 5.316064 4.728607 0.00000 0.00000 AK088907 Rrm2 5.230384 2.677843 0.00001 0.00017 AK010768 Glipr1 5.099789 3.606215 0.00012 0.00051 AK077326 Tm4sf1 4.958723 3.841042 0.00005 0.00014 BC027838 Ereg 4.932776 5.481379 0.00009 0.00035 AK017282 Prr16 4.836482 3.832481 0.00137 0.00237 AK133465 Ampd3 4.716253 4.337096 0.00001 0.00003 AK019924 Gcnt2 4.499549 3.182036 0.00003 0.00012 AK085754 Trf 4.460362 4.526262 0.00027 0.00020 AK014783 Irak3 4.400925 3.991471 0.00000 0.00001 AK132597 Prg4 4.393782 3.576796 0.00037 0.00073 AK044924 Ccna2 4.356286 2.566724 0.00005 0.00012 AK137203 Avpr1a 4.338242 2.591989 0.00114 0.00268 AK089687 Il13ra2 4.334973 3.433053 0.00009 0.00489 AK011243 Chaf1b 4.236358 2.54614 0.00000 0.00005 AK010166 Car13 4.174618 3.138488 0.00005 0.00052 AK035320 Gna14 4.169618 3.686905 0.00000 0.00001 AK020117 Hist1h1b 4.15125 2.965882 0.00009 0.00005 AK041985 Tmem255a 4.138532 3.666536 0.00050 0.00043 AY462056 Kng2 4.128916 3.196306 0.00005 0.00042 BC003748 Sntb1 4.126773 4.036997 0.00028 0.00013 AK013034 Cyp7b1 4.108213 4.236047 0.00000 0.00004 AK020144 Hmmr 4.099548 2.299273 0.00000 0.00006 BC062809 Figf 4.090241 2.712381 0.00040 0.00309 AK033321 Top2a 4.06593 2.559974 0.00000 0.00001 AK034466 Il4ra 4.018968 4.437014 0.00001 0.00003 AK077435 Stom 4.012598 2.138095 0.00004 0.00441 AK002590 Fas 3.936136 3.500189 0.00001 0.00002 AY773477 Cpa6 3.911952 3.079558 0.00039 0.00104 AK034534 Ckap2 3.910782 2.460517 0.00006 0.00055 AK050778 Kif11 3.903225 2.418473 0.00001 0.00003 AK150138 Gbp2 3.893997 2.406885 0.00434 0.00031 AK136911 Epas1 3.882268 3.862787 0.00000 0.00002 AK044764 Gpnmb 3.881549 3.73639 0.00569 0.00785 BC005501 Cd302 3.878927 3.763387 0.00104 0.00146 BC046640 Tmem173 3.86508 3.524008 0.00016 0.00008 BC053042 Fabp5 3.852989 2.591685 0.00000 0.00001 AK010121 Hist1h3f 3.848258 2.474341 0.00223 0.01874 AK028919 Ncaph 3.845509 2.186252 0.00005 0.00051 AK040174 Ect2 3.807371 2.345197 0.00013 0.00046 BC023918 Prr11 3.806128 2.306216 0.00002 0.00010 BC109166 Npas2 3.803827 3.858367 0.00000 0.00000 AK141381 Slc16a3 3.758652 2.841889 0.00010 0.00015 AK018553 Slc6a14 3.688266 2.189831 0.00008 0.00026 AK027921 Epb4.1l3 3.667832 2.730453 0.00001 0.00038 BC147143 Hist1h2bk 3.62179 2.190252 0.00070 0.00788 AK018587 Batf 3.552031 2.931136 0.00001 0.00001 AK152518 Zc3h12a 3.548046 3.949195 0.00035 0.00016 AK051873 Nrp2 3.519498 3.695869 0.00001 0.00011 AK085188 Tk1 3.513158 2.192858 0.00004 0.00008 AK052772 Mki67 3.479479 2.485127 0.00000 0.00008 AK002451 Cks2 3.420331 2.214901 0.00016 0.00031 ENSMUST00000119358 Gm12891 3.420331 2.194852 0.00016 0.00198 AK035110 Robo2 3.417219 2.895939 0.00010 0.00003 BC057985 Orm2 3.402009 2.341387 0.00319 0.03604 BC053042 Fabp5 3.378628 2.432083 0.00382 0.01370 AK044746 Adamts5 3.375166 2.870561 0.00034 0.00061 AK021258 Bub1 3.365665 2.342344 0.00005 0.00044 AK144181 Adm 3.351067 4.19217 0.00092 0.00027 AK134045 Plk1 3.347549 2.048398 0.00000 0.00002 AK052980 Ltbp2 3.339131 3.230122 0.00024 0.00038 AK083728 Esco2 3.321932 2.333977 0.00115 0.00373 AK014340 Moxd1 3.303451 3.281951 0.00003 0.00001 BC125011 Hist1h2af 3.27506 2.122775 0.00004 0.00113 AY455829 Ncapg2 3.273902 2.049313 0.00001 0.00006 AK137073 Diap3 3.269065 2.170774 0.00060 0.00251 AK050456 Depdc1a 3.247834 2.096459 0.00002 0.00037 BC023743 Olfml2b 3.199891 2.557307 0.00079 0.00130 AK002451 Cks2 3.197963 2.194852 0.00000 0.00198 AK003838 Pbk 3.196375 2.431016 0.00014 0.00119 XR_141327 Gm10309 3.187629 3.02827 0.00056 0.00473 NM_144938 C1s 3.155822 3.758609 0.00045 0.00028 AK048550 Il1r1 3.154684 2.505077 0.00002 0.00033 AK005646 Asf1b 3.154585 2.351757 0.00002 0.00005 AK042697 Nusap1 3.133519 2.16863 0.00244 0.00616 BC116724 Serpina3c 3.131995 2.023954 0.00049 0.00470 AK135524 Pdzrn4 3.112787 2.321498 0.00064 0.00023 BC028559 Kif23 3.100701 2.350365 0.00005 0.00015 AK132030 Clu 3.092802 3.081445 0.00000 0.00002 AK012040 Ckap2l 3.066297 2.149343 0.00018 0.00009 AK036316 Lrrn3 3.041489 2.129354 0.00003 0.00041 BC120800 Hist1h3c 3.019196 2.592406 0.00012 0.00118 AK141392 Ifitm2 3.016995 2.586935 0.00001 0.00000 AK132236 Sdc1 2.991175 2.628846 0.00005 0.00011 AK143672 Atp2b4 2.946065 2.663231 0.00008 0.00010 AK019885 Tnfrsf9 2.941514 2.715401 0.00008 0.00007 AK033205 Brip1 2.931556 2.089569 0.00402 0.00730 AK040324 B4galnt1 2.922828 2.60693 0.00002 0.00091 AK049856 Slc11a2 2.917666 2.743064 0.00000 0.00001 AK009085 Anxa8 2.903387 2.245385 0.00053 0.00189 AK011276 Ppap2b 2.900832 3.025726 0.00007 0.00019 AK003065 S100a4 2.843178 2.13308 0.00055 0.00735 AK144785 Fosl1 2.842498 3.244133 0.00177 0.00307 BC068159 Iqgap3 2.837056 2.108396 0.00005 0.00000 AK011502 Egln3 2.832465 2.576058 0.00012 0.00026 AK047165 Socs3 2.821183 2.59929 0.00001 0.00005 AK086930 Layn 2.821033 2.091929 0.00041 0.00252 AK043023 Prnd 2.802805 3.518696 0.00139 0.00036 AK030851 Alcam 2.789025 2.175434 0.00019 0.00122 AK075871 2310008H04Rik 2.770158 2.83781 0.00000 0.00001 AK005501 Plac1 2.732455 3.174842 0.00271 0.00124 BC021433 Dram1 2.731856 2.090148 0.00007 0.00187 AK133234 Tgfbr3 2.712935 2.663818 0.00033 0.00018 AK079862 Ccl20 2.684432 3.071001 0.00081 0.00053 AF529169 AF529169 2.682965 2.993022 0.00001 0.00007 BC034832 Apon 2.675724 2.272804 0.00034 0.00003 AK032722 Rnf150 2.652922 2.442335 0.00000 0.00000 AK164473 Mis18bp1 2.646057 2.024664 0.00041 0.00097 AK014612 Fbxo32 2.633732 2.730948 0.00008 0.00007 AK041009 Map3k8 2.616559 2.331679 0.00004 0.00003 AK005043 Tlr2 2.607269 2.793053 0.00028 0.00054 AK149744 Cd14 2.596057 2.629516 0.00019 0.00064 AK051920 Abca1 2.594416 2.349839 0.00003 0.00002 AK143374 Cebpb 2.59341 2.627848 0.00000 0.00001 BC022176 Ptx3 2.593308 2.460028 0.00092 0.00101 AK012027 Angpt1 2.545211 2.241095 0.00015 0.00074 AK020346 Scel 2.542338 2.221579 0.00054 0.00053 AK046595 Fam102b 2.535551 2.161411 0.00076 0.00328 AK143383 Smim3 2.508378 2.206243 0.00005 0.00014 AK028996 Man1a 2.423081 2.258443 0.00011 0.00064 AK002546 Tmem176a 2.422503 2.208782 0.00007 0.00012 AK003466 Ier3 2.415157 2.59353 0.00033 0.00019 AK151469 B4galt5 2.412835 2.157225 0.00053 0.00260 BC025635 Cd44 2.398681 2.33368 0.00027 0.00111 AK015393 Artn 2.391486 2.311297 0.00074 0.00257 AK089215 Nfkbia 2.384698 2.252251 0.00005 0.00007 AK079570 Emb 2.36529 2.120357 0.00198 0.00358 AK033516 Slc16a1 2.353408 2.06689 0.00022 0.00027 AK149748 Icam1 2.332983 2.282829 0.00003 0.00029 AK005062 Dusp6 2.33199 2.427764 0.00146 0.00510 AK002219 Gpx3 2.331732 2.493474 0.00216 0.00147 AK156348 Prss23 2.31478 2.362845 0.00001 0.00000 AK028271 Cdh11 2.299023 2.205559 0.00459 0.00476 AK031556 Traf3ip2 2.293935 2.009266 0.00008 0.00006 AK014528 Ptpn2 2.290971 2.296846 0.00000 0.00024 AK049923 Ptgs2 2.285227 2.254839 0.00014 0.00135 AK089921 Bckdhb 2.280026 2.223992 0.00000 0.00001 BC018793 Fam46c 2.277305 2.599259 0.00017 0.00023 AK004883 Egfr 2.276502 2.201963 0.00026 0.00060 AB112350 Napepld 2.246405 2.198311 0.00002 0.00073 AK009690 Stx6 2.234425 2.374847 0.00000 0.00000 AK002443 Dgat2 2.224601 2.07968 0.00036 0.00112 AK004925 Nek6 2.217727 2.278691 0.00000 0.00000 AK157231 Mocs1 2.217636 2.054902 0.00218 0.00417 AK154578 Acvr1b 2.216657 2.173707 0.00004 0.00013 AK087680 Tnfaip2 2.200294 2.073475 0.00016 0.00269 AK147751 Lonrf3 2.189143 2.209183 0.00015 0.00018 AK137142 Arg2 2.13924 2.680021 0.00026 0.00479 AK040020 Osmr 2.130696 2.121305 0.00000 0.00000 AK007408 Tmem176b 2.121146 2.091643 0.00001 0.00002 AK018527 Clmp 2.119316 2.015781 0.00003 0.00012 AK186355 Fam63b 2.11788 2.091318 0.00050 0.00033 AF459018 C1rb 2.113374 2.220932 0.00006 0.00003 AF065903 Fgf2 2.106408 2.105303 0.00019 0.00011 AK052998 Macrod1 2.057018 2.32974 0.00006 0.00000 AK159139 Hspa1b 2.007872 9.390988 0.00089 0.00017 BC023391 D930015E06Rik 2.005007 2.193157 0.00099 0.00052

Supplementary Table S5. List of genes upregulated in common by Ad-Mt2, Ad-Mtf1, Ad- Zip8 infection in chondrocytes (fold change ≥ 2; P < 0.05)

P value Fold change Fold changeFold changeP value P value Gene accession Gene symbol (Ad- (Ad-Mt2) (Ad-Mtf1) (Ad-Zip8) (Ad-Mt2) (Ad-Mtf1) Zip8) BC034858 Saa2 96.60043 24.2647 55.08124 0.00012 0.00008 0.00006 AK144158 Cxcl3 87.32795 12.05285 56.65844 0.00000 0.00006 0.00000 AK148467 Mmp3 78.21605 20.27975 63.6611 0.00003 0.00003 0.00001 AK150430 Hp 68.07137 25.51925 42.06763 0.00000 0.00000 0.00000 BC052495 Saa1 53.60302 13.97818 26.95952 0.00000 0.00010 0.00003 BC019212 Saa4 35.64665 12.46459 12.76148 0.00000 0.00001 0.00000 AK003101 Ccl5 34.08537 8.899312 13.56748 0.00001 0.00015 0.00008 AK143562 Lbp 24.57527 12.28706 21.84639 0.00000 0.00001 0.00001 AK003905 Steap4 22.33843 12.52124 21.59643 0.00065 0.00072 0.00063 BC029976 C3 22.25393 12.40415 21.14663 0.00031 0.00029 0.00033 AK150728 Mmp13 22.18268 13.30671 21.26615 0.00051 0.00036 0.00052 AK010170 Slpi 20.56522 9.436339 15.56371 0.00000 0.00006 0.00000 AK014895 Slc39a8 20.4415 7.184332 30.42593 0.00000 0.00006 0.00000 BC055885 Saa3 19.62024 14.39575 19.19776 0.00098 0.00093 0.00099 AK132590 Ccl2 19.52451 4.855447 16.15223 0.00000 0.00028 0.00003 AK149531 Cfb 17.4254 6.563163 13.12618 0.00000 0.00001 0.00000 AJ132103 Vnn3 15.51554 4.869322 14.56365 0.00000 0.00004 0.00000 AK002773 Vnn1 15.45479 9.015094 14.63372 0.00051 0.00021 0.00060 AK140312 Cxcl1 13.94237 3.593797 14.04267 0.00001 0.00029 0.00001 ENSMUST0000017 Gm20419 13.66814 6.629757 8.713394 0.00002 0.00009 0.00002 4425 AK003143 Fabp4 12.95102 4.462663 6.870779 0.00104 0.00822 0.00360 BC010546 Ifi204 10.88619 3.72209 6.310496 0.00005 0.00034 0.00004 AK008517 Xdh 10.44445 4.756782 10.05781 0.00002 0.00001 0.00013 AK003407 Ifitm3 9.200959 3.954263 5.771282 0.00003 0.00035 0.00010 AK051475 Chi3l1 9.146325 5.927864 8.597485 0.00061 0.00064 0.00071 AK050355 Slfn9 8.905243 4.476949 3.389684 0.00000 0.00004 0.00013 AF065919 Nos2 8.52496 2.268262 6.776468 0.00069 0.00094 0.00072 AK078827 Ccl7 8.259001 2.758231 8.610187 0.00025 0.00423 0.00073 AK041059 Bst1 8.249263 2.303911 8.110897 0.00010 0.00250 0.00040 AF272948 Gpr84 8.140849 3.775507 9.013507 0.00001 0.00002 0.00001 AK080147 Il13ra1 7.473545 2.854076 5.319193 0.00000 0.00001 0.00010 AF140672 Ifi202b 7.043477 2.711639 4.652537 0.00011 0.00637 0.00039 AK140360 Ch25h 6.935945 2.228542 3.911698 0.00001 0.00231 0.00053 AK139833 A4galt 6.907491 2.297404 5.022715 0.00001 0.00153 0.00003 BC117110 Hist1h2ab 6.807732 4.058616 3.312814 0.00006 0.00023 0.00203 EF660528 AW112010 6.493383 3.058779 6.519243 0.00017 0.00261 0.00034 XR_105655 Gm3601 6.404353 3.72299 4.199841 0.00013 0.00014 0.00006 XR_105655 Gm3601 6.390864 3.774777 4.064342 0.00015 0.00011 0.00015 AK053190 Cebpd 6.235834 3.822443 5.932958 0.00006 0.00003 0.00002 AK087960 Srgn 6.177983 3.054898 5.226686 0.00020 0.00043 0.00012 BC065803 Hist1h2ag 6.00027 3.995715 3.037499 0.00004 0.00016 0.00162 AK009161 Angptl4 5.9353 2.345213 5.672122 0.00275 0.01985 0.00273 AK033934 Fgf7 5.748685 2.090315 5.521054 0.00002 0.00202 0.00002 BC013651 Serpina3n 5.672865 4.238974 4.712867 0.00001 0.00001 0.00001 AK075570 Sod3 5.670892 3.132767 5.376531 0.00014 0.00057 0.00009 AK151131 Ccl9 5.647404 4.182777 4.05048 0.00005 0.00002 0.00019 AK004651 Mmp9 5.61343 2.287531 4.604836 0.00017 0.00671 0.00070 BC033044 Nfkbiz 5.542515 2.906147 5.11664 0.00002 0.00010 0.00000 AK149774 Lcn2 5.427273 4.188388 5.166223 0.00187 0.00247 0.00187 AK018830 Fam111a 5.426406 3.307999 3.349733 0.00003 0.00014 0.00009 AK155016 Bcl3 5.316064 3.085611 4.728607 0.00000 0.00001 0.00000 AK088907 Rrm2 5.230384 3.17838 2.677843 0.00001 0.00007 0.00017 AK010768 Glipr1 5.099789 3.589245 3.606215 0.00012 0.00055 0.00051 AK077326 Tm4sf1 4.958723 2.152427 3.841042 0.00005 0.00263 0.00014 AK017282 Prr16 4.836482 3.009399 3.832481 0.00137 0.00593 0.00237 AK133465 Ampd3 4.716253 2.04609 4.337096 0.00001 0.00087 0.00003 AK019924 Gcnt2 4.499549 2.170602 3.182036 0.00003 0.00109 0.00012 AK085754 Trf 4.460362 2.618377 4.526262 0.00027 0.00103 0.00020 AK014783 Irak3 4.400925 2.162217 3.991471 0.00000 0.00032 0.00001 AK132597 Prg4 4.393782 2.528544 3.576796 0.00037 0.00349 0.00073 AK044924 Ccna2 4.356286 2.983986 2.566724 0.00005 0.00015 0.00012 AK137203 Avpr1a 4.338242 2.083971 2.591989 0.00114 0.00759 0.00268 AK011243 Chaf1b 4.236358 3.357223 2.54614 0.00000 0.00009 0.00005 AK010166 Car13 4.174618 2.304785 3.138488 0.00005 0.00090 0.00052 AK020117 Hist1h1b 4.15125 3.174702 2.965882 0.00009 0.00019 0.00005 AK041985 Tmem255a 4.138532 2.75318 3.666536 0.00050 0.00003 0.00043 AY462056 Kng2 4.128916 2.072079 3.196306 0.00005 0.00460 0.00042 AK013034 Cyp7b1 4.108213 2.264848 4.236047 0.00000 0.00003 0.00004 AK020144 Hmmr 4.099548 2.599013 2.299273 0.00000 0.00008 0.00006 BC062809 Figf 4.090241 2.202339 2.712381 0.00040 0.00796 0.00309 AK033321 Top2a 4.06593 2.946578 2.559974 0.00000 0.00000 0.00001 AK002590 Fas 3.936136 2.614915 3.500189 0.00001 0.00007 0.00002 AK034534 Ckap2 3.910782 2.71332 2.460517 0.00006 0.00034 0.00055 AK050778 Kif11 3.903225 2.983621 2.418473 0.00001 0.00001 0.00003 AK136911 Epas1 3.882268 2.533606 3.862787 0.00000 0.00001 0.00002 AK044764 Gpnmb 3.881549 2.833224 3.73639 0.00569 0.01892 0.00785 BC005501 Cd302 3.878927 2.13382 3.763387 0.00104 0.00448 0.00146 BC046640 Tmem173 3.86508 2.004502 3.524008 0.00016 0.00158 0.00008 BC053042 Fabp5 3.852989 2.421103 2.591685 0.00000 0.00002 0.00001 AK010121 Hist1h3f 3.848258 2.754168 2.474341 0.00223 0.00753 0.01874 AK028919 Ncaph 3.845509 2.839674 2.186252 0.00005 0.00009 0.00051 AK040174 Ect2 3.807371 2.55311 2.345197 0.00013 0.00018 0.00046 BC023918 Prr11 3.806128 2.243597 2.306216 0.00002 0.00033 0.00010 AK027921 Epb4.1l3 3.667832 3.134705 2.730453 0.00001 0.00001 0.00038 BC147143 Hist1h2bk 3.62179 2.868462 2.190252 0.00070 0.00082 0.00788 AK085188 Tk1 3.513158 2.398965 2.192858 0.00004 0.00008 0.00008 AK052772 Mki67 3.479479 3.036617 2.485127 0.00000 0.00000 0.00008 AK035110 Robo2 3.417219 2.040298 2.895939 0.00010 0.00011 0.00003 BC053042 Fabp5 3.378628 2.170133 2.432083 0.00382 0.01862 0.01370 AK021258 Bub1 3.365665 2.413469 2.342344 0.00005 0.00030 0.00044 AK134045 Plk1 3.347549 2.580292 2.048398 0.00000 0.00017 0.00002 AK083728 Esco2 3.321932 2.59992 2.333977 0.00115 0.00232 0.00373 AK014340 Moxd1 3.303451 2.457193 3.281951 0.00003 0.00005 0.00001 BC125011 Hist1h2af 3.27506 2.428823 2.122775 0.00004 0.00094 0.00113 AY455829 Ncapg2 3.273902 2.484263 2.049313 0.00001 0.00019 0.00006 AK137073 Diap3 3.269065 3.183627 2.170774 0.00060 0.00019 0.00251 AK050456 Depdc1a 3.247834 2.568832 2.096459 0.00002 0.00027 0.00037 BC023743 Olfml2b 3.199891 2.944911 2.557307 0.00079 0.00084 0.00130 AK002451 Cks2 3.197963 2.046897 2.214901 0.00000 0.00006 0.00031 AK003838 Pbk 3.196375 2.680772 2.431016 0.00014 0.00029 0.00119 XR_141327 Gm10309 3.187629 2.113415 3.02827 0.00056 0.00117 0.00473 AK005646 Asf1b 3.154585 2.131435 2.351757 0.00002 0.00009 0.00005 AK042697 Nusap1 3.133519 2.136376 2.16863 0.00244 0.00792 0.00616 BC116724 Serpina3c 3.131995 2.162867 2.023954 0.00049 0.00328 0.00470 BC028559 Kif23 3.100701 2.633978 2.350365 0.00005 0.00007 0.00015 AK132030 Clu 3.092802 3.025618 3.081445 0.00000 0.00000 0.00002 AK012040 Ckap2l 3.066297 2.316945 2.149343 0.00018 0.00006 0.00009 AK036316 Lrrn3 3.041489 2.006738 2.129354 0.00003 0.00084 0.00041 BC120800 Hist1h3c 3.019196 2.780675 2.592406 0.00012 0.00026 0.00118 AK019885 Tnfrsf9 2.941514 2.451291 2.715401 0.00008 0.00015 0.00007 AK033205 Brip1 2.931556 2.71277 2.089569 0.00402 0.00221 0.00730 AK049856 Slc11a2 2.917666 2.072238 2.743064 0.00000 0.00000 0.00001 AK144785 Fosl1 2.842498 2.526481 3.244133 0.00177 0.00144 0.00307 BC068159 Iqgap3 2.837056 2.074824 2.108396 0.00005 0.00151 0.00000 AK047165 Socs3 2.821183 2.10329 2.59929 0.00001 0.00081 0.00005 AK043023 Prnd 2.802805 2.341532 3.518696 0.00139 0.00167 0.00036 AK164473 Mis18bp1 2.646057 2.07785 2.024664 0.00041 0.00079 0.00097 AK020346 Scel 2.542338 6.514211 2.221579 0.00054 0.00000 0.00053 AK003466 Ier3 2.415157 2.117775 2.59353 0.00033 0.00093 0.00019

Supplementary Table S6. List of upregulated (A; fold change ≥ 1.5; P < 0.05) and downregulated (B; fold change ≤ 0.7; P < 0.05) genes in Mt1 and Mt2 double knock-out chondrocytes

A. Upregulated genes

Gene accession Gene symbol Fold change P value ENSMUST00000082757 Gm24149 1.654107846 0.03911 ENSMUST00000160674 Csprs 1.607281917 0.02799 ENSMUST00000158208 Gm23542 1.597215156 0.04046 ENSMUST00000157157 Gm23169 1.529631707 0.03051 ENSMUST00000083298 Gm22973 1.516099739 0.01847

B. Downregulated genes Gene accession Gene symbol Fold change P value

ENSMUST00000157410 Gm22885 0.699910489 0.01012 NM_001253736 Pdlim2 0.698135994 0.04401 NM_174877 Zar1 0.696245338 0.00974 NM_146230 Acaa1b 0.691004495 0.03671 NM_001104550 Vmn2r98 0.687512254 0.02345 NR_046286 H2-T10 0.686804787 0.03181 ENSMUST00000122525 Gm25783 0.686075062 0.02781 NM_001085544 Gm14744 0.685545662 0.00888 NM_016917 Slc40a1 0.684751772 0.03491 NM_001199556 AW551984 0.676299566 0.02028 NM_011709 Wap 0.674007554 0.04370 NM_207000 H2afy2 0.673774624 0.00812 NM_001172121 Rbms3 0.664566281 0.02823 ENSMUST00000103554 Tcrg-V4 0.658566942 0.00718 NM_001025570 Prrx1 0.657788185 0.04792 XM_006544510 Gm20830 0.657392848 0.02867 NM_007925 Eln 0.652755826 0.00199 NM_001085554 Gm10922 0.651269694 0.03356 XR_373437 2310016D23Rik 0.644116997 0.04849 NM_001024618 Xirp2 0.639790437 0.03373 ENSMUST00000175448 Gm25694 0.636132999 0.04232 ENSMUST00000175033 Gm26197 0.626347849 0.04257 NM_001289926 2010111I01Rik 0.623196628 0.03176 ENSMUST00000158411 Gm22158 0.619886075 0.02454 NM_053169 Trim16 0.61724679 0.02435 ENSMUST00000158789 Gm22010 0.615862134 0.04440 ENSMUST00000157395 Gm22762 0.60120198 0.02577 AK019583 4930425F17Rik 0.600917566 0.03995 NM_010644 Klk1b26 0.59125293 0.03932

Supplementary Table S7. List of upregulated (A; fold change ≥ 1.5; P < 0.05) and downregulated (B; fold change ≤ 0.7; P < 0.05) genes in chondrocytes infected with Ad-Mt1 (800 MOI)

A. Upregulated genes Gene accession Gene symbol Fold change P value ENSMUST00000157865 Gm24400 1.939973 0.007698 NR_039557 Mir5098 1.823237 0.033173 ENSMUST00000082398 mt-Ta 1.728461 0.026295 NM_001170705 Dthd1 1.667953 0.001416 ENSMUST00000082398 mt-Ta 1.605091 0.047754 NM_023294 Ndc80 1.566291 0.034971 ENSMUST00000158812 Gm23199 1.561098 0.049675 ENSMUST00000103290 Trbj1-7 1.555524 0.011044 NM_001198914 Myb 1.536439 0.042221 ENSMUST00000161449 Gm16092 1.536274 0.038035 ENSMUST00000178642 Gm25934 1.534987 0.027492 ENSMUST00000101958 Gm23940 1.532945 0.011842 ENSMUST00000099047 Gm10719 1.524715 0.041349 NM_028440 Abracl 1.524482 0.002723

B. Downregulated genes

Gene accession Gene symbol Fold change P value XM_006519849 LOC102636283 0.697216 0.030602 NM_001101613 Gm11567 0.694775 0.04915 ENSMUST00000158251 Gm25861 0.693696 0.01546 NM_146464 Olfr1196 0.692357 0.01081 NM_053214 Myo1f 0.690199 0.019734 ENSMUST00000138741 Gmfg 0.689187 0.029902 ENSMUST00000177795 Igkv4-62 0.68894 0.021154 ENSMUST00000158089 Gm22726 0.686763 0.020427 ENSMUST00000102076 Gm22750 0.684936 0.01145 ENSMUST00000083437 Gm22739 0.682818 0.013598 ENSMUST00000158774 Gm23312 0.68127 0.023607 NM_001142410 Gm14124 0.681019 0.03773 ENSMUST00000104459 Gm24981 0.679904 0.029791 NM_007979 F9 0.679041 0.037731 AK133679 Gm20255 0.676931 0.001541 NM_029863 9230110F15Rik 0.674097 0.038208 ENSMUST00000082521 n-R5s56 0.668768 0.041929 NM_001100465 Rhox2h 0.665263 0.028254 NR_045904 2310008N11Rik 0.664383 0.022328 ENSMUST00000157673 Gm26415 0.659645 0.015804 ENSMUST00000103654 Trav9-2 0.655054 0.027718 NM_001034103 Gm5941 0.651809 0.015303 ENSMUST00000175114 Gm25958 0.647249 0.016674 ENSMUST00000167409 Trav9d-4 0.636421 0.042627 NM_001110779 Gm7714 0.631719 0.032073 ENSMUST00000122529 Gm26425 0.629188 0.048335 ENSMUST00000122793 n-R5s19 0.626496 0.018942 ENSMUST00000175033 Gm26197 0.60512 0.000494 ENSMUST00000103667 Trav16 0.603729 0.004764 NM_001126491 Gm14459 0.59359 0.030392 ENSMUST00000174979 Gm25675 0.563703 0.006778 ENSMUST00000104171 Gm25308 0.553464 0.013316 NR_029572 Mir186 0.546289 0.047407 NM_010374 Gzmf 0.536169 0.005012

A D MT1 ZIP8 ZIP8 MT2 GAPDH HIF-2 NAMPT 0 5 10 25 50 MT1 MT1 TNF- (ng/ml, 24 h) MT2 MT2

MT1 GAPDH GAPDH MT2 25 50 25 50 100

GAPDH 100 Ad-C None None Ad-C None 0 10 20 50100 None Zip8- Zip8- C-siRNA IL-6 C-siRNA siRNA (nM) siRNA (nM) (ng/ml, 24 h) Ad-Epas1 (800 MOI) Ad-Nampt (800 MOI) B MT1 MT2 C IL-1(1 ng/ml, 24 h) 15 10 20 ** ** None Vehicle CaEDTA 8 * 15 MT1 MT2 10 6 10

4 DAPI 5 5 2 MT1 MT2 Relative mRNA levels Relative mRNA 0 0 0 25 50100 25 50100

Zip8- Mtf1- DAPI None None None TPEN Vehicle Vehicle siRNA siRNA Vehicle C-siRNA Zip8- Mtf1- C-siRNA C-siRNA (nM) (nM) CaEDTA siRNA siRNA IL-1(24 h, 1 ng/ml) IL-1 (24 h, 1 ng/ml) IL-1(24 h, 1 ng/ml) IL-1(1 ng/ml, 24 h)

Figure S1. Upregulation of MT1 and MT2 in primary cultured articular chondrocytes. (A) Representative RT-PCR gel images of MT1 and MT2 mRNA in chondrocytes treated with the indicated concentrations of TNF-α or IL-6 (n =5).(B) mRNA levels of the indicated genes in chondrocytes treated with IL-1β in the absence or presence of control (C) siRNA (100 nM) or the indicated concentrations of Zip8 siRNA or Mtf1 siRNA or treated with TPEN (1 μM) or CaEDTA (10 mM) (n ≥ 5). (C) Immunofluorescence microscopy of MT1/MT2 proteins and DAPI in chondrocytes treated with IL-1β in the absence or presence of CaEDTA or 100 nM C-siRNA, Zip8 siRNA, or Mtf1 siRNA (n = 5). (D) mRNA levels of the indicated genes in chondrocytes infected with control virus (Ad-C) or Ad-Epas1 in the absence or presence of C-siRNA (100 nM) or the indicated concentrations of Zip8 siRNA or Mtf1 siRNA (n = 4). Values are presented as means ± SEM (*P < 0.01, **P < 0.001). A B 100 150

80

100 WT → 60 Ad-Mt2 → KO → KO/TBHP Ad-Mt1 → ← WT/TBHP → Ad-C ← TBHP 40 → Cell count Cell count 50

20

0 0 102 103 104 102 103 104 DCF fluorescence DCF fluorescence

Figure S2. MTs modulate the intracellular oxidant level. (A) Representative intracellular ROS levels measured in chondrocytes infected with Ad-C, Ad-Mt1 or Ad-Mt2 at MOI of 800. Infected chondrocytes were loaded with 20 μM DCF-DA for 30 min and ROS levels examined via flow cytometry (n = 4). Tert-butyl hydroperoxide (TBHP, 50 μM) was used as a positive control. (B) Representative intracellular ROS levels in chondrocytes isolated from WT or Mt1/Mt2 double KO mice left untreated or treated with TBHP (50 μM) for 1 h (n =4). A Meniscus Ligament Synovium B Ad-C Ad-Mt2 3 C *

M

Ad-C 2

C 1

Mt2 M (arbitrary score, 0-3) score, (arbitrary Ad- C inflammation Synovial 0 MT1/MT2 Synovitis Ad-C Ad-Mt2 C D 6 3 NS NS * 4 * 2

2 NS 1 NS Synovitis (0-3) Synovitis OARSI grade (0-6) 0 0 Ad-C Ad-Mt1 Ad-Mt2 Ad-Mt1 Ad-C Ad-Mt1 Ad-Mt2 Ad-Mt1 Mt2 Mt1 Mt2 Mt1 Ad-Mt2 Ad-Mt2 Mt2 Mt2 Ad-C Ad-C Ad- Ad- Ad- Ad- ;Ad- ;Ad- Mt1 Mt1 Ad- Ad-

Figure S3. MT2 overexpression in knee joint tissues promotes OA pathogenesis in WT mice, irrespective of MT1 expression. (A) Representative image of MT1/MT2 immunostaining in meniscus, ligament, and synovium of WT mice 3 weeks after IA injection of Ad-C or Ad-Mt2 (n = 12). C, cartilage; M, meniscus. (B) Representative images of safranin-O/hematoxylin staining and scoring of synovial inflammation in WT mice 3 weeks after IA injection of Ad-C or Ad-Mt2 (n = 12). (C and D) WT mice were IA-injected with Ad-C (1 × 109 PFU), Ad-Mt1 (0.5 × 109 PFU) with Ad-C (0.5 × 109 PFU), Ad-Mt2 (0.5 × 109 PFU) with Ad-C (0.5 × 109 PFU), or Ad-Mt1 (0.5 × 109 PFU) with Ad-Mt2 (0.5 × 109 PFU) for 3 weeks. Representative safranin-O staining image of cartilage and OARSI grade (n = 14) are shown (C). Representative safranin-O/hematoxylin staining images of synovium and scoring of synovial inflammation (n = 14) are shown (D). Values are presented as means ± SEM (*P < 0.0001; ns, not significant). Scale bar: 50 μm. I8MF xs A dCo Ad- or Ad-C (A) axis. ZIP8-MTF1 S4 Figure B F D A OARSI grade KO WT KO WT Synovitis Cartilage 0 1 2 3 4 5 6 3 weeks WT KO WT dCAd- Ad-C

Ad-C Ad- Ad-C Ad-C NS T vrxrsini on ise rmtsO ahgnssi ieidpnetyo h zinc- the of independently mice in pathogenesis OA promotes tissues joint in overexpression MT2 . Ad-Zip8 NS

Ad-Mtf1 Zip8 8 weeks

Ad-C fl/fl NS Ad-Zip8 8 weeks NS

Ad-Mtf1 3 weeks Ad- 8 weeks Zip8 Synovial inflammation Zip8 0 1 2 3 Mt2 3 weeks NS Ad-Zip8 Zip8 (1 NS Mt2 CKO Ad- × Ad-Mtf1 10 Mtf1 Ad- 9 Osteophyte size (m2) F)wsI-netdit T cartilage-specific WT, into IA-injected was PFU) Mtf1 0.00 0.05 0.10 0.15 Mtf1 CKO 8 weeks Ad-C C KO WT SNS NS Ad-Zip8 E KO WT Ad-Mtf1 dCAd- Ad-C OARSI grade (0-6) 0 1 2 3 4 5 6 Osteophyte maturity Ad- Ad-C dCAd- Ad-C 0 1 2 3 F/F * F/F

8 weeks Ad-C NS i8Mtf1 Zip8 NS CKO Ad-Zip8 Mt2 3 weeks NS 8 weeks

Ad-Mtf1 Zip8 Zip8

Subchondral bone Synovial inflammation (arbitrary score, 0-3) thickness (m) 0 1 2 3 120 160 200 40 80 dCAd- Ad-C F/F Zip8 * Ad- F/F Ad-

8 weeks Ad-C Mtf1 K or cKO Mtf1 NS i8Mtf1 Zip8 SNS NS CKO Ad-Zip8 Mt2

Ad-Mtf1 Mtf1 cKO mice. Left: Representative images of safranin-O staining to detect cartilage destruction and safranin-O/hematoxylin staining to detect synovial inflammation. Right: Scoring of cartilage destruction and synovitis (n ≥ 6). (B and C) WT or Mt1/Mt2 double-KO mice were IA-injected with Ad-C, Ad-Zip8 or Ad-Mtf1, and sacrificed 3 weeks after the first injection of adenovirus. Representative images of safranin-O staining of cartilage tissue (B) and safranin-O/hematoxylin staining of synovial tissue (C) are shown (n ≥ 7). (D and E) WT and Mt1/Mt2 double KO mice were IA-injected with Ad-C, Ad-Zip8 or Ad-Mtf1, and sacrificed 8 weeks after the first injection of adenovirus. Representative images of safranin-O staining of joint tissue (D) and cartilage/subchondral bone tissues (E) are shown (n ≥ 6). (F) OA manifestations, including OARSI grade, synovial inflammation, osteophyte size and maturity, and subchondral bone sclerosis, were scored in WT and Mt1/Mt2 double KO mice IA-injected with Ad-C, Ad-Zip8,orAd-Mtf1 (n ≥ 6). Values are presented as means ± SEM (*P < 0.0001; NS, not significant). Scale bar: 50 μm A B MT1 MT1 MT2 MT2 MMP3 MMP3 MMP9 MMP13 MMP12 ADAMTS5 MMP13 SOX9 Type II collagen ADAMTS5 Aggrecan SOX9 GAPDH Type II collagen Mt1 Mt2 Aggrecan Mt2 Ad-C None Ad- Ad- GAPDH Ad- MOI Mt1; 400 400 800 800 200 1600 Ad-C None Ad-Mt1 Ad-Mt2 Ad- C D MT1 MT1 MT2 MT2 MMP3 MMP3 MMP13 MMP13 ADAMTS5 ADAMTS5 SOX9 SOX9 Type II collagen Type II collagen Aggrecan Aggrecan GAPDH GAPDH 25 25 25 50 50 50 50 25 25 50 25 50 None #1 #2 #1 #2 None -siRNA C-siRNA Mt1- Mt2- C-siRNA C-siRNA C-siRNA

siRNA siRNA -siRNA -siRNA (nM) (nM) Mt1 Mt1 Mt1/Mt2 Ad-C Ad-Mt2

Figure S5. Transcriptional regulation of matrix-degrading and cartilage matrix molecules by MT1 and MT2 in chondrocytes. (A) Relative mRNA levels of matrix- degrading enzymes and cartilage matrix molecules in chondrocytes infected with Ad-C (800 MOI) or the indicated MOI of Ad-Mt1 or Ad-Mt2 (n ≥ 5). (B) mRNA levels of the indicated genes in chondrocytes infected with Ad-C, Ad-Mt1 or Ad-Mt2, co-infected with Ad-Mt1 and Ad-Mt2 or left uninfected (n ≥ 5). (C) Relative mRNA levels of the indicated genes in chondrocytes transfected with control (C) siRNA, Mt1 siRNA, or Mt2 siRNA (n ≥ 5). (D) Chondrocytes pretreated with 50 nM control siRNA (C-siRNA) or the indicated concentrations of Mt2 siRNA were infected with Ad-C or Ad-Mt2 at an MOI of 800 for 2 h and incubated for 24 h. The indicated mRNAs were detected using RT-PCR (n ≥ 5). A B MT2 MT2 ZIP8 MTF1 MMP3 MMP3 MMP13 MMP13 SOX9 SOX9 Type II collagen Type II collagen Aggrecan Aggrecan GAPDH GAPDH Con Con Mtf1 Zip8 Ad-C Ad-C None None None None siRNA siRNA Ad-Mt2 Ad-Mt2 C D E 50 MT2 10 HIF-2 HIF-2 NAMPT MT2 MT2 40 8 MMP3 MMP3 MMP9 MMP9 30 6 MMP12 MMP12 MMP13 20 4 MMP13 SOX9 SOX9 10 2 Type II collagen Type II collagen Relative mRNA levels Relative mRNA Aggrecan Aggrecan 0 0 GAPDH GAPDH 400 800 400 800 200 200 Con Con Ad-C Ad-C Ad-C Ad-C None None None None None None Epas1 Ad-Mt2 Ad-Mt2 Nampt siRNA siRNA Ad-Mt2 Ad-Mt2

Figure S6. MT2-induced regulation of matrix-degrading enzymes and cartilage matrix molecules is independent of the zinc-ZIP8-MTF1 axis, HIF-2α,andNAMPT.(AandB) Primary culture articular chondrocytes pretreated with 100 nM control siRNA (C- siRNA) or siRNAs targeting Zip8 (A) or Mtf1 (B) were infected with Ad-C or Ad-Mt2 at an MOI of 800. The indicated mRNAs were detected via RT-PCR (n ≥ 5). (C) Chondrocytes were either infected with Ad-C or Ad-Mt2 or left uninfected. MT2 and HIF-2α mRNA levels were quantified via qRT-PCR (n ≥ 5). (D and E) Chondrocytes transfected with 50 nM control siRNA (C-siRNA) or siRNAs targeting HIF-2α (D) or NAMPT (E) were infected with Ad-C or Ad-Mt2 at an MOI of 800. The indicated mRNAs were detected using RT-PCR (n ≥ 5). A MT2 MT2 MT2 MT2 STAT1 ATF2 IRF1 MMP3 STAT2 MMP3 MMP3 MMP3 MMP9 MMP9 MMP9 MMP9 MMP12 MMP12 MMP12 MMP12 MMP13 MMP13 MMP13 MMP13 ADAMTS5 ADAMTS5 ADAMTS5 ADAMTS5 ACTIN ACTIN ACTIN ACTIN 25 50 25 50 10 50 25 50 100 100 100 100 Ad-C Ad-C Ad-C Ad-C None None None None Stat1&2-siRNA (nM) Atf2-siRNA (nM) Irf1-siRNA (nM) DAPT (nM) C-siRNA C-siRNA C-siRNA Ad-Mt2 (800 MOI) Ad-Mt2 (800 MOI) Ad-Mt2 (800 MOI) Ad-Mt2 (800 MOI) B 3 * * 2

1 (arbitrary score, 0-3) score, (arbitrary Synovial inflammation Synovial

0 Ad-C Ad-Mt2 Ad-Mt2;SC-514 Ad-Mt2;T-IIA --+ SC-514 - - + T-IIA Ad-Mt2

Figure S7.NF-κB and AP-1 mediate MT2-induced OA pathogenesis. (A) mRNA levels of the indicated genes were determined via RT-PCR in chondrocytes infected with Ad-C or Ad-Mt2 (800 MOI) in the absence or presence of control siRNA (C-siRNA; 100 nM) or the indicated concentrations of siRNAs specific for STAT1, STAT2, ATF2, or IRF1. Alternatively, chondrocytes were treated with DAPT to inhibit γ-secretase. (B) Safranin-O/hematoxylin staining to detect synovial inflammation and scoring of synovitis in mice IA-injected with Ad-C or Ad-Mt2 with or without SC-514 or T-IIA (n = 12). Values are presented as means ± SEM (*P < 0.01, **P < 0.001). Scale bar: 50 μm.