Mice
The Nsd1fl/fl mice [18], Prrx1-Cre mice [34] and Setd2fl/fl mice [34] were mentioned before. K36M/+ mice were provided by Dr. Kai Ge (National Institutes of Health). Nsd2fl/fl mice were provided by Dr. Jun Qin (Shanghai Institute of Nutrition and Health, CAS). All mice analyzed were maintained on the C57BL/6 background. All mice were monitored in a specific pathogen–free environment and treated in strict accordance with protocols approved by the Shanghai Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.
Cell culture
Primary chondroprogenitor cells were obtained from the femoral condyles and tibial plateau of newborn mice. Cartilages were digested with 1mg/ml collagenaseⅡ (Sigma, C6885) for two hours at 37℃ and the digestions were discarded. The left tissues were digested with half concentration of collagenaseⅡ overnight at 37℃ and the digestions were filtered with 70µm cell strainer (Falcon, 352350) the next day. The cells were plated into α-MEM medium (Corning, 10-022-CVR) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin.
Micromass culture
Micromass culture was performed when primary chondrocytes reached 80%-90%. Digested and suspended chondrocytes to 1×107cells/ml, plated a droplet of 12.5µl cell suspension to the central of 12-well-plate, let the plate stand at 37℃ for two hours and then gently added the chondrogenic differentiation medium, which contains DMEM, 10ng/ml TGFβ3 (Peprotech, 100-36E), 100nM dexamethasone (Sigma, D1756), 50µg/ml L-ascorbic acid 2-phosphate (Sigma, A8960), 1mM sodium pyruvate (Sigma, 25-000-CIR), 40µg/ml proline (Sigma, P5607) and 1% ITS (Cyagen, ITSS-10201-10). Micromass at different time was acidified with 0.1N HCl and then stained with 1% alcian blue (Sigma, A5268).
Human articular cartilage samples
Human cartilage samples were obtained from individuals undergoing total knee arthroplasty, and were conducted in accordance with the informed consent of the patients and approval of ethics committee of Shanghai Sixth People's hospital and Zhejiang Provincial People’s Hospital.
µCT analysis
Mouse knee joints were collected, removed the soft tissues and fixed in 70% ethanol. Samples were scanned with instrument Scanco µCT80 (SCANCO Medical, Swiss) or Skyscan 1272 scanner (Bruker, Germany) at a resolution of 10µm.
Histology and immunohistochemistry
Knee joints from mice were fixed in 4% paraformaldehyde for 48 hours, decalcified in 10% EDTA and embedded in paraffin and cut into 8µm thick sections. Immunohistochemistry was performed using Vector Rabbit kit according to the manufacturer’s instructions. Images were captured using a microscope (Olympus BX51, Tokyo, Japan).
Immunofluorescence
Sections were blocked in PBS with 10% horse serum for 1 hour and then stained overnight with specific antibody. Secondary antibodies were used according to the species of primary antibody. DAPI (sigma, D8417) was used for counterstaining. Slides were mounted with anti-fluorescence mounting medium (Dako, S3023) and images were acquired with Olympus BX51 microscope or Leica SP8 confocal microscope.
Senescent chondrocyte model
Chondrogenic cell line (ATDC5) was treated with the DNA-damaging agent etoposide (“DNA damage-induced senescence”, DIS). We confirmed the induction of the senescence phenotype in these cells by evaluation senescence-associated β-galactosidase (SA-β-Gal) staining.
Antibodies
H3K36M (Arigo, ARG57228), NSD1 (Bioss, bs-8170R), COL2 (Abcam, ab34712), H3K36me1 (Abcam, ab9048), H3K36me2 (Abcam, ab9049), H3K36me3 (Abcam, ab9050), SOX9 (Millipore, AB5535), OSR2 (sc-393516), MMP3 (Abcam, ab52915), MMP13 (Abcam, ab39012), ADAMTS5 (Thermo Fisher, PA5-27165), H3K4me1 (Abcam, ab176877), H3K4me2 (Abcam, ab7766), H3K4me3 (Abcam, ab213224), H3K9me1 (Abcam, ab176880), H3K9me2 (Abcam, ab176882), H3K9me3 (Active Motif, 61013), H3K27me1 (Abcam, ab194688), H3K27me2 (Abcam, ab24684), H3K27me3 (Abcam, ab6002), H3K79me1 (Abcam, ab2886), H3K79me2 (Abcam, ab3594).
Western blot
Tissues or cells were harvested and lysed with EBC buffer (1% NP-40, 10% glycerol, 135 nM NaCl, 20mM Tris pH8.0) containing protease inhibitors. Then lysates were separated through running SDS-PAGE gel and blotting on PVDF membrane (Bio-Rad). After incubation with specific antibodies, we used enhanced chemiluminescence kit (Millipore) to detect the protein signals.
Real-time PCR analysis
Total RNA was isolated from different tissues and cells with TRIzol Reagent (Sigma, T9424) and reverse-transcribed with the PrimeScript RT Reagent Kit (Takara, RR037A). The real-time reverse transcriptase (RT)-PCR reaction was performed with the Bio-Rad CFX Connect Real-Time System. The primer sets used were mouse Gstk1: sense 5’-GGTCCTATGCAGATACCAACAC-3’ and anti-sense 5’-GTACTGGCCTTTTCGGGGAA-3’; mouse Gstm7: sense 5’-ATGAT GCGGCTTTACTCCGAG-3’ and anti-sense 5’-GCCCCAAATAGCCATCTTTGTG- 3’; mouse Gstt1: sense 5’-CCGTCGCGCCATTTATATCTT-3’ and anti-sense 5’-CCCTCTTCATGGGGTTCACC-3’; mouse Cth: sense 5’-TTCCTGCCTAGTT TCCAGCAT-3’ and anti-sense 5’-GGAAGTCCTGCTTAAATGTGGTG-3’; mouse Mgst1: sense 5’-CTCAGGCAGCTCATGGACAAT-3’ and anti-sense 5’-GTTAT CCTCTGGAATGCGGTC-3’; mouse Galnt12: sense 5’-TCAACATCTATCTGAGC GACCG-3’ and anti-sense 5’-CTTGGGCAGGTTATCATAATCGT-3’; mouse Galnt15: sense 5’-GCTCCACAACACTGGATTTGG-3’ and anti-sense 5’-GTGTG CTCCAGGTTCTGTTG-3’; mouse Extl1: sense 5’-TTCTGGCTGGCGTTGTCAG − 3’ and anti-sense 5’-GGGTTCGTCTCAGACTGGGA-3’; mouse Gcnt1: sense 5’-ACTTGTTTCGGAGGAGACTTTTC-3’ and anti-sense 5’-GGGTCACCCTGTAA AATCTTGGT-3’; mouse Aldh3a1: sense 5’-AATATCAGTAGCATCGTGAACCG-3’ and anti-sense 5’-GGAGAGCCCCTTAATCGTGAAA-3’; mouse Acot2: sense 5’-GTTGTGCCAACAGGATTGGAA-3’ and anti-sense 5’-GCTCAGCGTCGCATTT GTC-3’; mouse Car9: sense 5’-TGCTCCAAGTGTCTGCTCAG-3’ and anti-sense 5’-CAGGTGCATCCTCTTCACTGG-3’; mouse Nos2: sense 5’-GTTCTCAGCCC AACAATACAAGA-3’ and anti-sense 5’-GTGGACGGGTCGATGTCAC-3’; mouse Acss2: sense 5’-AAACACGCTCAGGGAAAATCA-3’ and anti-sense 5’-ACCGTA GATGTATCCCCCAGG-3’; mouse Hsd11b1: sense 5’-CAGAAATGCTCCAG GGAAAGAA-3’ and anti-sense 5’-GCAGTCAATACCACATGGGC-3’; mouse Lipg: sense 5’-ATGCGAAACACGGTTTTCCTG-3’ and anti-sense 5’-GTAGCTGGTAC TCCAGTGGG-3’; mouse Scd1: sense 5’-TTCTTGCGATACACTCTGGTGC-3’ and anti-sense 5’-CGGGATTGAATGTTCTTGTCGT-3’; mouse Scd2: sense 5’-GCATTTGGGAGCCTTGTACG-3’ and anti-sense 5’-AGCCGTGCCTTGTATG TTCTG-3’; mouse Scd3: sense 5’-GTTGCCACTTTACTGAGATACGC-3’ and anti-sense 5’-GAAGCCCTCGCCCATACTT-3’; mouse Enpp1: sense 5’-CTGGT TTTGTCAGTATGTGTGCT-3’ and anti-sense 5’-CTCACCGCACCTGAATTTGTT − 3’; mouse Aldoc: sense 5’-AGAAGGAGTTGTCGGATATTGCT-3’ and anti-sense 5’-TTCTCCACCCCAATTTGGCTC-3’; mouse Col2: sense 5’-CGGTCCTACGG TGTCAGG-3’ and anti-sense 5’-GCAGAGGA CATTCC CAGTGT-3’; mouse Sox9: sense 5’-TTCCTCCTCCCGGCATGAGTG-3’ and anti-sense 5’-CAACTTTGCC AGCTTGCACG-3’; mouse Acan: sense 5’-AATCCCCAAATCCCTCATAC-3’ and anti-sense 5’-CTTAGTCCA CCCCTCCTCAC-3’; mouse Osr2: sense 5’-CTCAC CAATTACTCCTTCCTGC-3’ and anti-sense 5’-GCACCGCACTGAGACCATAG-3’; mouse Mmp3: sense 5’-ACATGGAGACTTTGTCCCTTTTG-3’ and anti-sense 5’-TTGGCTGAGTGGTAGAGTCCC-3’; mouse Mmp13: sense 5’-CTTCTTCTTG TTGAGCTGGACTC-3’ and anti-sense 5’-CTGTGGAGGTCACTGTAGACT-3’; mouse Adamts5: sense 5’-GGAGCGA GGCCATTTACAAC-3’ and anti-sense 5’-CGTAGACAAGGTAGCC CACTTT-3’; human NSD1: sense 5’-AGCAAAGAAA TGAAGTGGACGG-3’ and anti-sense 5’-TGAATGTGTGTTAATCAACGGA-3’, human OSR2: sense 5’-TCCGCCTAAGATGGGAGACC-3’ and anti-sense 5’-GGTAAAGTGTCTGCC GCAAAA-3’.
ChIP-Seq and ChIP-PCR
Nsd1 fl/fl primary chondrocytes were infected with lentivirus expressing Egfp and Cre respectively. After puromycin treatment, Egfp and Cre expressing primary chondrocytes were fixed by 1% formaldehyde for 10 min and terminated by glycine for 5 min (final concentration = 0.125 M). After twice washing by pre-cooling PBS (protease inhibitor containing), cells were scraped and resuspended in SDS-lysis buffer respectively (50 mM Tris-HCl Ph 7.5, 10 mM EDTA, 1% SDS and protease inhibitor), and sonicated. Cells were centrifuged to obtain cell extracts, which then were added to pre-cleaning protein G agarose and rotated for 1 hour at 4℃. Extracts were centrifuged and supernatants were harvested to new tube. ChIP assay was performed using H3K36me1/2 antibodies. Normal IgG was used as negative control. ChIP-PCR was used to amplify various regions of the target gene genome, and primers used were Osr2 P1: sense 5’-AGTCCCGGGCCTCGTGTTCT-3’ and anti-sense 5’-TCTTGCCGAATGCGTA AATCT-3’. Osr2 P2: sense 5’-TGAAATGGAGAGGGAGGGAGCGGA-3’ and anti-sense 5’-ACGA AGTTTCTGCCCCTTCCCCGT-3’.
Lentiviruses and Infection
Lentiviruses expressing Egfp and mouse Osr2 were constructed by inserting the gene’s CDS into Plenti-vector. Virus package was according to the VSVG - delta 8.9 system. Mouse primary chondrocytes were cultured for two days, infected with lentivirus for forty-eight hours, digested and went on micromass culture.
Adenoviruses and Intra-articular injection
The CDS of Egfp and mouse Osr2 were cloned into pYr-adshutter-4. Then the LR recombination reactions were performed between the plasmid (pYr-sdshuttle-4-Egfp/Osr2) and the destination vector (pAd/BL-DEST) with LR Clonase II (Invitrogen, 12538120). Finally, HEK293A cells were transfected with PacI linearized adenovirus vectors (pAd-Egfp/Osr2) to form recombinant adenoviruses Ad-Egfp/Osr2. Cerium chloride gradient centrifugation was used to harvest purified and concentrated adenoviruses. Intra-articular (IA) injection of Ad-Osr2 (2×108 plaque-forming units in a total volume of 5 µl) was performed once weekly for 6 weeks into 4-week-old male mice; IA injection of Ad-Egfp was used as a control. Mice were sacrificed 4 days after the last IA injection for histological analyses.
Statistical analysis
All quantitative data were presented as the mean ± SD and analyzed using GraphPad Prism 6 software by unpaired t test (two-sided) or one-way ANOVA with Bonferroni or Sidak post hoc test. P < 0.05 was considered as statistically significant. Correlation assay was conducted with GraphPad.