Bulk RNA sequencing to compare the transcriptome differences between young and old chondrocytes.

(A) Healthy chondrocytes were isolated from knee joint cartilage from young and old donors without osteoarthritis (assessed by experienced surgeons). P0 cells were used for RNA sequencing analysis. (B) Volcano plot demonstrating 303 upregulated and 163 downregulated genes in old chondrocytes when compared to young cells. (C) Top 50 genes that are significantly differently expressed in young and old chondrocytes. (D) Activation Z score of top 10 transcription regulators that are activated (positive) or inhibited (negative) in aged versus young chondrocytes. A comprehensive list of gene names can be found in Table S3. (E) Ingenuity Pathway Analysis (IPA) of young versus old cells. The gray bars indicate that no activity pattern is identified in IPA despite the highly significant association of the genes within the pathway. Orange, positive z-score; white, zero z-score. OBs=osteoblasts; OCs=osteoclasts. (F) GATA4 IHC of healthy human cartilage tissue from young and aged donors. Bar=50 µm. (G) Relative protein levels of GATA4 in P1 chondrocytes from individual human donors were analyzed by western blot.

Impact of GATA4 overexpression on in vitro cartilage formation of young chondrocytes.

(A) Timeline depicting the study. (B) IHC to assess GATA4 protein levels in young cells overexpressing GFP control or GATA4. Scale Bar=50 µm. (C) RT-qPCR analysis of GATA4 gene expression in two groups. (D) Western blot to measure GATA4 protein levels. (E) Safranin O staining and (F) Collagen type II (COLII) IHC to examine the production of cartilage matrix. Scale Bar=50 µm. (G) RT-qPCR analysis of gene expression of cartilage matrix proteins aggrecan (ACAN) and collagen type II-α1 (COL2A1) and hypertrophy markers collagen type X-α1 (COL10A1) and India hedgehog (IHH). (H) RT-qPCR analysis of gene expression of proinflammatory cytokines, including interleukin (IL)-6, IL-8, and tumor necrosis factor-α (TNF-α) (n=6). (I) Concentrations of IL-6, IL-8, and chemokine (C-C motif) ligand 2 (CCL2) in condition medium (n=3). (J) RT-qPCR analysis of relative gene expression of matrix-degrading enzymes, including matrix metalloproteinases (MMP)-1,2,3,12, and 13, and a disintegrin and metalloproteinase (ADAMTS) 4 and 5 (n=6). (K) MMP-1 concentration in condition medium (n=3). Student’s two-tailed t-test with Welch’s correction for standard deviation and a p-value of 0.05 was used for all statistical analysis.

Assessment of activation of SMADs following GATA4 overexpression.

(A) Schematic showing the experiment. (B, C) Western blot to assess protein levels of phosphorylated SMAD1/5 (pSMAD1/5), phosphorylated SMAD2/3 (pSMAD2/3), and GATA4 in pellets derived from young (B)individual(Y1-3) or (C) pooled chondrocytes, which were infected with lentiviral vectors carrying GATA4 or control genes and then stimulated with (+) or without (-) TGF-β3 for two hours. Relative protein levels of (D)GATA4, (E)pSMAD1/5, and (F)pSMAD2/3 were semi-quantified using ImageJ (n=3). (G) The ratio of pSMAD1/5 compared to pSMAD2/3 was also calculated. Statistics were conducted using one-way Analysis of Variance (ANOVA) with Dunnett’s post hoc analysis.

Influence of GATA4 knockdown on in vitro cartilage formation of old chondrocytes.

(A) Schematic showing the study. (B) Safranin O staining and (C) COLII IHC to examine the production of cartilage matrix in the Scrambled Control or GATA4 siRNA group. Scale Bar=50 µm. (D) RT-qPCR analysis of relative gene expression of cartilage matrix proteins ACAN and COL2A1 and hypertrophy marker COL10A1(n=6). (E) RT-qPCR analysis of relative gene expression of proinflammatory cytokines IL-6 and IL-8 (n=6). (F) Concentrations of IL-6, IL-8, and chemokine (CCL2) in condition medium (n=3). (G) The relative protein levels of pSMAD1/5, pSMAD2/3, and phosphorylated p65 (p-P65) in two groups. (H) RT-qPCR analysis of relative gene expression of matrix-degrading enzymes, including MMP-1,2,3,12, and 13, and ADAMTS 4 and 5 (n=6). (I) MMP-1,2 and 13 concentrations in condition medium (n=3). Student’s two-tailed t-test with Welch’s correction for standard deviation and a p-value of 0.05 was used for all statistical analysis.

Assessment of the physiological roles of Gata4 through intraarticular overexpression.

(A) schematic of the study. Mice received one intraarticular injection of lentiviral vectors that carried mCherry or Gata4 gene one week before DMM surgery was performed. Knee joints were harvested 6 weeks post-surgery. Levels of Gata4 (B, C) and p-P65 (D, E) were assessed with IHC(B&D), and the staining was semi-quantitated with Image J (C&E). Cartilage degradation was assessed with (F) Safranin O/ fast green (FG) staining, and (G)OARSI score was calculated. (H)Knee hyperalgesia 6 weeks post-surgery. 507 g was the threshold baseline for non-surgery mice (dashed line). Student’s two-tailed t-test with Welch’s correction for standard deviation and a p-value of 0.05 was used for all statistical analysis.

GATA4 IHC of healthy human cartilage tissue from 3 young (Y1,Y2) and aged (O1,O2) donors.

Scale Bar=50 µm.

GATA4 IHC of healthy mouse cartilage tissue from young (left) and old (right) mice.

Scale Bar=50 µm.

(A) GATA4 overexpression of young, pooled chondrocytes in monolayer culture 48 hours after infection. The lentiviral control contained the EF1A promoter-driven expression of Green Fluorescent Protein (GFP), and the GATA4 lentivirus contained the EF1A promoter-driven overexpression of GATA4 with dTomato fluorescent protein. Scale Bar=100 µm. (B) Western blot to measure GATA4 protein levels. (C) RT-qPCR analysis of relative gene expression of cartilage matrix proteins aggrecan (ACAN) and collagen type II-α1 (COL2A1), and hypertrophy markers collagen type X- α1 (COL10A1) and India hedgehog (IHH).

Safranin-O staining for pellets derived from young, individual chondrocyte (Y1-3) transduced with GFP control lentivirus or GATA4 lentivirus.

Pellets were cultured in chondrogenic medium for 7 days. Scale Bar=50 μm.

Western blot to examine MMP-13 protein levels in pellets derived from two young chondrocyte lines (Y1 and Y2) overexpressing GFP control (GFP C) or GATA4.

Assessment of GATA4 siRNAs in monolayer.

(A) Schematic of the four different GATA4 siRNAs with corresponding target sequences assessed for GATA4 knockdown in monolayer culture of old pooled chondrocytes. (B) RT-qPCR assessing GATA4 levels after siRNA treatment (n=3).

Assessment of GATA4 small molecule inhibitor, NSC140905.

Pooled old human chondrocytes were pelleted and treated with the chondrogenic medium with or without supplanting NSC140905 for 14 days. (A) RT-qPCR analysis of relative gene expression of matrix-degrading enzymes, including MMP-1,13 and ADAMTS 4 and 5 (n=3). Student’s two-tailed t-test with Welch’s correction for standard deviation and a p-value of 0.05 was used for all statistical analysis. (B) Safranin-O/Fast green staining. Bar=50 μM.

(A) H&E staining and (B) p-P65 IHC to assess synovial inflammation in mice treated with lentiviral vectors carrying mCherry Control or Gata4. C. p-P65 IHC staining was also semi-quantitated.

Western blot to examine protein levels in chondrocytes treated with doxorubicin (100nM) or vehicle control for 3 days.

Information of chondrocyte donors.

Antibodies used for immunofluorescence (IF), Immunohistochemistry (IHC), or Western blot (WB).

Full names of genes shown in Figure 1.

Primers for qRT-PCR.

Information of Luminex assay kit.