Additional file 2 of Role of DNA dioxygenase Ten-Eleven translocation 3 (TET3) in rheumatoid arthritis progression

Additional file 2: Supplementary Table S1. TET3-mediated upregulated genes. Supplementary Table S2. TET3-mediated downregulated genes. Supplementary Table S3. Results of functional enrichment analysis. Supplementary Table S4. KEGG pathway analysis. Supplementary Table S5. Demographic, clinical, and biochemical features of RA and OA patients whose synovial tissues were used in the experiments

Additional file 1 of Role of DNA dioxygenase Ten-Eleven translocation 3 (TET3) in rheumatoid arthritis progression

Additional file 1: Supplementary Figure S1. Degeneration of TET3 mRNA induced by TNFα stimulation. Relative mRNA expression levels of TET3 in RA FLS (n = 3) treated with actinomycin D (Wako, 10 μg/mL), followed by stimulation with TNFα for 0, 0.5, 1, 2, and 6 hrs. Supplementary Figure S2. Relative mRNA expression levels of TET1/2/3 with or without TET3-knockdown. (A) RA FLS (n = 2) samples were used to study TET3 mRNA levels by qPCR. Data are mean ± SEM. (B) RA FLS (n = 3) samples were used to study TET3 protein levels by Western blotting. Supplementary Figure S3. Heat map of differentially expressed genes in all RA FLS with or without TNFα stimulation and TET3-knockdown. 2013 of all 21,448 genes were differentially expressed genes in 4 RA FLS groups (ANOVA F-test, P < 0.05) and analyzed. Red corresponds to gene upregulation and blue to gene downregulation. Supplementary Figure S4. Cell proliferation of FLS by TET3 expression. RA FLS (n=3) were transfected with control or TET3 siRNAs. Cell numbers of FLS were counted with Hemocytometer at day 1, 3, and 7. P value by the t-test

Additional file 3 of Role of DNA dioxygenase Ten-Eleven translocation 3 (TET3) in rheumatoid arthritis progression

Additional file 3

The hierarchy of gene expression in MSCs during differentiaion into osteoblasts, chondrocytes, and osteocytes.

<p>MSCs differentiate into osteoblasts or chondrocytes under the regulation of their master regulators, RUNX2 or SOX9, respectively. A part of chondrocytes also differentiate into osteoblasts.</p

Nano-fiber plugs induce the differentiation of MSCs derived from patients with RA or OA.

<p>MSCs derived from patients with RA (n = 3) or OA (n = 3) were cultured in MSCGM onto plastic plates (two-dimensional) or nano-fiber plugs (three-dimensional) for 7 (A) or 28 days (B). After RNA extraction, real-time PCR was performed to evaluate the gene expression level. Each line represents one patient. MSCs from healthy donors (n = 6), RA (n = 3) or OA (n = 3) patients were cultured in NFs for 7 days (C) or 28 days (D), and then analyzed as for their gene expression by real-time PCR. n.s.: not significant by ANOVA.</p

Nano-fiber plugs induce osteogenesis of human MSCs.

<p>Healthy donor-derived MSCs were seeded onto plastic plates (A-C) or injected into the nano-fiber plugs (D-I), and then cultured in MSCGM for 1 day (D), 7 days (E, G), 14 days (F, H) and 28 days (A-C, I). Then, the expression levels of RUNX2 (A, D, G), osteocalcin (B, E, H), and DMP-1 (C, F, I) were evaluated immunohistochemically. Representative results of three experiments are shown. (J) Healthy donor-derived MSCs were cultured in MSCGM onto plastic plates (two-dimensional) or nano-fiber plugs (three-dimensional) for 28 days. After RNA extraction, <i>MEPE</i> expression in the 4 groups of healthy donor-derived MSCswas evaluated by real-time PCR. *p<0.05 vs. two-dimensional culture by paired <i>t</i>-test. Scale bars, 50 μm.</p

Nano-fiber plugs induce morphological changes in MSCs.

<p>Healthy donor-derived MSCs were seeded onto nano-fiber plugs, and then cultured in MSCGM. Scanning electron micrographs taken on Days 7 (A) and 28 (B) are shown. All samples were tested three times, and representative results are shown. Scale bars, 50 μm.</p

MSCs cultured on nano-fiber plugs produce minerals and proteoglycan upon differentiation stimulation.

<p>Healthy MSCs were seeded onto plastic plates (A, D) or injected into the center of the nano-fiber plugs (B, C, E, F), and then cultured in OIM (A-C) or CM (D-F) for 28 days. The samples were then fixed with formalin, and then stained with von Kossa stain (A, B, E) or Safranin O (C, D, F). Representative results of three experiments are shown. Scale bars, 50 μm.</p

Characteristics of hMSCs derived from healthy donors or patients with RA/OA.

<p>Expanded bone marrow cells collected from healthy donors (n = 6) or patients with RA (n = 3), OA (n = 3) were analyzed as for their characteristics. Representative histograms of the cell surface markers on bone marrow cells derived from healthy donors (A), patients with RA (B), OA (C) were shown. Red lines show isotype controls. Gene expression of <i>RUNX2</i> (D) and <i>SOX9</i> (E) in hMSCs was analyzed by real-time PCR. n.s.: not significant by ANOVA.</p

Nano-fiber plugs induce MSCs to produce minerals and proteoglycan.

<p>Healthy donor-derived MSCs (A-F) or healthy donor-derived skin fibroblasts (G, H) were seeded onto plastic plates (A, B) or injected into the center of the nano-fiber plugs (C-H), and then cultured in mesenchymal stem cell growth medium for 14 days (C, E), 28 days (A, B, F-H), or 56 days (D). The samples were fixed with formalin, embedded in paraffin, and stained with von Kossa stain (A, C, D, G) or Safranin O (B, E, F, H). Representative results of three experiments are shown. Scale bars, 50 μm.</p