(A) Low expression levels of Mir204/Mir211 in FLS of CIA mice. (B) qRT-PCR analysis of the relative Mir204/Mir211 expression in CIA FLS 48 hr after transfection. Please see Figure 1—source data 1. …
Numerical data obtained during experiments represent in Figure 1.
(A) Transfection efficiency of Mir204/Mir211 in collagen-induced arthritis (CIA) fibroblast-like synoviocyte (FLS) at different time points. To determine transfection efficiency, Mir204/Mir211 …
(A) Effects of Mir204/Mir211 on apoptosis of CIA FLS. (B) Effects of Mir204/Mir211 on cell migration ability of CIA FLS. Representative photomicrographs show a wound scratch assay at specific time …
Numerical data obtained during experiments represent in Figure 2.
All data are displayed as a value relative to those in the NC group. Please see Figure 3—source data 1. Data are pooled from at least three independent experiments and are presented as mean ± SEM. …
Numerical data obtained during experiments represent in Figure 3.
(A) Western blot assays of the NF-κB pathway in transfected collagen-induced arthritis (CIA) fibroblast-like synoviocyte (FLS). (B) Immunofluorescence staining of p65 nuclear translocation in …
Numerical data obtained during experiments represent in Figure 4.
Original western blot files for Figure 4.
(A) CCK-8 analysis in four groups of transfected CIA FLS. (B) Mir204/Mir211 induce G0/G1 phase arrest of CIA FLS. (C) Expression levels of cell cycle regulatory molecule in transfected CIA FLS. (D) …
Numerical data obtained during experiments represent in Figure 5.
Original western blot files for Figure 5.
Primary FLSs are isolated from male WT and dKO C57 mice. FLSs are seeded in six-well plates at the density of 5×105, and β-galactosidase staining is performed.
(A) Diagrams describing the process of predicting target genes of Mir204/Mir211 (B) Diagram of putative Mir204/Mir211 binding sequence in Ssrp1 3’ untranslated region (UTR) from TargetScan. (C) High …
Numerical data obtained during experiments represent in Figure 6.
Original western blot files for Figure 6.
(A) Overexpression of Mir204/Mir211 decreases SSRP1 expression. (B) Mir204/Mir211 decrease Ki-67 levels by targeting Ssrp1. Please see Figure 7—source data 1. Data are pooled from four independent …
Numerical data obtained during experiments represent in Figure 7.
Original western blot files for Figure 7.
293T cells are divided into Ctrl group (untransfected), SSRP1 group (transfected with pcDNA3.1 - Ssrp1 plasmid), and negative control group (transfected with empty vector). 293T cells are …
(A) Severity of arthritis in CIA wild-type (WT) and CIA dKO mice. The arthritis scores, paw swelling, and body weight are documented by two independent blinded observers since the day of the second …
Numerical data obtained during experiments represent in Figure 8.
Representative photographs of mouse knee joints with TRAP staining and IHC staining for matrix metalloproteinase (MMP)-13 are shown.
(A) Ssrp1 knockdown ameliorates swelling degrees of hind paws in CIA Mir204/Mir211 dKO mice. Representative photomicrographs of knee joint sections in synovium and cartilage stained with hematoxylin …
Numerical data obtained during experiments represent in Figure 9.
Representative photographs of mouse knee joints with TRAP and IHC staining for matrix metalloproteinase (MMP)-13 are shown.
Primary fibroblast-like synoviocytes (FLSs) derived from collagen-induced arthritis (CIA) mice display decreased expression of Mir204/211. On the one hand, upon being challenged by IL-1β, FLS from …
Gene | Primer sequences |
---|---|
mmu-Mir204 forward primer | 5 '- GGGCTTCCCTTTGTCATCCTAT –3' |
mmu-Mir211 forward primer | 5 ' - GGGCTTCCCTTTGTCATCCTT –3' |
Universal U6 Forward primer | 5 ' - GCAAATTCGTGAAGCGTTCCATA –3' |
Universal PCR reverse primer | 5 ' - AACGAGACGACGACAGAC –3' |
Name | Gene | Forward primer sequence | Reverse primer sequence |
---|---|---|---|
β-Actin | Actb | 5'-GTGACGTTGACATCCGTAAAGA-3' | 5'-GCCGGACTCATCGTACTCC-3' |
IL-1β | Il1b | 5'-GAAATGCCACCTTTTGACAGTG-3' | 5'-TGGATGCTCTCATCAGGACAG-3' |
IL-4 | Il4 | 5'-GGTCTCAACCCCCAGCTAGT-3' | 5'-GCCGATGATCTCTCTCAAGTGAT-3' |
IL-6 | Il6 | 5'-CTGCAAGAGACTTCCATCCAG-3' | 5'-AGTGGTATAGACAGGTCTGTTGG-3' |
IL-8 | Il8 | 5'-TCGAGACCATTTACTGCAACAG-3' | 5'-CATTGCCGGTGGAAATTCCTT-3' |
IL-10 | Il10 | 5'-GCTGGACAACATACTGCTAACC-3' | 5'-ATTTCCGATAAGGCTTGGCAA-3' |
IL-23 | Il23 | 5'-CAGCAGCTCTCTCGGAATCTC-3' | 5'-TGGATACGGGGCACATTATTTTT-3' |
TGF-β1 | Tgfb1 | 5'-CCACCTGCAAGACCATCGAC-3' | 5'-CTGGCGAGCCTTAGTTTGGAC-3' |
Cyclin D1 | Ccnd1 | 5'-GCGTACCCTGACACCAATCTC-3' | 5'-ACTTGAAGTAAGATACGGAGGGC-3' |
p16 | Cdkn2a | 5'-CGCAGGTTCTTGGTCACTGT-3' | 5'-TGTTCACGAAAGCCAGAGCG-3' |
p21 | Cdkn1a | 5'-CCTGGTGATGTCCGACCTG-3' | 5'-CCATGAGCGCATCGCAATC-3' |
p57 | Cdkn1c | 5'-GCAGGACGAGAATCAAGAGCA-3' | 5'-GCTTGGCGAAGAAGTCGTT-3' |
SSRP1 | Ssrp1 | 5'-CAGAGACATTGGAGTTCAACGA-3' | 5'-GACGGCTCAATCGAAGCCTC-3' |
Gene | Protein | Company | Molecular weight (kDa) | Dilution ratio |
---|---|---|---|---|
Rela | NF-κB p65 | Cell Signaling Technology | 65 | 1:1000 |
Nfkbia | I-κBα | Cell Signaling Technology | 39 | 1:1000 |
Chuk | IKKα | Cell Signaling Technology | 85 | 1:1000 |
Ikbkb | IKKβ | Cell Signaling Technology | 87 | 1:1000 |
Pik3r1 | PI3K | Cell Signaling Technology | 85 | 1:1000 |
Akt1 | P-AKT | Cell Signaling Technology | 60 | 1:1000 |
Akt1 | AKT | Cell Signaling Technology | 60 | 1:1000 |
Trp53 | P53 | Cell Signaling Technology | 53 | 1:1000 |
Ssrp1 | SSRP1 | Cell Signaling Technology | 81 | 1:1000 |
Gapdh | GAPDH | Cell Signaling Technology | 37 | 1:1000 |