Journal: International Journal of Molecular Sciences

Article Title: MicroRNA-766-3p Contributes to Anti-Inflammatory Responses through the Indirect Inhibition of NF-κB Signaling

doi: 10.3390/ijms20040809

Figure Lengend Snippet: Generalizability of the suppression of inflammatory stimuli in miR-766-3p-transfected cells. ( A ) NHMCs were transfected with miRNA mimics (30 nM) and exposed to TNF-α or IL-1β for 24 h. The expression of the indicated genes was examined by a qPCR. ( B , C ) C28/I2 cells ( B ) and NHMCs ( C ) were co-transfected with pNF-κB-Luc along with miRNA mimics (C28/I2: 5 nM, NHMC: 30 nM). After incubation, cells were treated with TNF-α or IL-1β for 6 h and subjected to a luciferase assay to evaluate the activity of NF-κB. The luciferase activity was normalized by the number of viable cells and then normalized to the respective values in the vehicle samples (upper bar graphs). The lower scatterplots show the frequency of NF-κB inhibition by miR-766-3p. Assays were performed in quadruplicate. Data are expressed as the mean ± SEM. Asterisks indicate statistically significant differences ( p < 0.05).

Article Snippet: The human synovial fibroblast cell line MH7A was obtained from Riken Cell Bank (Ibaraki, Japan) [ ], the human chondrocyte cell line C28/I2 was obtained from Merck Millipore (Darmstadt, Germany), and normal human mesangial cells (NHMCs) were obtained from Lonza (Basel, Switzerland).

Techniques: Transfection, Expressing, Incubation, Luciferase, Activity Assay, Inhibition

Journal: Nature Communications

Article Title: Regulation of senescence-associated secretory phenotypes in osteoarthritis by cytosolic UDP-GlcNAc retention and O-GlcNAcylation

doi: 10.1038/s41467-024-55085-1

Figure Lengend Snippet: a – c Fold change (FC) heatmap of the expression levels of transporters for UDP-GlcNAc and UDP-GalNAc in human OA cartilage and OA-relevant conditions. Public transcriptome datasets analyzed were generated from ( a ) human OA cartilage (GSE16464, GSE43923, GSE64394, GSE113825, GSE117999, GSE178557, and GSE186220), ( b ) IL-1β-treated chondrocytes (GSE6119, GSE104793, and GSE163080), and ( c ) cartilage from OA models of mice (GSE26475, GSE53857, GSE101573, GSE110268, and GSE143447) and rats (GSE28958). d, e SA-β-Gal staining and quantification of SA-β-Gal positivity (left), representative images of immunofluorescence and quantification of BrdU incorporation (middle), and relative mRNA expression levels of CDK inhibitors or Lmnb1 (right) in ( d ) C28/I2 chondrocytes treated with 100 nM of doxorubicin ( n = 5) or in ( e ) mouse chondrocytes treated with 50 μg/mL of bleomycin ( n = 5). f, g Relative mRNA expression levels of transporters for UDP-GlcNAc and UDP-GalNAc in ( f ) C28/I2 chondrocytes treated with doxorubicin ( n = 5) or in ( g ) mouse chondrocytes treated with bleomycin ( n = 5). h Quantification of UDP-GlcNAc levels in the whole cell (left) and the ER, Golgi apparatus, and cytosol (right) of C28/I2 chondrocytes treated with vehicle, DON, or doxorubicin ( n = 5). i Quantification of UDP-GlcNAc levels in the whole cell (left) and cytosol (right) of mouse chondrocytes treated with vehicle, DON, or bleomycin ( n = 6). j Sulfated GAG (sGAG) release of C28/I2 chondrocytes treated with vehicle or doxorubicin measured using an sGAG assay ( n = 5). k sGAG release of mouse chondrocytes treated with bleomycin ( n = 5). Scale bars: d (left), e (left), 50 μm, d (middle), e (middle), 25 μm. d – k Data represent means ± s.e.m. P values are from two-tailed t test ( d – g, j, k ) or one-way ANOVA followed by Dunnett’s post-hoc test ( h, i ). Source data are provided as a Source Data file.

Article Snippet: Human chondrocyte cell line C28/I2 (Merck, SCC043) was maintained in DMEM/F-12 supplemented with 10% FBS, 15 mM HEPES, 100 U/mL penicillin, and 100 μg/mL streptomycin in a humidified 37 °C and 5% CO 2 atmosphere .

Techniques: Expressing, Generated, Staining, Immunofluorescence, BrdU Incorporation Assay, Two Tailed Test