Journal: Scientific Reports

Article Title: Hypoxia-inducible factor-1 alpha maintains mouse articular cartilage through suppression of NF-κB signaling

doi: 10.1038/s41598-020-62463-4

Figure Lengend Snippet: Regulation of catabolic genes by HIF-1α. ( A ) Protein levels of Hif1a, and mRNA levels of Mmp13 , and Hif2a in WT mouse primary chondrocytes transduced with GFP or HIF1A adenoviral vectors under the hypoxic condition (3% O 2 ). GFP or HIF1A was transduced at a multiplicity of infection (MOI) of 100. The cells were treated with or without 10 ng/mL IL-1β for 2 days. Bars show the mean ± SD of three samples per group. * P < 0.05. ( B ) Protein levels of Hif1a, and mRNA levels of Mmp13 , and Hif2a in WT mouse primary chondrocytes transfected with siRNA against GFP or Hif1a under the hypoxic condition. The cells were treated with or without 10 ng/mL IL-1β for 2 days. Bars show the mean ± SD of three samples per group. * P < 0.05. ( C ) Protein levels of Hif1a, and mRNA levels of Mmp13 , and Hif2a in Hif1a fl/fl primary chondrocytes transduced with GFP or Cre adenoviral vectors under the hypoxic condition. GFP or Cre was transduced at a MOI of 100. The cells were treated with or without 10 ng/mL IL-1β for 2 days. Bars show the mean ± SD of three samples per group. * P < 0.05.

Article Snippet: For immunohistochemistry, we incubated the sections with antibodies to HIF1A (1:100; NB100–105, Novus Biologicals), MMP13 (1:100; MAB8887, Merck Millipore), HIF2A (1:100; sc-28706, Santa Cruz Biotechnology)), IκBα (1:100; sc-371, Santa Cruz Biotechnology), p-IκBα S32/36 (1:100; sc-101713, Santa Cruz Biotechnology), Rela (1:100; #8242, Cell Signalling Technology), C1qtnf3 (1:100; ab36870, Abcam).

Techniques: Transduction, Infection, Transfection

Journal: Scientific Reports

Article Title: Hypoxia-inducible factor-1 alpha maintains mouse articular cartilage through suppression of NF-κB signaling

doi: 10.1038/s41598-020-62463-4

Figure Lengend Snippet: Regulation of cartilage degradation by HIF-1α. ( A ) Amount of glycosaminoglycans released into the medium determined by the dimethylmethylene blue assay during 3 days culture of Hif1a fl/fl (Cntl) and Col2a1-Cre ERT2 ;Hif1a fl/fl (KO) femoral heads treated with or without 10 ng/mL IL-1β under the hypoxic condition (3% O 2 ). Tamoxifen induction was performed three days before sacrifice. Bars show the mean ± SD of three samples per group. * P < 0.05. ( B ) Immunofluorescence of Mmp13, Hif2a, and Hif1a in the Cntl and KO femoral head cartilage after 3-day culture with or without 10 ng/mL IL-1β. Scale bars, 50 µm.

Article Snippet: For immunohistochemistry, we incubated the sections with antibodies to HIF1A (1:100; NB100–105, Novus Biologicals), MMP13 (1:100; MAB8887, Merck Millipore), HIF2A (1:100; sc-28706, Santa Cruz Biotechnology)), IκBα (1:100; sc-371, Santa Cruz Biotechnology), p-IκBα S32/36 (1:100; sc-101713, Santa Cruz Biotechnology), Rela (1:100; #8242, Cell Signalling Technology), C1qtnf3 (1:100; ab36870, Abcam).

Techniques: Dimethylmethylene Blue Assay, Immunofluorescence

Journal: Scientific Reports

Article Title: Hypoxia-inducible factor-1 alpha maintains mouse articular cartilage through suppression of NF-κB signaling

doi: 10.1038/s41598-020-62463-4

Figure Lengend Snippet: Regulation of OA development by HIF-1α. Plain radiographs of the entire bodies at 8 weeks of age ( A ), and knee joints 8 weeks after OA surgery ( B ) of Cntl and cKO littermates. Scale bars, 10 mm and 1 mm, respectively. ( C ) Safranin O and TUNEL staining’s of Cntl and KO knee joints at 8 weeks after OA surgery (n = 9 and 11, respectively). Tamoxifen induction was performed at 7 weeks. Inset boxes in top images indicate the regions of high magnification Safranin O and TUNEL images. Inset boxes in middle images indicate the regions of immunofluorescence in ( G ). White dotted lines in TUNEL images indicate the cartilage area. Scale bars, 400 µm and 100 µm, respectively. ( D ) OARSI (Osteoarthritis Research Society International) score of OA development. Lines show the mean ± SD. * P < 0.05. ( E ) Rate of TUNEL-positive cells in the cartilage area. Bars show the mean ± SD. * P < 0.05. ( F ) mRNA levels of Hif1a in articular cartilage tissues of Cntl and KO. Bars show the mean ± SD. * P < 0.05. ( G ) Immunofluorescence of Hif1a, Mmp13, Hif2a, IκBα, Ser 32 and 36 dual phosphorylated IκBα, Rela, and C1qtnf3. Scale bars, 20 µm. The percentage of positive cells in the immunofluorescence is shown below. * P < 0.05.

Article Snippet: For immunohistochemistry, we incubated the sections with antibodies to HIF1A (1:100; NB100–105, Novus Biologicals), MMP13 (1:100; MAB8887, Merck Millipore), HIF2A (1:100; sc-28706, Santa Cruz Biotechnology)), IκBα (1:100; sc-371, Santa Cruz Biotechnology), p-IκBα S32/36 (1:100; sc-101713, Santa Cruz Biotechnology), Rela (1:100; #8242, Cell Signalling Technology), C1qtnf3 (1:100; ab36870, Abcam).

Techniques: TUNEL Assay, Immunofluorescence

Journal: Scientific Reports

Article Title: Hypoxia-inducible factor-1 alpha maintains mouse articular cartilage through suppression of NF-κB signaling

doi: 10.1038/s41598-020-62463-4

Figure Lengend Snippet: Suppression of catabolic gene expression by C1qtnf3. ( A ) mRNA levels of C1qtnf3 in WT mouse primary chondrocytes transduced with GFP or HIF1A adenoviral vectors under the hypoxic condition (3% O 2 ). GFP or HIF1A was transduced at a multiplicity of infection (MOI) of 100. ( B ) mRNA levels of C1qtnf3 in Hif1a fl/fl primary chondrocytes transduced with GFP or Cre adenoviral vectors under the hypoxic condition. GFP or Cre was transduced at a MOI of 100. The cells were treated with or without 10 ng/mL IL-1β for 2 days. (C) mRNA levels of C1qtnf3 , Mmp13 , and Hif2a in ATDC5 cells transfected via lentivirus with GFP or C1qtnf3. The cells were treated with or without 10 ng/mL IL-1β for 2 days. ( D ) mRNA levels of C1qtnf3 , Mmp13 , and Hif2a in WT mouse primary chondrocytes transduced with GFP or C1qtnf3 adenoviral vectors at a MOI of 10. The cells were treated with or without 10 ng/mL IL-1β for 2 days. ( E ) mRNA levels of C1qtnf3 , Mmp13 , and Hif2a in ATDC5 cells transfected via retrovirus with siRNA against GFP or C1qtnf3. The cells were treated with or without 10 ng/mL IL-1β for 2 days. ( F ) Protein levels of IκBα, and Ser 32 and 36 dual phosphorylated IκBα in ATDC5 cells transfected via lentivirus with GFP or C1qtnf3. The cells were treated with or without 10 ng/mL IL-1β for 24 hours. In all panels, bars show the mean ± SD of three samples per group. * P < 0.05.

Article Snippet: For immunohistochemistry, we incubated the sections with antibodies to HIF1A (1:100; NB100–105, Novus Biologicals), MMP13 (1:100; MAB8887, Merck Millipore), HIF2A (1:100; sc-28706, Santa Cruz Biotechnology)), IκBα (1:100; sc-371, Santa Cruz Biotechnology), p-IκBα S32/36 (1:100; sc-101713, Santa Cruz Biotechnology), Rela (1:100; #8242, Cell Signalling Technology), C1qtnf3 (1:100; ab36870, Abcam).

Techniques: Gene Expression, Transduction, Infection, Transfection