human chondrocyte growth medium  (Cell Applications Inc)

Journal: JCI Insight

Article Title: Activation of SLIT2/ROBO1/LRP6 axis aggravates cartilage degradation via β -catenin signaling in TMJOA

doi: 10.1172/jci.insight.193632

Figure Lengend Snippet: ( A ) Western blot detection of the effects of SLIT2 overexpression in primary chondrocytes from 3-week-old C57 and SLIT2-Tg mice. Primary chondrocytes for each independent isolation batch were pooled from 3 mice, and the process was repeated 3 times. ( B ) Relative quantification of COL2A1, MMP13, and IL-1β proteins. ( C ) qRT-PCR analysis of the effect of SLIT2 overexpression in primary chondrocytes from 3-week-old C57 or SLIT2-Tg mice. ( D ) Western blot detection of COL2A1, MMP13, and MMP3 after 48 hours of treatment with rhSLIT2 at 0, 10, 20, 50, and 100 ng/mL in SW1353 cells. ( E ) Relative quantification of COL2A1, MMP3, MMP13 and SOX9 proteins. # P < 0.05, ## P < 0.01, ### P < 0.001 compared with the 0 ng/mL group. ( F ) Western blot detection of the effect of SLIT2 knockdown on human chondrocytes treated with NC or Si-SLIT2 sequence for 48 hours. ( G ) Relative quantification of MMP3, MMP13, and SOX9 proteins. ( H ) qRT-PCR detection of the effect of SLIT2 knockdown on SW1353 cells treated with NC or Si-SLIT2 sequence for 24 hours. ( I ) A heatmap illustrating differentially expressed genes related to TMJOA from RNA-seq analysis between the NC and Si-SLIT2 groups. ( J ) GO analysis of 12 key terms from the top terms. All data are shown as mean ± SD. Statistical significance was assessed by 2-tailed Student’s t test ( B , C , G , and H ) and 1-way ANOVA with Dunnett’s multiple-comparison test ( E ). * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: HEK293T cells and the human chondrocyte cell line SW1353 were purchased from Procell Life Science & Technology Co., Ltd. and cultured in DMEM containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S). rhSLIT2 (R&D Systems) or IL-1β (PeproTech) was added when the cells reached the appropriate density.

Techniques: Western Blot, Over Expression, Isolation, Quantitative Proteomics, Quantitative RT-PCR, Knockdown, Sequencing, RNA Sequencing, Comparison

Journal: JCI Insight

Article Title: Activation of SLIT2/ROBO1/LRP6 axis aggravates cartilage degradation via β -catenin signaling in TMJOA

doi: 10.1172/jci.insight.193632

Figure Lengend Snippet: ( A ) GSEA was performed using gene sets related to the Wnt/β-catenin pathway from the GO and KEGG databases to assess differences between the NC and Si-SLIT2 groups. ( B and C ) IHC images of active β-catenin in condylar cartilage, and quantitative analysis of the percentage of active β-catenin–positive cells ( n = 4). ( D and E ) IHC images of p-LRP6 in condylar cartilage, and quantitative analysis of the percentage of p-LRP6–positive cells ( n = 4). ( F and G ) Western blot detection of the effect of ROBO1 knockdown for GSK-3β and active β-catenin proteins in SW1353 chondrocytes, and relative quantification of p-GSK-3β/t-GSK-3β and active β-catenin proteins. ( H and I ) IF staining images of β-catenin proteins after being treated with rhSLIT2 and/or Si-ROBO1, and relative quantification of fluorescence intensity of β-catenin proteins. ( J and K ) Western blot analysis assessing the effect of LRP6 knockdown on rhSLIT2-induced catabolism in SW1353 chondrocytes, and relative quantification of MMP3, MMP13, and SOX9 proteins. ( L and M ) Western blot analysis showing the impact of LRP6 knockdown on rhSLIT2-induced nuclear translocation of β-catenin in SW1353 cells, and relative quantification of β-catenin proteins in the cytoplasm and nucleus. Scale bars: 50 μm. All data are shown as mean ± SD. Statistical significance was assessed by 2-tailed Student’s t test ( C , E , and G ), 2-way ANOVA with Šidák’s post hoc analysis ( I and K ), and 1-way ANOVA with Dunnett’s multiple-comparison test ( M ). * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: HEK293T cells and the human chondrocyte cell line SW1353 were purchased from Procell Life Science & Technology Co., Ltd. and cultured in DMEM containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S). rhSLIT2 (R&D Systems) or IL-1β (PeproTech) was added when the cells reached the appropriate density.

Techniques: Western Blot, Knockdown, Quantitative Proteomics, Staining, Fluorescence, Translocation Assay, Comparison

Journal: JCI Insight

Article Title: Activation of SLIT2/ROBO1/LRP6 axis aggravates cartilage degradation via β -catenin signaling in TMJOA

doi: 10.1172/jci.insight.193632

Figure Lengend Snippet: ( A and B ) Western blot analysis of p-LRP6 with the effect of rhSLIT2 and/or Si-ROBO1 in SW1353 cells, and relative quantification of p-LRP6 proteins. ( C and D ) IF staining images of the effect of rhSLIT2 and Si-ROBO1 on the expression of p-LRP6 in SW1353 cells, and relative quantification of fluorescence intensity of p-LRP6 proteins. Scale bar: 50 μm. ( E and F ) Western blot analysis examining the effect of LRP6 knockdown on ROBO1 expression in SW1353 cells, and relative quantification of ROBO1 proteins. ( G ) Visualization of representative potential binding sites between LRP6 and ROBO1 using PyMOL. ( H ) Co-IP experiments detected the interaction between ROBO1 and LRP6 in SW1353 cells. ( I and J ) Co-IP experiments using overexpression plasmids for LRP6 and ROBO1 to validate their interaction in SW1353 chondrocytes. ( K ) Co-IP analysis of the effect of rhSLIT2 on the interaction between LRP6 and ROBO1 in SW1353 chondrocytes transfected with overexpression plasmids. ( L ) IF staining showing the effect of rhSLIT2 on the colocalization of ROBO1 and LRP6 in SW1353 cells. Scale bars: 10 μm (left) and 1 μm (right). ( M ) IF staining intensity curve of ROBO1 and LRP6 in IF images. ( N ) Quantification of colocalization using Pearson’s correlation and overlap coefficients. All data are shown as mean ± SD. Statistical significance was assessed by 1-way ANOVA with Dunnett’s multiple-comparison test ( B and D ) and 2-tailed Student’s t test ( F and N ). NS, not significant. ** P < 0.01; *** P < 0.001.

Article Snippet: HEK293T cells and the human chondrocyte cell line SW1353 were purchased from Procell Life Science & Technology Co., Ltd. and cultured in DMEM containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S). rhSLIT2 (R&D Systems) or IL-1β (PeproTech) was added when the cells reached the appropriate density.

Techniques: Western Blot, Quantitative Proteomics, Staining, Expressing, Fluorescence, Knockdown, Binding Assay, Co-Immunoprecipitation Assay, Over Expression, Transfection, Comparison