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Fig. 2 | BMC Musculoskeletal Disorders

Fig. 2

From: Pigment Epithelium-Derived Factor (PEDF) mediates cartilage matrix loss in an age-dependent manner under inflammatory conditions

Fig. 2

PEDF ectopic expression upregulates catabolic gene expression in normal human articular chondrocytes (NHACs). a Lentiviral constructs were generated that contained the human SERPINF1 gene, which encodes for the PEDF protein, and a tdTomato moiety that was separated from the SERPINF1 gene by a P2A self-cleaving peptide (LV-PEDF). A lentiviral construct encoding the gene for green fluorescent protein (GFP) and a tdTomato moiety (LV-GFP) was also constructed. b tdTomato expression is comparable between the two expression vectors. c Western Blot analysis confirmed elevated PEDF protein production in the cytoplasm and nucleus of NHACs transduced with LV-PEDF. α-tubulin was used as a loading control for cytoplasmic proteins. TATA binding protein (TBP) was used as a loading control for nuclear proteins. LV-GFP: GFP-encoding lentivirus; LV-PEDF: PEDF-encoding lentivirus. d ELISA analysis of PEDF protein secretion into the media of NHACs transduced with LV-GFP or LV-PEDF following 4 days in culture. Data are represented as fold change relative to LV-GFP samples. e Quantitative RT-PCR analysis of MMP-1, MMP-3 and MMP-13 on NHACs transduced with LV-GFP or LV-PEDF and cultured in the presence or absence of 1 ng/mL IL-1β for 4 days. Data are represented as fold change relative to LV-GFP-transduced cells in the absence of IL-1β within each gene. Gene expression was normalized to TATA-binding protein mRNA expression levels. f ELISA analysis of MMP-1, MMP-3 and MMP-13 protein secreted into the media of NHACs transduced with LV-GFP or LV-PEDF and cultured in the presence or absence of 1 ng/mL IL-1β for 4 days. Data are presented as fold change relative to that found in the media of LV-GFP-transduced cells cultured in the absence of IL-1β. All data are plotted as mean ± SEM. * p < 0.05 (Mann–Whitney test)

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