Journal: Journal of Dental Sciences
Article Title: Periodontal ligament fibroblasts utilize isoprenoid intermediate farnesyl diphosphate for maintaining osteo/cementogenic differentiation abilities
doi: 10.1016/j.jds.2024.04.025
Figure Lengend Snippet: The isoprenoid metabolic process is most severely downregulated metabolic pathway in PPARγ-knockdown PDLFs. (A) Gene ontology analysis of the downregulated genes in PDLFs transfected with siRNA for PPARG by comparing with PDLFs transfected with control siRNA. Raw RNA-seq data were obtained from NCBI's Gene Expression Omnibus (GEO) under accession number GSE178607 . (B) PDLF-1 cells were cultured in mineralization-inducing medium for 3 and 6 days in the presence of troglitazone (10 μM) or rosiglitazone (10 μM). The gene expression levels of AKR1C3 , ALDH3A2 , AKR1C1 , and SDC3 , which were categorized in “isoprenoid metabolic process” and identified as the downregulated genes in PPARG-knockdown PDLFs, were evaluated. (C) PDLFs were cultured in mineralization-inducing medium for 6 days in the presence of troglitazone (10 μM) or rosiglitazone (10 μM) and FPP (20 μM) or GGPP (20 μM). ALP activities were normalized by cell numbers. (D) PDLFs were cultured in mineralization-inducing medium for 12 days in the presence of FPP (0, 10, 20 μM), and calcium deposition was visualized and quantified by Alizarin Red S staining. (E) Demineralized 1.5-month-old maxilla sections were stained without the primary antibody or with mouse IgG, α-FDPS, α-GGPS1, or α-FDFT1 antibodies, ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001 indicated significantly higher expression levels compared with those in DMSO-treated PDLFs at each day point (B), non-treated PDLFs in either DMSO, troglitazone, or rosiglitazone treatment group (C), and non-treated PDLFs (D). Tro = troglitazone. Ros = rosiglitazone. P = pulp. D = dentin. Pdl = periodontal ligament tissue. Ab = alveolar bone. Scale bars correspond to 100 μm.
Article Snippet: The sections were incubated with rabbit IgG (DA1E, Cell Signaling Technologies, Danvers, MA, final concentration 10 μg/mL), rabbit anti-farnesyl diphosphate farnesyltransferase 1 (FDFT1) (13128-1-AP, Proteintech, Rosemont, IL, final concentration 10 μg/mL), rabbit anti-farnesyl pyrophosphate synthetase (FDPS) (16129-1-AP, Proteintech, final concentration 10 μg/mL), and rabbit anti-geranylgeranyl diphosphate synthase 1 (GGPS1) (14944-1-AP, Proteintech, final concentration 10 μg/mL) at 4 °C overnight.
Techniques: Knockdown, Transfection, Control, RNA Sequencing, Gene Expression, Cell Culture, Staining, Expressing
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