Source Data for the Effects of Decanoic Acid and Dexamethasone on Human Chondrocyte Inflammatory and Transcriptional Responses
Description
This repository contains the source data supporting the figures and tables in the associated manuscript investigating the effects of decanoic acid, dexamethasone, and combined decanoic acid plus dexamethasone treatment on inflammatory responses, gene expression, and PPARγ activity in human chondrocytes. The deposited files include replicate-level quantitative reverse-transcription polymerase chain reaction data and the underlying experimental data used to generate the manuscript figures. Depending on the assay, the figure source files contain treatment labels, compound concentrations, experimental identifiers, replicate measurements, time points, and the numerical values used to calculate the plotted means and variability. The qRT-PCR workbook includes GAPDH-normalized ΔCt measurements and corresponding ΔΔCt values for SOX9, RUNX2, COL2A1, ACAN, MMP13, and COL1A1. Human osteoarthritis chondrocytes were evaluated after treatment with medium control, decanoic acid, dexamethasone, or combined decanoic acid plus dexamethasone at the time points reported in the manuscript. The final temporal qRT-PCR analysis was restricted to experimental contexts containing 0.05–0.10 mM decanoic acid. COL2A1 and ACAN analyses were restricted to low-passage cultures, whereas all eligible passage groups were retained for SOX9, RUNX2, MMP13, and COL1A1. The qRT-PCR workbook is organized as follows: README — Describes the dataset, normalization procedures, inclusion criteria, and workbook organization. DeltaCt_Used — Contains the replicate-level ΔCt observations included in the final analysis. Columns identify the experiment, experiment date, culture day, time group, passage, density, treatment, decanoic acid concentration, dexamethasone concentration, replicate number, target gene, and ΔCt value. DeltaDeltaCt_Used — Contains the corresponding ΔΔCt values calculated relative to the mean medium-control ΔCt for the same experiment, culture day, and target gene. Inclusion_Audit — Documents the experiment, treatment, dose, passage, and replicate contexts retained in the final analysis. Data_Dictionary — Defines the column headings and variables contained in the workbook. Additional deposited files provide the numerical source data used for the manuscript figures, including the relevant assay measurements, treatment groups, concentrations, biological or technical replicate labels, and calculated plotting values. File and worksheet names are retained wherever possible to allow the deposited data to be readily matched to the corresponding figures and panels in the manuscript. These data are provided to support transparency, reproducibility, and independent evaluation of the reported findings. No source values were reconstructed from plotted figures or manuscript summary statistics.
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Single-donor primary human synovial chondrocytes derived from normal (HSC-N; male, 52 years of age) and OA tissue (HSC-OA; female, 79 years of age) were obtained commercially from Articular Engineering (Northbrook, IL). All other culturing reagents were purchased from ThermoFisher Scientific (Waltham, MA) unless otherwise stated. For experiments, cells were seeded at 3,000 cells/cm2 in the tissue culture plates described below and then expanded in DMEM/F12 containing 20% FBS at 37 C and 5% CO2. When 100% confluent, the cultures were equilibrated for 24 hours in DMEM/F12 containing 5% FBS before treatment, with treatments performed in the 5% FBS media. HSC-N were utilized at passages 2–3 and HSC-OA at passages 5–7. Acute-phase inflammatory stimulation and PGE2 release were measured in HSC-N cultures. In brief, cells were seeded into 96-well tissue culture plates as described above and then exposed to dexamethasone sodium phosphate (Dex; 0.01-100 nM in a 10-fold serial dilution), decanoic acid (DA; 6-500 μM in a 3-fold serial dilution), or the Dex dilution series with DA incorporated at set amounts across the range (1, 2, 10, 12.5, 19, or 50 μM DA), and then activated with Interleukin-1 beta (IL-1) at a final concentration of 0.1 ng/ml. These concentrations were selected from preliminary concentration-response studies to span weak to near-saturating inhibition responses. Supernatants were collected 24 hours after stimulation, and PGE2 was measured via competitive ELISA (Cayman Chemical, Ann Arbor, MI). Long-term transcriptional regulation was measured in resting HSC-OA cultures using quantitative real-time PCR. In brief, cells were seeded into 24-well tissue culture plates and maintained under treatment for up to 28 days (0.1 μM Dex, 50-100 μM DA, or in the corresponding combination), with the medium refreshed every 2–3 days. Total RNA was isolated using Qiagen RNeasy plus columns (Germantown, MD) at 7, 10-14, and 20-28 days, and amplification from complementary DNA was performed for Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Collagen type II alpha 1 (Col2a1), Collagen type I alpha 1 (Col1a1), Aggrecan (ACAN), SRY-Box Transcription Factor 9 (SOX9), Runt-Related Transcription Factor 2 (RunX2), and Matrix Metalloproteinase 13 (MMP13) using validated primers and reagents from Qiagen. GAPDH-normalized ΔCt or ΔΔCt values were then calculated, with results presented as mean ± SD. Regulatory PPARγ activation was then assessed in resting HSC-OA cultures. In brief, cells were seeded into 10 cm2 dishes as described above, treated for the times indicated with 0.1 μM Dex, 100 μM DA, 250 μM, or combinations thereof, and nuclear protein extracts were isolated. Rosiglitazone (Rosig; 10 μM) obtained from Sigma-Aldrich (St. Louis, MO) served as a positive control for these experiments. Nuclear DNA-binding activity was then measured using a capture assay from Active Motifs (Carlsbad, CA).