Dataset of effect of Wogonin, a natural flavonoid, on the viability and activation of NF-κB and MAPKs in IL-1β-stimulated human OA chondrocytes

This article contains data related to the article “Wogonin, a plant derived small molecule exerts potent anti-inflammatory and chondroprotective effects through activation of ROS/ERK/Nrf2 signaling pathways in human Osteoarthritis chondrocytes” (Khan et al. 2017) [1]. The data are related to effects of Wogonin on the viability and IL-1β-stimulated activation of NF-κB and ERK1/2, JNK1/2 and p38 MAPKs in human OA chondrocytes. Gene expression data representing the chondrogenic phenotype and the efficiency of Nrf2 knockdown in monolayer culture of human OA chondrocytes were shown. Moreover, mass spectrometric calibration curve of Wogonin used to quantify the intracellular uptake were also presented. The data are presented in the form of figures and significance of these has been given in the research article (Khan et al. 2017) [1].


a b s t r a c t
This article contains data related to the article "Wogonin, a plant derived small molecule exerts potent anti-inflammatory and chondroprotective effects through activation of ROS/ERK/Nrf2 signaling pathways in human Osteoarthritis chondrocytes" ) [1]. The data are related to effects of Wogonin on the viability and IL-1β-stimulated activation of NF-κB and ERK1/2, JNK1/2 and p38 MAPKs in human OA chondrocytes. Gene expression data representing the chondrogenic phenotype and the efficiency of Nrf2 knockdown in monolayer culture of human OA chondrocytes were shown. Moreover, mass spectrometric calibration curve of Wogonin used to quantify the intracellular uptake were also presented. The data are presented in the form of figures and significance of these has been given in the research article (Khan et  For the analysis of protein expression, Western blot analysis using specific antibodies were used [2]. For the analysis of mRNA expression, Sybrgreen and Taqman assay were performed [2]. We confirm that data provided in the article are original and have not been published elsewhere.
Value of the data 1. The datasets provide evidence in the favor of chondrogenic phenotype of OA chondrocytes in monolayer culture. 2. The data of cell cycle analysis using propidium iodide staining provides proof for the nonproliferative nature of monolayer culture of human OA chondrocytes. 3. The datasets provide the efficiency of nucleofection in human OA chondrocytes to deplete the expression of gene of interest. This data set may assist researchers to choose the appropriate method of transfection in chondrocytes, the cell type difficult to transfect. 4. These data will be of value to the scientific community working in the area of osteoarthritis and nutraceuticals since they illustrate the therapeutic targets of OA beyond NF-κB and MAPKs. 5. The data about mass-spectrometric calibration curve of Wogonin are promising and may be valuable for the uptake studies of flavonoids in the cells.

Data
These data were related to the article "Wogonin, a plant derived small molecule exerts potent antiinflammatory and chondroprotective effects through activation of ROS/ERK/Nrf2 signaling pathways in human Osteoarthritis chondrocytes" ) [1]. The dataset presents here, illustrate the chondrogenic phonotype of cultured OA chondrocytes (Fig. 1) and represents the effects of natural flavonoid Wogonin on the viability ( Fig. 2A and B), and IL-1β-stimulated activation of NF-κB and ERK1/2, JNK1/2 and p38 MAPKs in human OA chondrocytes ( Fig. 3A and B). Fig. 4 shows the evidence of effective depletion of target gene expression using specific siRNA in chondrocytes. Fig. 5 presents four point calibration curve generated to quantify the intracellular concentration of Wogonin in chondrocytes.

Total RNA isolation and real time PCR
Total RNA from chondrocytes were isolated using Trizol s reagent and cDNA was synthesized from 1 mg of total RNA using high-capacity cDNA reverse transcription kit (Life Technologies) [2]. The mRNA expression of COL2A1 and ACAN was quantified using TaqMan Gene Expression Assays and relative expression levels were calculated using the 2 À ΔΔC T method [2].     4. Genetic ablation of Nrf2 using specific siRNA in chondrocytes: OA chondrocytes were transfected with siRNA specific for Nrf2 (50, 100 nM) using nucleofection. Forty-eight hours after the transfections, RNA was isolated form OA chondrocytes and gene expression of Nrf2 was measured by quantitative PCR using the SYBR s green assay (Life Technologies). Bar graph represents mean7 SD from two subjects. *pr 0.05, as compared to control siRNA.

Viability assay
Primary human OA chondrocytes were serum starved overnight, and then treated with Wogonin (10-50 mM) for 24 h in serum free medium and viability was determined using MTT assay and cell cycle analysis using propidium iodide staining [2]. A total of 20,000 cells were acquired in BDAccuri C6 Flowcytometer and percent apoptotic cells were determined by analyzing a sub-G1 population (o 2n DNA content) using FlowJo software.

Western immunoblotting
Chondrocytes (1 Â 10 6 /well of 6-well plate) were pretreated with Wogonin (50 mM) for 2 h followed by treatment with IL-1β (10 ng/ml) for 15-30 min. After treatments, OA chondrocytes were harvested, washed with cold PBS and lysed in ice-cold RIPA buffer and lysate were prepared [2]. Equivalent amounts of lysate protein (20 mg) were resolved by 10% SDS-PAGE and transferred to a PVDF membrane (Bio-Rad, USA) and the blots were incubated with primary antibodies diluted in 2% blocking buffer for overnight at 4°C. Blots were then incubated with horse radish peroxidaseconjugated secondary antibody, followed by washing with TBST. Blot were developed using Luminata Western HRP substrate (EMS Millipore) and the antibody reactive proteins were visualized by chemiluminescence and imaged using the Pxigel imaging system (Syngene, Frederick, MD).

Calibration curve of Wogonin
The calibration curve of Wogonin was generated using LC-MS/MS analysis using a UHPLC system connected to a triple quadrupole mass spectrometer (LC-MS 8040; Shimadzu, Kyoto, Japan) equipped with an electrospray ionization source. The quantitation was performed using the ESI ion source in multiple reaction monitoring and negative ion mode (MRM-) by monitoring transition pairs m/z 283.00 (precursor ion)/162.9 (product ion). The following instrument settings were used for MRM analysis: heat block temperature, 400°C; DL temperature, 250°C; nebulizing gas (N 2 ), 3 L/min; drying gas (N 2 ), 15 L/min; collision energy, 35.0; dwell time, 100 ms. Four point calibration curve was prepared using pure Wogonin dissolved in methanol at the concentrations of 0.001, 0.01, 0.1 and 1 mg/ml.