Journal: Redox Report
Article Title: Nuclear receptor subfamily 4, group A, member 1 inhibits extrinsic apoptosis and reduces caspase-8 activity in H2O2-induced human HUC-F2 fibroblasts
Figure Lengend Snippet: Effect of si-NR4A1 on mRNA expression and cell growth. (A) HUC-F2 cells were cultured with 10 nM siRNA and 0.2% Lipofectamine 2000 in α-MEM containing 10% fetal bovine serum and 150 IU/ml to 100 μg/ml penicillin to streptomycin medium for 24, 48, and 72 hours in 5% CO 2 . Real-time PCR analysis showed that NR4A1 expression is reduced by 70.4, 87.2, and 83.7%, respectively. (B) HUC-F2 cells were transfected with si-NR4A1 for 48 hours and treated with 200μM H 2 O 2 for 2–8 hours. Expression levels of NR4A1 and NR4A3 were determined using real-time PCR. Normalization of data was achieved by quantitating the cycle time at an arbitrary fluorescence intensity in the linear exponential phase using StepOnePlus Real-Time system software by calculating the ratio of the concentration of each enzyme relative to that of β-actin and GAPDH cDNA, respectively. (C) Effects of the H 2 O 2 concentration on the HUC-F2 cells treated with si-NR4A1 were determined with the MTT assay. Specifically, HUC-F2 cells were transfected with si-NR4A1 or si-NC, incubated in 5% CO 2 for 48 hours, and treated with H 2 O 2 . After 24 hours, cell viability was assessed with the MTT assay. Results are presented as mean ± standard deviation of three samples in each treatment group. The differences between the groups were analyzed by ANOVA with Fisher's PLSD post hoc method. * P
Article Snippet: Cells were cultured in Alpha-Modified Eagle's Medium (α-MEM) (Cellgro, Manassas, VA, USA) containing 10% foetal bovine serum (FBS) (MP Biomedicals Inc., Solon, OH, USA), 150 IU/ml penicillin (Meiji Seika Kaisha, Tokyo, Japan), and 100 mg/ml streptomycin (Meiji Seika Kaisha) in humidified air at 37°C with 5% CO2 .
Techniques: Expressing, Cell Culture, Real-time Polymerase Chain Reaction, Transfection, Fluorescence, Software, Concentration Assay, MTT Assay, Incubation, Standard Deviation