Fig. 1The effect of lipopolysaccharide (LPS) on nuclear factor-E2-related factor 1 (NFE2L1) mRNA expression in MC3T3-E1 cells. Cells were treated with 0, 2, 5, or 10 µg/mL LPS for 24 hours. Levels of mRNA were analyzed by (A) semi-quantitative polymerase chain reaction (PCR) and (B) quantitative real time-PCR. The expression level of each mRNA was normalized to the β-actin levels. aP<0.05 compared with the control group.
Fig. 2Nuclear factor-E2-related factor 1 (NFE2L1) mRNA expression after transient transfection with control siRNA (siCONT) or NFE2L1 siRNA (siNFE2L1) in MC3T3-E1 cells. Levels of mRNA were analyzed by semi-quantitative (A) polymerase chain reaction (PCR) and (B) quantitative real time-PCR. The expression level of each mRNA was normalized to the β-actin levels. aP<0.05 compared with the siCONT group.
Fig. 3Measurement of reactive oxygen species (ROS) with H2DCF-DA in MC3T3-E1 cells. Intracellular ROS in the transfectants of control siRNA (siCONT) and nuclear factor-E2-related factor 1 siRNA (siNFE2L1) were compared under control (no stimulation) and stimulations by lipopolysaccharide (LPS, 10 µg/mL) for 10 minutes. aP<0.05 compared with the control group or siCONT cells.
Fig. 4The effect of nuclear factor-E2-related factor 1 (NFE2L1) knockdown on antioxidant gene mRNA expression in lipopolysaccharide (LPS) treated cells. Antioxidant genes are as follows: (A) metallothionein 1 (MT1), (B) metallothionein 2 (MT2), (C) glutamate-cysteine ligase catalytic subunit (GCLC), (D) glutamate-cysteine ligase modifier subunit (GCLM), (E) NAD(P)H dehydrogenase, quinone 1 (NQO1), and (F) glutathione peroxidase 1 (GPx1). MC3T3-E1 cells were transfected with control siRNA (siCONT) or NFE2L1 siRNA (siNFE2L1) followed by 24-hour treatment of 10 µg/mL LPS. Controls received culture medium only. Quantitation of mRNA levels was analyzed by quantitative real-time polymerase chain reaction. The expression level of each mRNA was normalized to the β-actin levels. aP<0.05 compared with the control group or siCONT cells.
Table 1Primers Used
![enm-31-336-i001.jpg]()
Gene |
Forward (5'-3') |
Reverse (5'-3') |
β-Actin
|
CCGCGAGCACAGCTTCTT |
CCCACGATGGAGGGGAATAC |
NFE2L1
|
GGAGAGCTTCCCTGCACAGT |
TTACTTCCATAGCCTGCATTTCC |
MT1
|
ATGGACCCCAACTGCTCCT |
ACAGCCCTGGGCACATTT |
MT2
|
CCGATCTCTCGTCGATCTTCAACC |
CAGGAGCAGCAGCTTTTCTTGCAG |
GCLC
|
GCACGGCATCCTCCAGTTCCT |
TCGGATGGTTGGGGTTTGTCC |
GCLM
|
GGCTTCGCCTCCGATTGAAGA |
TCACACAGCAGGAGGCCAGGT |
NQO1
|
GCATTGGCCACACTCCACCAG |
ATGGCCCACAGAGAGGCCAAA |
GPx1
|
TGCTCATTGAGAATGTCGCGTCTC |
AGGCATTCCGCAGGAAGGTAAAGA |