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. 2014 May 22;5(5):e1239. doi: 10.1038/cddis.2014.194

Figure 5.

Figure 5

JunB upregulates endogenous IRE1a gene expression. (a) Real-time PCR assay of IRE1a mRNA level in C2C12 cell lines infected with Ad-JunB expression plasmid or control Ad-GFP. It is found that IRE1a mRNA was upregulated in C2C12 cell lines infected with Ad-JunB (MOI=50) adenovirus when compared with control C2C12 cells. The normalized values were then calibrated against the control value. The units are arbitrary and the left bar indicates a relative level of IRE1a mRNA of 1; *P<0.05. (b) Exogenous expression of JunB increased the IRE1a gene expression in C2C12 cells. Cell lysates from untreated C2C12 cells (CTR) or from C2C12 cells infected with Ad-JunB (MOI=50) or Ad-GFP were subjected to SDS-PAGE and detected by anti-IRE1a antibody, or anti-JunB, or anti-tubulin, respectively. Tubulin is served as an internal control. (c) Semi-quantification of protein relative levels of JunB and IRE1α in the C2C12 infected with various adenoviruses, as indicated in b. Levels were normalized against those of tubulin by MJ Opticon Monitor Analysis Software (Bio-Rad); data were expressed as means±S.D. (n=3). Every treatment group was compared with control groups, respectively, *P<0.05. (d) siRNA against JunB mRNA efficiently inhibited the expression of endogenous JunB in C2C12 cells. Cells were infected with either siJunB or control siRNA (CTR), and total RNA was collected for real-time PCR. Expression of JunB was normalized against the GAPDH endogenous control. The normalized values were then calibrated against the control value, here set as 1. *P<0.05. (e) Repression of JunB largely abolished IRE1a expression in BMP2-induced C2C12 cells. C2C12 cells were treated with BMP2 (300 ng/ml), then infected with either control siRNA (CTR) or JunB siRNA (siJunB) adenovirus. Total RNA was extracted from the cells and the mRNA levels of IRE1a and GAPDH were measured by real-time PCR at various time points. The units are arbitrary and the relative level of IRE1a mRNA on day 0 was set to 1. All experiments were repeated three times. Every siJunB treatment group was compared with control groups, respectively, *P<0.05