Fig 1.
Effects of MBP on the progesterone secretion.
(A) MLTC-1 and (B) Y1 cells were exposed to 0 ~ 104 nM MBP in the presence of 100 U/L hCG or 10 μM forskolin for 24 h respectively, the secretion of progesterone was determined in triplicate. The amounts of progesterone measured in control cells were MLTC-1: 8.85±0.21 ng/ml, Y1:8.01±0.32 ng/ml, respectively. *p<0.05 and **p<0.01 compared with cells exposed to hCG or forskolin alone.
Fig 2.
Effects of hCG/For on the expressions of StAR, p450scc, 3β-HSD, vimentin, β-actin, and tubulin.
MLTC-1 and Y1 cells were exposed to 100 U/L hCG or to 10 μM forskolin for 24 h, respectively. (A) Western blots analysis and relative protein levels of (B) StAR, (C) p450SCC, (D) 3β-HSD, (E) vimentin, (F) β-actin, and (G) tubulin. *p<0.05 and **p<0.01 compared with medium control cells.
Fig 3.
Effects of MBP on the expressions of StAR, p450scc, 3β-HSD, vimentin, β-actin, and tubulin.
MLTC-1 and Y1 cells were treated by 1000 nM MBP for 24 h. (A) Western blots analysis and relative protein levels of (B) StAR, (C) p450SCC, (D) 3β-HSD, (E) vimentin, (F) β-actin, and (G) tubulin. **p<0.01 compared with medium control cells.
Fig 4.
Effects of MBP and hCG/For on the expressions of StAR, p450scc, 3β-HSD, vimentin, β-actin, and tubulin.
MLTC-1 and Y1 cells were exposed to1000 nM MBP in the absence or presence of 100 U/L hCG or 10 μM forskolin for 24 h, respectively. (A) Western blots analysis and relative protein levels of (B) StAR, (C) p450SCC, (D) 3β-HSD, (E) vimentin, (F) β-actin, and (G) tubulin. **p<0.01 compared with medium control cells; ##p<0.01 compared with cells treated by MBP alone; ΔΔp<0.01 compared with cells treated by hCG or For alone.
Fig 5.
Effects of vimentin on the MBP-induced progesterone secretion.
(A and B), MLTC-1 and Y1 cells were transfected by con-siRNA or vimentin-siRNA for 12 h, respectively, qRT-PCR (top) and Western blots (bottom) analysis of the expressions of vimentin mRNA and protein. (C and D), after MLTC-1 and Y1 cells were transfected by con-siRNA or vimentin-siRNA for 12 h, they were exposed to100 U/L hCG or 10 μM forskolin in the absence or presence of 1000 nM MBP for 24 h, respectively. The secretion of progesterone was determined in triplicate. The amounts of progesterone measured in control cells were MLTC-1 (hCG): 8.32±0.22 ng/ml, MLTC-1 (hCG+MBP): 10.10±0.42 ng/ml, Y1 (For): 8.64±0.33 ng/ml, and Y1 (For+MBP): 10.31±0.21 ng/ml respectively. **p<0.01 compared with cells transfected by con-siRNA.
Fig 6.
MBP improved the expression of vimentin by DNA demethylation.
(A-D), MLTC-1 cells were exposed to 1000 nM MBP for 24 h, the methylation status of vimentin promoter was determined in triplicate by qMSP (A and B). Annotation, methylated (M); unmethylated (U); the percentage of methylation in a sample was estimated using the following formula: methylation (%) = (M/M+U) ×100% = [1/(1+U/M)] ×100% = [1/(1+2∆Ct)] ×100%. (C and D), Western blots analysis and relative protein levels of DNMT1, DNMT3a, and DNMT3b. *p<0.05 and **p<0.01 compared with medium control cells. (E-G), MLTC-1 and Y1 cells were exposed to 1000 nM MBP in the presence or absence of 200 μM SAM for 24 h, respectively. (E) qRT-PCR analysis in triplicate of vimentin mRNA. (F) Western blots analysis and (G)relative protein levels of vimentin. *p<0.05 and **p<0.01 compared with medium control cells; ##p<0.01 compared with cells exposed to MBP alone.
Fig 7.
Effects of MBP on the activation of NF-κB.
MLTC-1 and Y1 cells were exposed to 1000 nM MBP for 0, 12, or 24 h, respectively. (A) Western blots analysis and (B)relative protein levels of p-IKKβ, p-IκBα, and p-RelA. *p<0.05 and **p<0.01 compared with medium control cells.
Fig 8.
Functions of NF-κB in the transcriptional activation of vimentin.
(A) MLTC-1 cells were co-transfected by Con-siRNA or RelA-siRNA plus pGL3-vimentin-Luc construct (wild type, WT; or mutant, MT) for 12 h. Luciferase reporter assay analysis of the effects of NF-κB on the transcriptional activity in vimentin promoter. (B and C) MLTC-1 cells were transfected by Con-siRNA or RelA-siRNA for 12 h. (B) Western blots analysis and (C)relative protein levels of vimentin. **p<0.01 compared with cells transfected by Con-siRNA. (D) After MLTC-1 and Y1 cells were pre-treated by 0 or 10 μM BAY11-7082 for 12 h, they were exposed to 0 or 1000 nM MBP for 24 h. (D, top) qRT-PCR analysis in triplicate of vimentin mRNA, *p<0.05 and **p<0.01 compared with medium control cells; ##p<0.01 compared with cells exposed to MBP alone. (D, bottom) Western blots analysis of the expression of p-RelA. (E) After MLTC-1 and Y1 cells were pre-transfected by Con-siRNA or RelA-siRNA for 12 h, they were exposed to 1000 nM MBP for 24 h. (E, top) qRT-PCR analysis in triplicate of vimentin mRNA, **p<0.01 compared with medium control cells; ##p<0.01 compared with cells exposed to MBP plus Con-siRNA.
Fig 9.
Effects of NF-κB/vimentin signaling on the MBP-induced progesterone secretion in vitro.
(A) MLTC-1 and Y1 cells were treated as described in Fig 8E, the secretion of progesterone was determined in triplicate (top), and the expression of p-RelA was determined by Western blot (bottom). The amounts of progesterone measured in control cells were MLTC-1: 8.23±0.24 ng/ml, Y1: 9.42±0.37 ng/ml, respectively. (B) After MLTC-1 cells were co-transfected by Con-siRNA or RelA-siRNA plus Vector or vimentin-Flag construct for 12 h, they were exposed to 1000 nM MBP for 24 h. The secretion of progesterone was determined in triplicate (top), and the expressions of Flag and p-RelA was determined by Western blot (bottom). The amounts of progesterone measured in control cells were MLTC-1 (vector): 9.02±0.31 ng/ml and MLTC-1 (vimentin-FLag): 10.56±0.53 ng/ml, respectively. *p<0.05 and **p<0.01 compared with medium control cells; ##p<0.01 compared with cells exposed to MBP plus Con-siRNA.
Fig 10.
Effects of NF-κB/vimentin on DBP-induced testosterone secretion in vivo.
The pubertal male, Sprague-Dawley rats (10 per guoup) were orally administered DBP at the doses of 1 mg/kg·day for 30 days. (A) The serum testosterone level was determined in triplicate. (B) The methylation status of vimentin promoter regions was determined in triplicate by qMSP. (C) qRT-PCR analysis in triplicate of the expression of vimentin mRNA. (D) Western blots analysis and (E) relative protein levels of p-RelA, vimentin, DNMT1, DNMT3a, and DNMT3b. *p<0.05 and **p<0.01 compared with rats exposed to no DBP. (F-H) After Con-siRNA, RelA-siRNA, or vimentin-siRNA was injected into the interstitial tissue of testis, respectively, the rats were orally administered DBP as described above. (F) The serum testosterone level was determined in triplicate. The amounts of testosterone measured in control animals were 16.36±0.22 ng/ml. (G) Western blots analysis and (H) relative protein levels of p-RelA and vimentin. **p<0.01 compared with Con-siRNA group; ##p<0.01 compared with DBP plus Con-siRNA group; ΔΔp<0.01 compared with DBP plus vimentin-siRNA group.