Figure legends
Fig 1. Alterations in the maternal adrenal and placental
functions, and the level of CORT in fetal male rats with PDE.(A) Maternal serum CORT
concentration; (B) Maternal serum ACTH concentration; (C) The mRNA
expression of maternal adrenal steroidogenic genes; (D) Fetal serum
CORT concentration; (E) Placental
Mdr1a, Mdr1b, 11β-HSD1, 11β-HSD2
mRNA expression; (G, H) Placental Mdr1a, Mdr1b, 11β-HSD1, 11β-HSD2
protein expression; (F, I) The ratio of placental 11β-HSD1/11β-HSD2 mRNA
and protein expression; (J) Correlations between maternal serum
CORT concentration and fetal serum
CORT
concentration. n=8 placentas or
adrenals for qRT-PCR and n=3 for Western blot in each group. Comparisons
between groups were performed using Student’s t -test or Pearson
correlation analysis. Mean ± S.E.M.,*P <0.05,**P <0.01 vs . respective
controls. CORT, corticosterone; PDE, prenatal dexamethasone exposure;
ACTH, adrenocorticotropic hormone; Mdr1a, multidrug-resistance protein
1a; 11β-HSD1, hydroxysteroid
11-beta dehydrogenase 1; StAR, steroidogenic acute regulatory protein;
P450scc, cytochrome P450 cholesterol side-chain cleavage; 3β-HSD,
3β-hydroxysteroid dehydrogenase; P450c21, steroid 21-hydroxylase;
P450c11, steroid 11β-hydroxylase.
Fig 2. Alterations in
serum CORT level, histone acetylation and expression levels of IGF1,
adrenal steroidogenesis,
and related indicators in fetal male rats with PDE.(A, D, E, H) The mRNA expression
of fetal adrenal steroidogenic genes, IGF1 signal pathway, GRα and
Sirt3; (B, C) StAR protein expression (yellow represents StAR positive
cells, 400×); (F) Clustering of miRNAs differentially expressed in fetal
adrenal; (G) Adrenal miR-370-3p expression; (I) Enrichment of H3K9ac,
H3K14ac and H3K27ac levels of IGF1 promoter region; preimmune IgG served
as the negative control. n=8 adrenals for qRT-PCR and n=5 for IHC/ChIP
assay in each group. Comparisons between groups were performed using
Student’s t -test Mean ± S.E.M.,*P <0.05,**P <0.01 vs . respective
controls. CORT, corticosterone; IGF1, insulin-like growth factor 1; PDE,
prenatal dexamethasone exposure; GRα, glucocorticoid receptor alpha;
H3K9ac, histone 3 lysine 9 acetylation; StAR, steroidogenic acute
regulatory protein; P450scc, cytochrome P450 cholesterol side-chain
cleavage; 3β-HSD, 3β-hydroxysteroid dehydrogenase; P450c21, steroid
21-hydroxylase; P450c11, steroid
11β-hydroxylase; IGF1R, IGF1
receptor; Akt1, protein kinase B.
Fig 3. Alterations in serum CORT level, histone acetylation and
expression levels of IGF1, adrenal steroidogenesis, and related
indicators s in male offspring rats with PDE.(A) Adult serum
CORT concentration; (B, D, E, G) Adrenal GRα,
Sirt3, IGF1 signal pathway, StAR and P450scc mRNA expression; (F)
miR-370-3p expression; (C) Adrenal StAR protein expression (yellow
represents StAR positive cells, 400×); (H) Enrichment of H3K9ac, H3K14ac
and H3K27ac levels of IGF1 promoter region; preimmune IgG served as the
negative control. n=8 adrenals for qRT-PCR and n=5 for IHC/ChIP assay in
each group. Comparisons between groups were performed using Student’st -test. Mean ± S.E.M.,*P <0.05,**P <0.01 vs . respective
controls. CORT, corticosterone; IGF1, insulin-like growth factor 1; PDE,
prenatal dexamethasone exposure; GRα, glucocorticoid receptor alpha;
StAR, steroidogenic acute regulatory protein; P450scc, cytochrome P450
cholesterol side-chain cleavage; H3K9ac, histone 3 lysine 9 acetylation;
IGF1R, IGF1 receptor; Akt1, protein kinase B.
Fig 4. Effects of chronic
stress on CORT level, the histone acetylation and expression levels of
IGF1, steroidogenesis, and related indicators in male rats with PDE.(A) Serum
CORT concentration; (B-D,F) Adrenal GRα, Sirt3,
IGF1, StAR and P450scc mRNA expression; (E) miR-370-3p expression; (G)
Enrichment of H3K27ac level of IGF1 promoter region; preimmune IgG
served as the negative control. n=8 adrenals for
qRT-PCR and n=5 for ChIP assay in
each group. Mean ± S.E.M., *P <0.05,**P <0.01 vs . respective
controls. CORT, corticosterone; IGF1, insulin-like growth factor 1; PDE,
prenatal dexamethasone exposure; GRα, glucocorticoid receptor alpha;
StAR, steroidogenic acute regulatory protein; P450scc, cytochrome P450
cholesterol side-chain cleavage;
H3K27ac, histone 3 lysine 27
acetylation.
Fig 5. Effects of
dexamethasone on the histone acetylation and expression levels of IGF1
and steroidogenesis in NCI-H295R cells.(A) IGF1 mRNA expression; (B, E)
StAR mRNA expression; (C) IGF1 and StAR protein expression; (D)
Intracellular cortisol concentration; (F) Enrichment of H3K9ac, H3K14ac,
H3K27ac levels of IGF1 promoter region; preimmune IgG served as the
negative control. n=6 for qRT-PCR from triplicate independent
experiments and n=5 for Western blot in each group. Mean ± S.E.M.,*P <0.05,**P <0.01 vs control;#P <0.05,##P <0.01 vs 500 nM
dexamethasone by one-way ANOVA or Student’s t -test. IGF1,
insulin-like growth factor 1; StAR, steroidogenic acute regulatory
protein; H3K9ac, histone 3 lysine 9 acetylation.
Fig 6. Effects of
low physiological concentrations of cortisol on the histone acetylation
and expression levels of IGF1, steroidogenesis and related indicators in
NCI-H295R cells. (A, C-E, G)
StAR, GRα, miR-370-3p, Sirt3 and IGF1 expression; (B) Intracellular
cortisol concentration; (G) Enrichment of H3K9ac,
H3K14ac, H3K27ac levels of IGF1 promoter region; preimmune IgG served as
the negative control; (H) StAR, Sirt3 and IGF1 protein expression. n=6
for qRT-PCR from triplicate independent experiments and n=5 for Western
blot/ChIP assay in each group. Comparisons between groups were performed
using Student’s t -test and ANOVA. Mean ± S.E.M.,*P <0.05,**P <0.01 vs . respective
controls. IGF1, insulin-like growth factor 1; StAR, steroidogenic acute
regulatory protein; GRα, glucocorticoid receptor alpha; H3K9ac, histone
3 lysine 9 acetylation.
Fig 7. Effects of
low physiological concentrations of cortisol on IGF1 and StAR expression
through the GRα/miR-370-3p/Sirt3 pathway in NCI-H295R cells. To confirm
the roles of GRα, miR-370-3p, and Sirt3 in the gene regulation of
cortisol, we incubated the cells with
cortisol (300 nM) in the absence or presence of GRα
plasmid, miR-370-3p mimics, Sirt3 siRNA, IGF1 plasmid, respectively, for
24 h and then harvested. (A) StAR
mRNA expression; (B, E, J) Enrichment of H3K27ac level of IGF1 promoter
region; preimmune IgG served as the negative control; (C, F, K) IGF1
expression; (D) Sirt3 expression; (G) Sequence of the WT or mutated
miR-370-3p binding site within the 3’-UTR of Sirt3 mRNA; (H) The
association between miR-370-3p and Sirt3 by luciferase assay; (I)
miR-370-3p expression. n=6 for qRT-PCR from triplicate independent
experiments and n=5 for Western blot/ChIP assay in each group. Mean ±
S.E.M.,*P <0.05,**P <0.01 vs control;#P <0.05,##P <0.01 vs 75 nM cortisol
by one-way ANOVA or Student’s t -test. IGF1, insulin-like growth
factor 1; StAR, steroidogenic acute regulatory protein; GRα,
glucocorticoid receptor alpha; H3K27ac, histone 3 lysine 27 acetylation.
Fig 8. The GRα/miR-370-3p/Sirt3 pathway mediated the inhibitory
effect of low physiological concentrations of cortisol on the H3K27ac
and expression levels of IGF1. GRα, glucocorticoid receptor alpha; GRE,
glucocorticoid receptor element; IGF1, insulin-like growth factor 1;
H3K27ac, histone 3 lysine 27 acetylation.