Transfection of fully differentiated 3T3-L1 adipocytes with C/EBPα or C/EBPβ siRNA decreases 11β-HSD1 mRNA levels, whilst CHOP siRNA has no effect.

<p>(<b>A</b>) Real-time PCR measurement of levels of mRNA encoding C/EBPα (left panel), C/EBPβ (centre panel) and CHOP (right panel), 24 h after transfection of fully differentiated 3T3-L1 adipocytes with scrambled RNA (Scr; as control) or siRNAs (32 pmol) targeting C/EBPα, β or CHOP. Data are normalized to TBP and expressed relative to levels in cells transfected with scrambled RNA (arbitrarily set to 100%). Values are mean±SEM of 3 different experiments (independent adipocyte differentiations) with each siRNA treatment tested in triplicate. *, Significantly different from scrambled RNA, p≤0.05. (<b>B</b>) Representative western blots showing levels of the 42 kDa (p42) and 30 kDa (p30) isoforms of C/EBPα (left panel; 20 µg protein/lane), 38 kDa LAP*, 35 kDa LAP and 20 kDa LIP isoforms of C/EBPβ (centre panel; 40 µg protein/lane) and CHOP (right panel; 20 µg protein/lane), 24 h after transfection of 3T3-L1 adipocytes with scrambled RNA or respective siRNAs targeting C/EBPα, β or CHOP. Blots were stripped and reprobed with β-tubulin antibody, as loading control. In C/EBPβ and CHOP westerns all samples were analysed in the same gel but not all in adjacent lanes. (<b>C</b>) Real-time PCR measurement of mRNA encoding 11β-HSD1, 24 h after transfection of 3T3-L1 mature adipocytes with scrambled RNA or siRNA (32 pmol) targeting C/EBPα, β or CHOP. Data are normalized to TBP and expressed relative to levels in cells transfected with scrambled RNA (arbitrarily set to 100%). Values are mean±SEM of 3 to 4 different experiments (independent adipocyte differentiations) with each siRNA treatment tested in triplicate. *, Significantly different from scrambled RNA, p≤0.05.</p

Tunicamycin-induced ER stress reduces 11β-HSD1 expression in 3T3-L1 adipocytes.

<p>(<b>A–E</b>) Real-time PCR measurement of levels of mRNA encoding (<b>A</b>) 11β-HSD1, (<b>B</b>) C/EBPβ, (<b>C</b>) CHOP, (<b>D</b>) calreticulin and (<b>E</b>) GRP78 in untreated differentiated 3T3-L1 adipocytes (white bars) or treated with tunicamycin (4 µg/ml, black bars) for 6 or 16 h. Data are normalized to TBP and expressed relative to levels of control cells (arbitrarily set to 100%). (<b>F</b>) Representative western blot (40 µg protein/lane) and quantification showing levels of C/EBPβ-LAP (38 kDa LAP* +35 kDa LAP) and -LIP (20 kDa) isoforms. Inset shows C/EBPβ-LIP:LAP ratio in untreated 3T3-L1 adipocytes (C, white bar) or treated with tunicamycin for 0.5 h (0.5 h, grey bar) or 2 h (2 h, black bar). Blots were stripped and reprobed with β-tubulin antibody, as loading control. All samples were analysed in the same gel but not in adjacent lanes. C/EBPβ levels were quantified relative to β-tubulin and are expressed in arbitrary units (AU). Values are mean±SEM of 2–3 independent adipocyte differentiations with each treatment tested in duplicate or triplicate. *, Significantly different from control. *, p≤0.05; **, p<0.001; ***, p<0.0001.</p

Sholl plots in stressed and non-stressed control and MR-tg mice.

<p>(A) The average dendritic length measured at specific distance points from the soma in all experimental groups. Analysis was based on the same cell groups as used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142012#pone.0142012.s002" target="_blank">S2 Fig</a>. Data are expressed as mean ± SEM. (B) Representative image of a CA3 pyramidal neuron analyzed at various bins representing the distance from the soma. We restricted the analysis to the apical dendrites only. Calibration bar: 20 μm.</p

Object in context.

<p>Left: schematic representation to indicate the setup of the object-in-context experiment. A) Mice were initially habituated in a context that had no object. B1) During training, mice were placed in the same context but with two identical objects and then placed in a novel context with two identical novel objects (B2). (C) Finally the mice were placed in the latter context but with one object being replaced by an object from the first context. (D) The discrimination index as observed from the object in context experiment. Data are expressed as mean ± SEM with p-values based on post-hoc LSD. n = 9 mice per group. *: significant, p ≤ 0.0125.</p

Neuroendocrinological parameters to determine the effectiveness of the CUS paradigm.

<p>These parameters included: (A) body weight gain (g), (B) thymus weight (mg / g body weight), (C) adrenals weight (mg / g body weight and (D) corticosterone levels. Data are expressed as mean ± SEM with p-values based on post-hoc LSD. n≥20 mice per group for A,B and C and n = 6–8 for D. *: significant, p ≤ 0.0125.</p

Hsd11b1^−/− peritoneal mast cells are hyper-responsive to degranulation induced by K/BxN serum.

<p>Release of β-hexosaminidase from peritoneal cells (2×10⁶) was measured following 15 min incubation with either (A) 10 µM ionomycin or (B) K/BxN serum (diluted 1∶8 or 1∶2 in Tyrode’s buffer). Black bars, <i>Hsd11b1^+/+</i>; white bars, <i>Hsd11b1^−/−</i>. Data are net degranulation in treated cells above levels measured in untreated cells (cells only are set to zero; see methods for details), and are mean ± SEM; n = 12−13, *p<0.05. (C) Representative micrographs (captured at 40× magnification) of enriched peritoneal CD117⁺ cells from <i>Hsd11b1^+/+</i> (+/+, left panels) or <i>Hsd11b1^−/−</i> mice (−/−, right panels) following 21 h incubation with buffer (top panels) or K/BxN serum (1∶8 dilution) (lower panels). Arrowheads indicate degranulated mast cells.</p

Expression of 11β-HSD1, C/EBPα, β, δ and CHOP in adipose tissue of HF diet-fed mice compared to control diet-fed mice.

<p>(A) Real-time PCR measurements of levels of mRNA encoding 11β-HSD1, C/EBPα, β, δ, and CHOP in adipose tissue of HF-fed (black bars) and control diet-fed (white bars) mice. Levels of mRNA, normalized to TBP, are expressed relative to levels in control diet-fed mice, arbitrarily set to 100%. (B-E) Representative western blots (50 µg protein/lane) and quantification of blots probed with; (B) C/EBPα antibody showing levels of the 42 kDa (p42) and 30 kDa (p30) isoforms of C/EBPα as well as total C/EBPα (p42+p30) levels (the intermediate immunoreactive protein indicated by * is likely to be a proteolytic product of p42); (C) C/EBPβ antibody showing levels of LAP (38 kDa LAP* +35 kDa LAP) and the 20 kDa LIP isoforms of C/EBPβ as well as total C/EBPβ (LAP+LIP) levels (inset shows LIP:LAP ratio); (D) C/EBPδ antibody and (E) CHOP antibody showing levels of respective proteins in adipose tissue of mice fed HF diet (HF, black bars) or control diet (C, white bars). Blots were stripped and reprobed with β-tubulin antibody, as loading control. In C/EBPβ and CHOP westerns, all samples were analysed in the same gel but not all in adjacent lanes. C/EBP levels were quantified relative to β-tubulin levels using ImageJ analysis and are expressed in arbitrary units (AU). All values are mean±SEM; n = 16/group (A) or 4/group (B-E). *, p≤0.05; **, p<0.001.</p

Inhibition of mTOR in 3T3-L1 adipocytes reduces C/EBPβ-LIP:LAP ratio and increases 11β-HSD1 expression.

<p>Real-time PCR measurement of levels of mRNA encoding (<b>A</b>) C/EBPβ and (<b>B</b>) 11β-HSD1 in 3T3-L1 adipocytes (white bars) or following treatment with 100 nM (grey bars) or 500 nM (black bars) rapamycin for 24 h. Data are normalized to TBP and expressed relative to levels of control cells (arbitrarily set to 100%). (<b>C</b>) Representative western blot (40 µg protein/lane) and quantification showing levels of C/EBPβ-LAP (38 kDa LAP* +35 kDa LAP) and -LIP (20 kDa) isoforms and inset showing C/EBPβ-LIP:LAP ratio in untreated 3T3-L1 adipocytes (C, white bars) or following treatment with 100 nM (100, grey bars) or 500 nM (500, black bars) rapamycin for 24 h. Blots were stripped and reprobed with β-tubulin antibody, as loading control. All samples were analysed in the same gel but not in adjacent lanes. C/EBPβ levels were quantified relative to β-tubulin and are expressed in arbitrary units (AU). Values are mean±SEM of up to 3 independent adipocyte differentiations, each treatment tested in triplicate. *, Significantly different from control. *, p≤0.05; **, p<0.001.</p

Altered C/EBPβ-LIP:LAP ratio <i>in vivo</i> affects 11β-HSD1 expression in adipose tissue.

<p>(<b>A</b>) Representative western blot (40 µg protein/lane) showing levels of C/EBPβ-LAP (38 kDa LAP* +35 kDa LAP) and -LIP (20 kDa) isoforms in the adipose tissue of C/EBPβ mutants C/EBPβ^(+/L) (+/L), C/EBPβ^ΔuORF (ΔuORF) and control (Con) mice. Blots were stripped and reprobed with β-tubulin antibody, as loading control. All samples were analysed in the same gel but not all in adjacent lanes. (<b>B</b>) Real-time PCR measurement levels of mRNA encoding 11β-HSD1 in adipose tissue of wild type control mice (Con, white bar), C/EBPβ^(+/L) (+/L, black bar) and C/EBPβ^ΔuORF (ΔuORF, grey bar). C/EBPβ^(+/L) mice are heterozygous for an allele of C/EBPβ in which the normal gene has been replaced by C/EBPβ-LIP (a “knock-in”) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037953#pone.0037953-Smink1" target="_blank">[31]</a> and C/EBPβ^ΔuORF is homozygous for the deletion of upstream ORF codon <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037953#pone.0037953-Wethmar1" target="_blank">[32]</a>. Adipose 11β-HSD1 mRNA levels, normalized to TBP, are expressed relative to levels in control mice (arbitrarily set to 100%) and are mean±SEM; n = 6–9/group. *, p≤0.05; **, p<0.001.</p

Decreased levels of glucocorticoid-sensitive transcripts in peritoneal cells from Hsd11b1^−/− mice.

<p>Real-time PCR measurement of (A) Carboxypeptidase A3 and (B) annexin 1 mRNA levels in total peritoneal cells from naïve male <i>Hsd11b1^−/−</i> (−/−; white bars) or <i>Hsd11b1^+/+</i> (+/+; black bars) mice. Data are mean ± SEM; n = 8, *p<0.05, **p<0.01. CPA; carboxypeptidase A3.</p