A pan-JAK inhibitor prevents the decrease in AQP1 expression following treatment with IFNγ.

<p>(A) Immunoblots showing expression of phosphorylated and total STAT1, phosphorylated and total STAT3, AQP1 and actin in CMT93 cells pretreated with a pan-JAK inhibitor (20 μM for 2 hr) or DMSO vehicle followed by treatment with or without IFNγ (10 ng/mL for 24 hr). (B) Densitometry graph of phospho-STAT1 normalized to total (t) STAT1 expression. (C,D) Densitometry graph of phospho-STAT3α and phospho-STAT3β each normalized to total (t) STAT3 expression. (E) Densitometry graph of AQP1 normalized to actin expression. *p < 0.05, **p < 0.01, ***p < 0.001 vs. DMSO + IFNγ; ^$$$p < 0.001 vs. DMSO alone and pan-JAK Inhibitor alone; ^#p < 0.05 vs. DMSO alone. Blots are representative of 4 separate experiments.</p

Exposure of CMT93 cells to 10 ng/mL IFNγ leads to a time-dependent reduction in AQP1 (A) mRNA and (B) protein expression compared to untreated control cells; *p < 0.05, **p < 0.01, n = 3 for each experiment.

<p>Exposure of CMT93 cells to 10 ng/mL IFNγ leads to a time-dependent reduction in AQP1 (A) mRNA and (B) protein expression compared to untreated control cells; *p < 0.05, **p < 0.01, n = 3 for each experiment.</p

Epithelial AQP1 expression is decreased following treatment of CMT93 epithelial cells with IFNγ.

<p>Confocal immunocytochemistry was performed to detect AQP1 in CMT93 cells treated with either vehicle or IFNγ (10 ng/mL for 24 hr). Constitutive AQP1 immunoreactivity was observed apically and laterally in control cells, which co-localized with E-cadherin. An overall decrease in AQP1 expression in the cell monolayers was observed after treatment with IFNγ. Much of the remaining AQP1 appeared to be re-localized from cell membranes to vesicular cytosolic structures. Images are representative of 4 monolayers per group.</p

Interferon-γ Suppresses Intestinal Epithelial Aquaporin-1 Expression via Janus Kinase and STAT3 Activation - Table 3

<p>Interferon-γ Suppresses Intestinal Epithelial Aquaporin-1 Expression via Janus Kinase and STAT3 Activation - Table 3 </p

IRF-2 appears to regulate baseline AQP1 expression and is not involved in the effects of IFNγ on AQP1 expression.

<p>(A) Immunoblots of IRF-2, AQP1 and actin levels in CMT93 cells pretreated with IRF-2 or scrambled siRNA (30 nM for 48 hr) or with transfection medium alone (Lipofectamine Control) in the presence or absence of IFNγ (10 ng/mL for 24 hr) (B) Densitometry graph of total IRF-2 normalized to actin expression. (C) Densitometry graph of AQP1 normalized to actin expression. ^#p < 0.05, ^###p < 0.001 vs. scrambled siRNA + media; **p < 0.01 vs. scrambled siRNA + IFNγ; blots are representative of 3 separate experiments.</p

Knockdown of STAT3 partially restores the IFNγ-induced decrease in AQP1 expression.

<p>(A) Immunoblots of phospho-STAT3, total STAT3, AQP1 and actin levels in CMT93 cells pretreated with STAT3 or scrambled siRNA (80 nM for 24 hr) or with transfection medium alone (Lipofectamine Control) in the presence or absence of IFNγ (10 ng/mL for 24 hr) (B) Densitometry graph of total STAT3 normalized to actin expression. (C) Densitometry graph of AQP1 normalized to actin expression. **p < 0.01 vs. scrambled siRNA + media; ^##p < 0.01 vs. scrambled siRNA + IFNγ blots are representative of 3 separate experiments.</p

Treatment of CMT93 cells with IFNγ leads to a decrease in IRF-2 protein expression.

<p>The same samples used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118713#pone.0118713.g006" target="_blank">Fig. 6</a> were run for these immunoblots. (A) Representative blots of CMT93 cells pretreated with STAT3 siRNA (80 nM, 24 hrs) and then IFNγ (10 ng/mL, 24 hrs) (B) Densitometry graph of total IRF-2 normalized to actin expression; blots are representative of 3 separate experiments.</p

Schematic of proposed mechanism of IFNγ-induced suppression of AQP-1.

<p>While IFNγ activates both STAT1 and STAT3 in a JAK-dependent manner, only activation of STAT3 plays a role in the reduced expression of AQP1. IRF2 may play a role in the constitutive expression of AQP1. IFNγ suppresses IRF2, but this does not appear to play a role in reduced AQP1 expression.</p