Mean body weight from 11–22 weeks of age.

<p>Autoimmune MRL/lpr mice were significantly lighter when compared to congenic controls. However, prolonged immunosuppression further reduced their body weight, but not in MRL +/+ mice.</p

Behavioural effects of chronic exposure to CY/sucrose solution.

<p>(A) Chronic immunosuppression reduced step-down latency in diseased MRL/lpr mice, but had the opposite effect on congenic controls. (B) In addition to the previously-documented substrain differences in sucrose consumption <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100684#pone.0100684-Sakic3" target="_blank">[24]</a>, CY had a significant detrimental effect on the performance in both groups. (C) Although a trend was noted, CY could not abolish previously-reported differences in overall floating in the forced swim test <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100684#pone.0100684-Sakic2" target="_blank">[23]</a>. (D) Prolonged immunosuppression improved spontaneous ambulatory activity in MRL/lpr mice, but had a detrimental effect on MRL +/+ controls. (E) Conversely, CY exposure significantly improved (but did not normalize) running wheel activity in MRL/lpr mice.</p

Daily fluid consumption from 11–22 weeks of age.

<p>(A) Despite fluctuations in daily water intake over the course of the study, Veh MRL +/+ mice consumed the most water in the 2-bottle test. They also drank significantly more than their CY +/+ counterparts, a trend that was not noted in the MRL/lpr substrain. (B) Lacing sweetened solution with CY significantly reduced daily consumption. However, autoimmune MRL/lpr mice consumed significantly less CY solution in comparison to MRL +/+ mice, particularly after the first week of treatment.</p

Weekly preference for sweet solutions.

<p>Initially, CY lpr mice displayed a higher preference for CY/sucrose in comparison to control CY +/+ group at 11 weeks of age. However, this difference was transient and became the lowest of all groups by the end of the study.</p

Weekly CY dose ingested.

<p>Although both substrains received similar CY dose at the starting of the treatment period, voluntary drinking in subsequent weeks was higher in less symptomatic MRL +/+ than in MRL/lpr mice.</p

The relative expression of PTEN, β-catenin and c-Jun in EHCC and their paired para-carcinoma tissues.

<p>(A, B, C) The mRNA expression level of PTEN, β-catenin and c-Jun in the primary EHCC and their paired para-carcinoma tissues determined by qRT-PCR. β-actin was used as the internal control (^★P<0.05, ^★★★P<0.001). (D) The inverse correlation of PTEN and miR-221 expression levels was examined by Spearman correlation analysis (R = -0.429, P = 0.032).</p

miR-221 expression in human EHCC tissues and CC cell lines, and the relationship between miR-221 expression and disease-free survival in EHCC patients.

<p>(A) The relative expression of miR-221 in 25 primary EHCC tissues compared to their matched normal adjacent bile duct tissues determined by qRT-PCR. U6 was used as the internal control (^★P<0.05). (B) The relative expression of miR-221 in CC cell lines (QBC939, HuCCT1, RBE and HCCC9810) compared to the HiBECs determined by qRT-PCR. U6 was used as the internal control (^★P<0.05, ^★★★P<0.001). (C) miR-221 predicts disease-free survival in EHCC patients. The Kaplan-Meier curve of disease-free survival in patients with low miR-221 expression (n = 14) and high miR-221 expression (n = 11). The median disease-free survival time was 22.00 months and 13.91 months in low- and high- miR-221 group respectively (P = 0.032).</p

PTEN is involved in miR-221 regulated EMT through β-catenin/cJun signaling pathway induced migration and invasion.

<p>(A) Both QBC939 and HuCCT1 cells were transfected with PTEN siRNAs then treated with or without miR-221 inhibitor to evaluate cell invasion and migration activities using a cell invasion assay in transwell chambers. Representative images of cells migrated into the lower chamber were shown (left panel, original magnification: X100), and quantitative data were also presented (right panel). (right panel, ^★★★P<0.001 indicates PTEN siRNAs, miR-221 inhibitor or a mixed PTEN siRNAs with miR-221 inhibitor <i>vs</i>. BC. ^$$$P<0.001 means a mixed PTEN siRNAs with miR-221 inhibitor <i>vs</i>. miR-221 inhibitor). The average numbers of cells per field of view from three different experiments are plotted. Negative control 1: mock control for siRNA containing only transfection reagent; Negative control 2: negative control for miR-221 inhibitor. (B) Morphological investigations of QBC939 and HuCCT1 cells. The cells were transfected with PTEN siRNAs then treated with or without miR-221 inhibitor. Cells transfected with siRNA containing only transfection reagent or scrambled oligonucleotide were used and named as Negative control 1 and Negative control 2 respectively (original magnification: ×200). (C) Both of the QBC939 and HuCCT1 cells were transfected with siRNA containing only transfection reagent, scrambled oligonucleotide, PTEN siRNAs and treated with or without miR-221 inhibitor and then the cells lysate were examined with indicated antibodies by Western blot. β-actin was used as loading control.</p

PTEN is a direct miR-221 target.

<p>(A) Putative miR-221 binding sequences in the 3’-UTR of PTEN mRNA. (B) 3’-UTR luciferase reporter assay showed a reduction of relative luciferase activity of wild-type PTEN 3’-UTR by pre-miR-221 in QBC939 and HuCCT1 cells (^★P<0.05). (C) qRT-PCR analysis of expression of miR-221 treated with miR-221 inhibitor in QBC939 and HuCCT1 cells (^★★★P<0.001). U6 was used as an internal control. (D) Western blot analysis of PTEN expression transfected with miR-221 inhibitor in QBC939 and HuCCT1 cells. β-actin expression levels were used as internal loading control.</p

β-catenin/c-Jun signaling pathway promotes miR-221 expression.

<p>(A) PTEN protein level with c-Jun siRNA or BIO treatments detected by western blot. Antibodies include: PTEN and β-actin. Negative control 1: mock control for siRNA containing only transfection reagent; Negative control 2: mock control for BIO with only 0.1% DMSO. (B) Both of the QBC939 and HuCCT1 cells were transfected c-Jun siRNAs and treated with or without BIO for desired time. Then the relative expression of miR-221 was determined by qRT-PCR. U6 was used as the internal control. (^★★★P<0.001 indicates c-Jun siRNAs, BIO or a mixed c-Jun siRNAs with BIO <i>vs</i>. NC. ^$$$P<0.001 means a mixed c-Jun siRNAs with BIO <i>vs</i>. BIO)</p