Data_Sheet_1_Prevalence and effect on prognosis of sarcopenia in patients with primary biliary cholangitis.docx

BackgroundSarcopenia adversely affects the treatment outcomes in Cirrhosis and NAFLD. However, such research is limited in primary biliary cholangitis (PBC) patients. This study was performed to examine the prevalence of sarcopenia and its impact on PBC patients’ prognoses.MethodsThis study enrolled confirmed PBC patients who had an abdominal CT scan. Sarcopenia was determined by the L3-skeletal muscle index with a Chinese population-based cut-off value. Laboratory test values and liver stiffness measurements values were obtained from the electronic medical records.ResultsIn total, 174 PBC patients with a median age of 54 (IQR, 48, 62) years old, were enrolled. 45 (25.9%) patients among them were diagnosed with sarcopenia. Univariate and multivariate logistic regression results illustrated that male gender (OR = 9.152, 95%CI = 3.131–26.751, p ConclusionThe current findings illustrate that comprehensive evaluation and management of sarcopenia may contribute to the improvement of treatment outcomes and life quality of PBC patients.</p

OTX2 regulates its own expression through the DHS 4 enhancer.

<p>(A) Luciferase assay for enhancer activity of DHS 4 in OTX2-expressing medulloblastoma cells treated with OTX2 siRNA. (B) Western blotting for ectopic and endogenous OTX2 following transfection of (B) OTX2-expressing or (C) OTX2-nonexpressing medulloblastoma cells with EGFP-tagged OTX2. (D) RT-qPCR demonstrating induction of the OTX2 target gene IL-6 in OTX2-nonexpressing medulloblastoma cells transfected with EGFP-tagged OTX2. (E) Chromatin immunoprecipitation of endogenous OTX2 in OTX2-expressing and -nonexpressing medulloblastoma cells. *<i>p</i><0.05 relative to scramble siRNA (A) or vector control (D), **<i>p</i><0.01 relative to scramble siRNA (A) or vector control (D), †p<0.05 relative to pGL3pro, ††p<0.01 relative to pGL3pro, Student's t-test. Error bars indicate standard deviation.</p

Validation of medulloblastoma DHS sites in a cohort of OTX2-expressing and -nonexpressing cells.

<p>Nuclei of medulloblastoma cell lines were treated with increasing concentrations of DNase (indicated as Units per reaction), and then the relative proportion of DNA remaining at the indicated regions were determined by qPCR. Open and solid markers indicate OTX2-expressing and -nonexpressing cell lines, respectively. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107156#pone.0107156.s002" target="_blank">Fig. S2</a> for detailed graphs.</p

DHS “R” mediates retinoic acid-induced OTX2 repression.

<p>(A) DNase sensitivity of DHS “R” in three OTX2-expressing (D283, D341, D721) and two OTX2-nonexpressing (D324, UW228) medulloblastoma cell lines. (B) Luciferase assays measuring enhancer activity of DHS “R” following treatment with 2 µM all-<i>trans</i> retinoic acid (ATRA) for 24 hours. (C) OTX2 mRNA level as determined by qPCR of medulloblastoma cells treated with 2 µM all-<i>trans</i> retinoic acid (ATRA) and/or 35 µM cycloheximide (CYHEX) for 8 hours. *<i>p</i><0.05 relative to DMSO, **<i>p</i><0.01 relative to DMSO, Student's t-test. Error bars indicate standard deviation.</p

Functional assessment of DHS sites.

<p>Luciferase assays measuring (A) orientation-sensitive promoter activity of proximal (<2 kb from the TSS) DHS sites in D283 cells, (B) enhancer activity of all DHS sites in D283 cells, (C) promoter activity of functionally-validated proximal DHS sites [underlined in (A)] in OTX2-expressing (D283, D341) and -nonexpressing (UW228) medulloblastoma cells, (D) enhancer activity of functionally-validated DHS sites [underlined in (B)] in OTX2-expressing (D283, D341) and -nonexpressing (UW228) cells, (E) insulator activity of DHS 3 in OTX2-expressing medulloblastoma cells, and (F) enhancer activity of minimal fragments and deletion mutants of DHS 4 in OTX2-expressing medulloblastoma cells. (G) Alignment of the critical Fragment A region of DHS 4 with the OTX2 position weight matrix <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107156#pone.0107156-Matys1" target="_blank">[28]</a>. *<i>p</i><0.05 relative to empty vector, **<i>p</i><0.01 relative to empty vector, †p<0.05 relative to DHS 1.pro, ††p<0.01 relative to DHS 1.pro, Student's t-test. Error bars indicate standard deviation.</p

Target genes regulated by RUNX3.

<p>a. The expression of cyclin D1, cyclin E, cdk2, cdk4, p-Rb, Rb, p27, MMP2, MMP9, TIMP-1 and TIMP-2 proteins were evaluated in 786-O-Ctrl and 786-O-RUNX3 cells by Western blot. b. The expression of cyclin D1, cyclin E, cdk2, cdk4, p-Rb, Rb, p27, MMP2, MMP9, TIMP-1 and TIMP-2 proteins were evaluated in HKC-Ctrl and HKC-siRUNX3 by Western blot. All examined gene expression levels quantitatively analyzed and expressed as the ratios over β-actin.</p

RUNX3 protein expression in CCRCC tissues and the matched noncancerous counterparts.

<p>a and b. Representative immunohistochemical photographs were taken at different magnifications in CCRCC tumor tissues and their matched noncancerous counterparts (×100 a1, ×200 a2, ×400 a3 and negative control a4 for noncancerous tissues; ×100 b1, ×200 b2, ×400 b3 and negative control b4 for tumor tissues). c. Expression protein levels of Runx3 in six CCRCC and the matched adjacent noncancerous tissues. d.Expression mRNA levels of RUNX3 in the CCRCC-derived cell lines and human kidney proximal tubular cell lines by real time RT-PCR. 18S was used as an internal control. ^*<i>P</i> <0.05 vs HKC cells. e. Expression protein levels of RUNX3 in the CCRCC-derived cell lines and human kidney proximal tubular cell lines by Western Blot. Tubulin was used as an internal control.</p

Effect of RUNX3 on tumorigenicity in nude mice and the cell-cycle analysis of 786-O cells.

<p>a. Average tumor weight was measurement of the excised tumors at the time of sacrifice. ^*<i>P</i> <0.05 vs 786-O-Ctrl cells. b. Average tumor size was estimated by physical measurement of the excised tumor at different time. ^*<i>P</i> <0.05 vs 786-O-Ctrl cells. d. 786-O-Ctrl cells and 786-O-RUNX3 cells were cultured in DMEM for 24 h. Cells were harvested and processed for FACS analysis.</p

Clinicopathological associations of RUNX3 expression in patients with CCRCC.

<p>Note: Interpretation of Runx3 staining was described in Section 2. Runx3 staining was graded as negative (−; score: 0–1), weak (+; score:2–4), and strong (++; score:5–8). Pearson's X² Test, ^**<i>P</i><0.001 vs non-cancerous tissues.</p