Free cholesterol induction of ER stress in adipocytes.

<p>Fully differentiated 3T3-L1 cells were incubated in the medium containing various concentration of ox-LDL (0-100µg/ml) with Sandoz 58035 for 48 hrs (A). Lane 1: control; lane 2: ox-LDL at 25μg/ml; lane 3: ox-LDL at 50μg/ml; lane 4: ox-LDL at 75μg/ml; lane 5: ox-LDL at 100μg/ml; Adipocytes were incubated with ox-LDL (50µg/ml) in a combination of Sandoz58035 for indicated times (0-48hrs) (B). Lane 1: control; lane 2: ox-LDL for 6h; lane 3: ox-LDL for 18h; lane 4: ox-LDL for 36h; lane 5: ox-LDL for 48h. GRP78 and CHOP expression was assessed by Western Blot andβ-actin was used as the housekeeping gene for normalization. Date is expressed as mean ±S.D from at least three independent determinations. * P<0.05, as compared with control; # P<0.01, as compared with control.</p

Chemical Chaperone Treatment Protects cells from secretion dysfunction.

<p>Differentiated 3T3-L1 adipocytes were incubated in the medium with ox-LDL(50µg/ml) ，Sandoz58035 and different concentration of TUDCA (0-400µM) for 48hrs. GRP78 and CHOP expression was determined by Western Blot and β-actin was used as the housekeeping gene for normalization(A). Lane 1: control; lane 2: TUDCA at 100µM; lane 3: TUDCA at 200µM; lane 4: TUDCA at 400µM. The mRNA expressions of resistin and visfatin were evaluated by real-time PCR (B).Resistin and visfatin release from 3T3-L1 adipocytes into supernatant medium were analyzed by ELISA (C). * P<0.05, as compared with control; # P<0.01, as compared with control.</p

Proliferation of tumor cells in three-dimensional type I collagen gel in the presence of FH535.

<p>Cells (HCC38, MDA-MB-231, T47D, Sk-Br3, MCF-7) were cultured in type I collagen gel as described in material and methods. Cell/gel matrices were fixed and embedded in paraffin. The paraffin section was serially cut at 5 m and mounted on slide glasses. Tissue sections were stained with anti-Ki67 antibody or control IgG followed by HRP-conjugated secondary antibody as visualizing by DAB staining with counter staining by DAPI to localize cells. The results were demonstrated by the (mean +/− standard deviation) of % of positive cells for DAB from three independent experiments. Insets showed the typical staining pattern of anti-Ki67 antibody in HCC38 and MDA-MB-231 cells in the presence or absence of FH535 at a concentration of 10 µM. Magnification x200. * <i>p</i><0.001 (by Student’s two-tailed paired <i>t</i>-test). n.s: not significance (by Student’s two-tailed paired <i>t</i>-test).</p

FH535 inhibited migration of MDA-MB231 and HCC38 cells to type I collagen.

<p>Cells were harvested, washed, and resuspended in RPMI-serum free media at a concentration of 5×10⁵ cells/ml. Type I collagen was used as a chemoattractant at a concentration of 3 g/ml. FH535 (0.01–1 µM) were added in both cell suspension and type I collagen solution and incubated for 4 hours at 37°C. Migrated cells were manually counted and expressed as mean +/− S.D. Experiments were repeated three times. * <i>p</i><0.001, ** <i>p</i><0.05 (by Student’s two-tailed paired <i>t</i>-test).</p

Gene expression profile of CSPG4 in breast cancer tissues.

<p>Gene expression data were retrieved from The Caner Genome Atlas (TCGA) project data portal (<a href="https://tcga-data.nci.nih.gov/" target="_blank">https://tcga-data.nci.nih.gov/</a>). * <i>p</i><0.001 (by ANOVA).</p

FH535 inhibited invasion of MDA-MB-231 and HCC38 cells into matrigel.

<p>Filters were coated with Matrigel. FH535 (0.01–1 µM) were added in both cell suspension and type I collagen solution and incubated overnight at 37°C. Antibodies (1 µg/ml) were incubated with cells. Migrated cells were manually counted and expressed as mean +/− S.D. Experiments were repeated three times. * <i>p</i><0.001, ** <i>p</i><0.05 (by Student’s two-tailed paired <i>t</i>-test).</p

FH535 inhibited expression of NEDD9 in MDA-MB-231 cells.

<p>(A) MDA-MB-231 or HCC38 cells were cultured in the presence or absence of FH535 (1 µM) overnight at 37°C. Cells were directly lysed in SDS sample buffer and separated on SDS-PAGE followed by transferred on to membranes. Membranes were blotted with anti-NEDD9, anti-FAK, anti-phospho FAK, anti-phospho Src, anti-Src, anti-phospho Erk1/2, anti-Erk1/2, anti-phospho p38, and anti-p38 antibodies. (B) MD-MB-231 cells were cultured as described above. Cells were directly lysed in 100 mM Tris-HCl (pH7.4) containing 1% Brij35, 0.14 M NaCl, 1 mM CaCl₂, 1 mM MgCl₂, 1 mM MnCl₂, and protease inhibitor cocktails. Cell lysates were precleared and immunoprecipitated with control, anti-β1, anti-CD44, or anti-CSPG antibodies for 4 hours. Bound proteins were released, separated, and transferred onto membranes. Membranes were blotted with anti-NEDD9 antibody.</p

Effect of FH535 on the expression of β-catenin and axin in MDA-MB-231 and HCC38 cells.

<p>Cells were cultured on type I collagen for 4 hours in the presence or absence of FH535 (1 µM) at 37°C. Cells were directly lysed in SDS-sample buffer. Proteins were separated on SDS-PAGE followed by transferred onto Immobilon-P membranes. Membranes were blotted with anti- β-catenin or anti-axin antibody. The membranes were then striped and blotted with anti-actin antibody as a loading control.</p

Positive Association of Fibroadenomatoid Change with HER2-Negative Invasive Breast Cancer: A Co-Occurrence Study

<div><p>Background</p><p>Risk assessment of a benign breast disease/lesion (BBD) for invasive breast cancer (IBC) is typically done through a longitudinal study. For an infrequently-reported BBD, the shortage of occurrence data alone is a limiting factor to conducting such a study. Here we present an approach based on co-occurrence analysis, to help address this issue. We focus on fibroadenomatoid change (FAC), an under-studied BBD, as our preliminary analysis has suggested its previously unknown significant co-occurrence with IBC.</p><p>Methods</p><p>A cohort of 1667 female patients enrolled in the Clinical Breast Care Project was identified. A single experienced breast pathologist reviewed all pathology slides for each case and recorded all observed lesions, including FAC. Fibroadenoma (FA) was studied for comparison since FAC had been speculated to be an immature FA. FA and Fibrocystic Changes (FCC) were used for method validation since they have been comprehensively studied. Six common IBC and BBD risk/protective factors were also studied. Co-occurrence analyses were performed using logistic regression models.</p><p>Results</p><p>Common risk/protective factors were associated with FA, FCC, and IBC in ways consistent with the literature in general, and they were associated with FAC, FA, and FCC in distinct patterns. Age was associated with FAC in a bell-shape curve so that middle-aged women were more likely to have FAC. We report for the first time that FAC is positively associated with IBC with odds ratio (OR) depending on BMI (OR = 6.78, 95%CI = 3.43-13.42 at BMI<25 kg/m²; OR = 2.13, 95%CI = 1.20-3.80 at BMI>25 kg/m²). This association is only significant with HER2-negative IBC subtypes.</p><p>Conclusions</p><p>We conclude that FAC is a candidate risk factor for HER2-negative IBCs, and it is a distinct disease from FA. Co-occurrence analysis can be used for initial assessment of the risk for IBC from a BBD, which is vital to the study of infrequently-reported BBDs.</p></div

Characteristics of IBC cases/controls in relationship to BBDs and risk/protective factors.

<p>*UN, Unknown, not used in Chi-square test.</p><p>Abbreviations: IBC = Invasive Breast Cancer; BBDs = Benign Breast Diseases; FA = Fibroadenoma; FAC = Fibroadenomatoid Change; FCC = Fibrocystic Changes; Num. = Number; Race, AA = African American, CA = Caucasian American; Current OC use = Current oral contraceptive use; BMI = Body Mass Index; HRT = Hormonal replacement therapy; Combo = Estrogen & Progesterone.</p><p>Characteristics of IBC cases/controls in relationship to BBDs and risk/protective factors.</p