Additional file 1: Figure S1. of Functional role of ALK-related signal cascades on modulation of epithelial-mesenchymal transition and apoptosis in uterine carcinosarcoma

(A) ChIP assay shows that N-myc is bound to the proximal region (−126 to +12 bp) of the ALK promoter. (B) The ALK promoter sequence containing two putative E-boxes (E1 and E2). (C) Various promoter constructs were used for evaluating transcriptional regulation of the ALK promoter by N-myc. Relative activity was determined based on arbitrary light units of luciferase activity normalized to pRL-TK activity. The activities of the reporter plus the effector relative to that of the reporter plus empty vector are shown as means ± SDs. The experiment was performed in duplicate. (TIF 725 kb

Additional file 2: Figure S2. of Functional role of ALK-related signal cascades on modulation of epithelial-mesenchymal transition and apoptosis in uterine carcinosarcoma

(A) Two independent Hec251 cell lines stably overexpressing ALK (H251-ALK#8 and #16) and mock cells were seeded at low density and monitored for growth. The cell numbers presented are means ± SDs. P0, P3, P5, and P7: 0, 3,5, and 7 days after passage. (B) Western blot analysis of expression of cyclin A, p21waf1, and p27kip1 at P6 of cell growth in stable ALK-overexpressing cell lines. (C) The pNF-κB reporter construct was transfected into H251-ALK#16 cells treated with 2.5 ng/ml TGF-β1 or 50 ng/ml HGF for 48 h. Relative activity was determined based on arbitrary light units of luciferase activity normalized to pRL-TK activity. The activities of the reporter plus the effector relative to that of the reporter plus empty vector are shown as means ± SDs. The experiment was performed in duplicate. (D) Various promoter constructs were used for evaluating transcriptional regulation of the ALK promoter by TNF-α. (E) The pNF-κB reporter construct, together with the ALK expression vector, were transfected into Ishikawa cells. (TIF 843 kb

Imatinib inhibits vascular smooth muscle proteoglycan synthesis and reduces LDL binding in vitro and aortic lipid deposition in vivo

The 'response to retention' hypothesis of atherogenesis proposes that proteoglycans bind and retain low-density lipoproteins (LDL) in the vessel wall. Platelet-derived growth factor (PDGF) is strongly implicated in atherosclerosis and stimulates proteoglycan synthesis. Here we investigated the action of the PDGF receptor inhibitor imatinib on PDGF-mediated proteoglycan biosynthesis in vitro, lipid deposition in the aortic wall in vivo and the carotid artery ex vivo. In human vSMCs, imatinib inhibited PDGF mediated 35S-SO 4 incorporation into proteoglycans by 31% (P < 0.01) and inhibited PDGF-mediated size increases in both chemically cleaved and xyloside associated glycosaminoglycan (GAG) chains by 19%, P < 0.05 and 27%, P < 0.05, respectively. Imatinib decreased PDGF stimulation of the 6:4 position sulphation ratio of disaccharides. The half maximal saturation value for LDL binding for proteoglycans from PDGF stimulated cells in the presence of imatinib was approximately 2.5-fold higher than for PDGF treatment alone. In high fat fed ApoE -/- mice, imatinib reduced total lipid staining area by ∼31% (P < 0.05). Carotid artery lipid accumulation in imatinib treated mice was also reduced. Furthermore, we demonstrate that imatinib inhibits phosphorylation of tyrosine 857, the autophosphorylation site of the PDGF receptor, in vSMCs. Thus imatinib inhibits GAG synthesis on vascular proteoglycans and reduces LDL binding in vitro and in vivo and this effect is mediated via the PDGF receptor. These findings validate a novel mechanism to prevent cardiac disease. © 2009 The Authors Journal compilatio

Abnormal Trabecular Bone Score, Lower Bone Mineral Density and Lean Mass in Young Women With Premature Ovarian Insufficiency Are Prevented by Oestrogen Replacement

Background: Low bone density (BMD) and fractures commonly affect women with premature ovarian insufficiency (POI). However, bone microarchitecture and body composition data are lacking. Objective: To assess and characterise musculoskeletal phenotype and effects of oestrogen replacement therapy (ERT) in women with POI. Method: Cross-sectional and longitudinal studies of 60 normal karyotype women with POI, aged 20-40 years, from 2005-2018. Dual x-ray absorptiometry (DXA)-derived spinal (LS) and femoral neck (FN) BMD, trabecular bone score (TBS), appendicular lean mass (ALM), total fat mass (TFM), and fracture prevalence were compared with 60 age-, and BMI-matched population-based controls. Longitudinal changes in bone and body composition variables and ERT effects were analysed using linear mixed models over a median duration of 6 years. Results: Women with POI were subdivided into spontaneous (s)-POI (n=25) and iatrogenic (i)-POI (n=35). Median(range) age of POI diagnosis was 34 (10-40) years with baseline DXA performed at median 1(0-13) year post-diagnosis. ERT was used by 82% women (similar for both POI groups). FN-BMD were lowest in s-POI (p<0.002). Low TBS was more common in s-POI [(44%), p=0.03], versus other groups. LS-BMD and ALM were lower in both s-POI and i-POI groups than controls (p<0.05). Fracture prevalence was not significantly different: 20% (s-POI), 17% (i-POI), and 8% (controls) (p=0.26). Longitudinal analysis of 23 POI women showed regular ERT was associated with ALM increment of 127.05 g/year (p<0.001) and protected against bone loss. However, ERT interruption was associated with annual reductions in FN BMD and TBS of 0.020g/cm2 and 0.0070 (p<0.05), respectively. Conclusion: Deficits in BMD, trabecular microarchitecture, and lean mass were present in women with POI. However, regular ERT protected against declines in bone variables, with an increase in ALM. Assessment of skeletal and muscle health, and advocating ERT adherence, is essential in POI to optimise musculoskeletal outcomes