Additional file 1: Figure S1. of Invasion of ovarian cancer cells is induced byPITX2-mediated activation of TGF-β and Activin-A

The expression of PITX2 is up-regulated in human ovarian cancer. (A) The level of PITX2 was observed by IHC in human ovarian tissue-sections with specific antibody followed by Alexa Fluor-488 (green) of low malignant potential (i; n = 20) and high malignant potential metastatic adenocarcinoma patients (ii; n = 20). The DAPI-stained nuclei and the merged images were also shown. Scale bar, 10 μm. (TIFF 28495 kb

Additional file 1 of Toe brachial index and not ankle brachial index is appropriate in initial evaluation of peripheral arterial disease in type 2 diabetes

Additional file 1: Table S1. Correlation of ABI and TBI across various ranges of ABI

In streptozotocin induced diabetic mice dmp acts like insulin.

<p>dmp activates insulin signaling in STZ mice. STZ induced BALB/c mice were starved for 12 h followed by oral administration of dmp (300μg kg^-1 bw) or vehicle. (A) Blood glucose level was detected at different times. *<i>p</i>< 0.05 <i>versus</i> STZ (vehicle), **<i>p</i>< 0.01 <i>versus</i> STZ (vehicle). (B) 4 h after dmp treatment, skeletal muscle tissues were collected from BALB/c mice (Con) or STZ mice or STZ mice fed with dmp, lysed and subjected to immunoblot using anti-pIR and anti-IR antibodies. (C) [¹⁴C] 2-DOG uptake (top) and [³H] Fatty acid uptake (bottom) by skeletal muscle or adipose tissue from above mentioned mice were determined in a liquid scintillation counter. *<i>P</i><0.05 <i>versus</i> Con; #<i>P</i><0.05 <i>versus</i> STZ (vehicle). (D) Skeletal muscle from STZ mice was incubated with insulin (100 nM) or dmp (250 nM) and [¹⁴C] 2-DOG uptake was measured at different time intervals. *<i>P</i><0.05 or **<i>P</i><0.01 <i>versus</i> STZ (vehicle). (E) dmp was orally administered to BL6 mice (5mg kg^-1 bw) and plasma dmp level was measured at different time intervals. All values are represented as mean ± s.e.m. (n = 5).</p

Structure of dmp.

<p>(A) FT-IR of DmpzH[VO(O2)2(dmpz)]. The symmetric O-O stretch, the symmetric metal-peroxo stretch, and the asymmetric metal-peroxo stretch, and occurred ca. 880, 600 and 500 cm^-1, respectively. (B) Raman spectra of DmpzH[VO(O2)2(dmpz)]. The spectra shows strong bands at ~988.66, ~881.00, ~599.30, ~542.43 cm^-1 which have been assigned to nV = O, nO-O (n1), nV-O2 (n3) and nV-O2 (n2) modes, respectively. (C) ORTEP plot with 35% probability ellipsoid of the anion of [VO(O2)2dmpz]–and dmpzH+ cation, with selected bond distances (Å): V1−O1, 1.5919 (1); V1−O2, 1.861(1); V1−O3, 1.853(1); V1−O4, 1.894(1);V1−O6, 1.1.588(1); V1−N1, 2.104(1); O1−O2, 1.480(2); O4−O3, 1.461(2); N1−N2, 1.360(2); N1−C3, 1.335(2); N2−C1,1.341(2).</p

Interaction between dmp and IR.

<p>(A) Binding of recombinant insulin with insulin receptor (IR) was studied by Surface Plasmon Resonance (SPR) using varied concentrations of insulin. (B) Binding of dmp to IR was studied by SPR where increasing concentrations of dmp from 1–50 μM were flowed over immobilized IR protein on CM5 chips. Binding affinity of dmp to IR is represented by KD value (1.17 μM). Rmax value is 8.880 (RU) which represents maximum binding capacity of dmp with IR. (C) Fluorescence spectra of IR-dmp. All steady-state fluorescence measurements were carried out using an excitation wavelength of 280 nm. The emission spectra were traced from 300 to 500 nm. The concentration of IR was 0.3μM whereas varied concentrations of dmp was used for fluorescence spectra, it was gradually increased from 0–0.6μM. Binding constant was calculated from Stern–Volmer equation Io/I = 1+Ksv [Q] [dmp]. Quenching constant was Xhalf = 1.0118 μM calculated accordingly where Io and I are fluorescence intensities in the absence or presence of the quencher (dmp) respectively and Ksv was quenching constant.</p

dmp induces expression of Ppar-γ and its target genes through the suppression of Wnt3a.

<p><i>In vitro</i> incubation of 3T3L1 cells were conducted in the absence (control) or presence of 250 nM dmp along with 0.5mM palmitate for 4 h. (A) RNA was extracted from cells and Ppar-γ1 and Ppar-γ2 mRNA level was measured by quantitative PCR. *<i>P</i><0.001 <i>versus</i> Con (B) dmp effects a dose dependent increase in PPARϒ expression. The cell lysates were used for immunoblotting with anti-PPARϒ antibody or anti-α tubulin antibody for loading control. (C) Wnt3a gene (top) and protein (bottom) expression levels were estimated. *<i>P</i><0.01 <i>versus</i> Con. (D) mRNA expressions of Wnt target genes, axin2 and wisp2 were estimated. *<i>P</i><0.01 <i>versus</i> Con. (E) dmp effect on mRNA expression of PPARϒ target genes Adpn, CD36 and aP2 in 3T3L1 adipocytes was observed. *<i>P</i><0.01 <i>versus</i> Con. (F) 3T3L1 adipocytes were incubated with 100 nM insulin (I) in the absence or presence of insulin receptor tyrosine kinase inhibitor HNMPA-(AM)3 and pIR protein levels are estimated by immunobloting. (G) Wnt3a, PPARϒ2 and Adpn protein levels were estimated in primary mice adipocytes from SD and HFD mice treated with or without dmp (250 nM) or dmp+HNMPA-(AM)3 (100 μM) (left). 3T3L1 adipocytes were preincubated with HNMPA-(AM)3 for 1 h followed by addition of 0.5mM palmitate in the absence or presence of dmp for 4 h to determine Wnt3a, PPARϒ2 and Adpn protein levels through immunoblot analysis. All values are represented as mean ± s.e.m. (n = 5).</p

dmp binding to IR augments insulin signalling pathway.

<p>(A) dmp fails to activate EGFR. L6 myotubes or 3T3L1 adipocytes were treated with or without 250 nM dmp for 4h. The cell lysates were analyzed by immunoblotting with anti-pEGFR and anti-EGFR antibodies. (B,C) dmp can induce IR phosphorylation in a dose dependent manner. L6 myotubes were treated with insulin (20–120 nm) or dmp (50–300 nm) for 4h and IR phosphorylation was monitored by ELISA (B) or immunoblotting with anti-pIR and anti-IR antibodies (C). (D) IR kinase activity was determined in L6 myotubes which were incubated with varied concentrations of insulin or dmp. (E) dmp stimulates IR and its downstream kinases phosphorylation. L6 myotubes or 3T3L1 adipocytes were treated with or without Insulin (100 nM) or dmp (250 nM) for 4h and the IR phosphorylation and its downstream signalling were monitored by immunoblotting. (F) L6 myotubes transfected with GFP-GLUT4 chimeric gene were incubated with insulin (100 nM) or dmp (250nM) for 4h. Cells on the cover slips were fixed in paraformaldehyde and observed under florescent microscope for GFP-GLUT4 translocation. (G) dmp like insulin promotes glucose uptake. L6 myotubes or skeletal muscle cells from soleus muscle of neonatal mice (2-3days) were incubated with 100 nm insulin or 250 nm dmp for 25 min. [¹⁴C] 2-DOG was then added, and the cells were further incubated for 5 min. [¹⁴C] 2-DOG uptake was measured by scintillation counting. *<i>P</i><0.05 <i>versus</i> Con; **<i>P</i><0.01 <i>versus</i> Con. (H) dmp augments fatty acid uptake. Primary culture adipocytes or 3T3L1 adipocytes were treated with 100 nm insulin or 250 nm dmp for 4h followed by incubation with [³H] Palmitate for 15 min. [³H] Palmitate uptake was measured in a liquid scintillation counter. *<i>P</i><0.05 <i>versus</i> Con; **<i>P</i><0.01 <i>versus</i> Con. (I) L6 myotubes were tranfected with IR siRNA(IR^KD) followed by estimation of IR gene and protein levels by qPCR (left) and immunoblotting (right) respectively. *<i>P</i><0.05 <i>versus</i> Con. (J) IR^KD L6 myotubes were incubated with dmp for 4h and [¹⁴C] 2-DOG uptake was measured according to the above description. **<i>P</i><0.01 <i>versus</i> Con. All values are represented as mean ± s.e.m. (n = 5).</p

dmp improves energy homeostasis in <i>db/db</i> mice.

<p>(A) BL6 mice were orally administrated with vehicle or dmp (300 μg kg^-1 bw) for 28 days. Body weight was recorded on the days mentioned in the figure. (B) <i>db/db</i> mice were orally administered with vehicle or dmp (300 μg kg^-1 bw) for 28 days. Weight of the abdominal fat was recorded. (C) Food intake was estimated in <i>db/db</i> (vechile) and dmp fed <i>db/db</i> mice. (D) Metabolic activities were measured by indirect colorimetry in BL6, <i>db/db</i> (vechile) and dmp fed <i>db/db</i> mice during day and night periods. The experimental animals were placed in metabolic cage and average hourly oxygen consumption (V̇O₂) and carbon dioxide production (VCO₂) were measured. Accordingly, RER and energy expenditure (EE) were calculated. *<i>P</i><0.05 <i>versus</i> BL6; #<i>P</i><0.05 <i>versus db/db</i> (vechile). (E) Western blot showing phosphorylation status of pAMPK and AMPK in the skeletal muscle of BL6, <i>db/db</i> (vechile) and dmp fed <i>db/db</i> mice. (F) Immunoblots showing abundance of PGC1α, NRF1 and tfam level in dmp treated mice. (G) Total DNA was extracted from muscle tissue and the content of mtDNA was calculated using real-time quantitative PCR by measuring the threshold cycle ratio (㥆Ct) of a mitochondrial-encoded gene COXII versus a nuclear encoded gene RIP140. *<i>P</i><0.05 <i>versus</i> BL6; #<i>P</i><0.05 <i>versus db/db</i> (vechile). (H) Complex IV activity (top) and ATP production (bottom) was measured in mitochondria isolated from skeletal muscle of BL6, <i>db/db</i> (vehicle) and dmp fed <i>db/db</i> mice. *<i>P</i><0.05 <i>versus</i> BL6; #<i>P</i><0.05 <i>versus db/db</i> (vechile). (I) <i>db/db</i> mice were orally administrated with vehicle or dmp (300 μg kg bw^-1) for 28 days. Blood glucose concentration (GTT) was measured before and after oral gavages of 1g glucose kg bw^-1 at the indicated time points. ITT was performed after injecting mice with 0.7U insulin kg bw^-1. Fasting insulin and fasting glucose was estimated and HOMA-IR was calculated. #<i>P</i><0.05 <i>versus db/db</i> (vechile). All values are represented as mean ± s.e.m. (n = 5).</p

Toxicity test of dmp.

<p>Rats were fed with dmp (5 mg kg bw^-1) for 14 days. (A) Hematological, (B) Biochemical analysis, (C) Liver and kidney histopathology were performed on 0 and 14 day to test toxic effect of dmp. (D) L6 myotubes and 3T3L1 adipocytes were incubated with 300 nM of dmp for 4 h and cell viability was assessed by MTT assay. (E) After incubation of 3T3L1 adipocytes with or without 300 nM dmp for 4 h, release of TNFα and IL6 in to the medium was estimated through ELISA. (F) ATP production was measured in L6 myotubes incubated in the absence or presence of 300nM dmp for 4 h. All values are represented as mean ± s.e.m. (n = 5)</p