Effect of YM-08 on the PGE₁-stimulated IL-6 release in MC3T3-E1 cells.

The cultured cells were pretreated with 10 μM of YM-08 for 60 min and then stimulated with 10 μM of PGE1 or vehicle for 48 h. IL-6 concentrations of the culture medium were determined by ELISA. Each value represents the mean ± SEM of triplicate determinations from three independent cell preparations. *p †p 1 alone.</p

Full-length gels and blots of Fig 4.

(PDF)</p

Effects of YM-08 on the PGE₁-stimulated phosphorylation of p38 MAPK in MC3T3-E1 cells.

The cultured cells were pretreated with 10 μM of YM-08 for 60 min and then stimulated with 10 μM of PGE1 or vehicle for 20 min. The cell extracts were then subjected to SDS-PAGE and subsequent Western blot analysis with antibodies against phospho-specific p38 MAPK, p38 MAPK, and actin. The histogram quantitatively represents the PGE1-induced levels obtained from laser densitometric analysis of three independent experiments. Each value represents the mean ± SEM of triplicate determinations from three independent cell preparations. *p †p 1 alone.</p

Effects of PD98059, SP600125 or SB203580 on the PGE₁-stimulated release of IL-6 in MC3T3-E1 cells.

Effects of PD98059, SP600125 or SB203580 on the PGE1-stimulated release of IL-6 in MC3T3-E1 cells.</p

Effects of SB203580 on YM-08’s amplification of PGE₁-stimulated IL-6 release in MC3T3-E1 cells.

The cultured cells were preincubated with 30 μM of SB203580 or vehicle for 60 min, subsequently pretreated with 10 μM of YM-08 or vehicle for 60 min, and then stimulated with 10 μM of PGE1 or vehicle for 48 h. IL-6 concentrations of the conditioned media were determined by ELISA. Each value represents the mean ± SEM of triplicate determinations from three independent cell preparations. *p †p 1 alone. ‡p 1 with YM-08 pretreatment.</p

Effect of VER-155008 on the PGE₁-induced expression levels of IL-6 mRNA in MC3T3-E1 cells.

The cultured cells were pretreated with 10 μM of VER-155008 for 60 min and then stimulated with 10 μM of PGE1 or vehicle for 2 h. The respective total RNA was then isolated and quantified by real-time RT-PCR. Each IL-6 mRNA value was divided by that of GAPDH mRNA (Vehicle: 0.007 ± 0.001; PGE1: 0.412 ± 0.047; VER-155008: 1.317 ± 0.138; PGE1+VER-155008: 3.339 ± 0.287). Each value represents the mean ± SEM of triplicate determinations from three independent cell preparations. *p †p 1 alone.</p

Effect of VER-155008 on the PGE₁-stimulated IL-6 release in MC3T3-E1 cells.

(A) Cultured cells were pretreated with 30 μM of VER-155008 (□,■) or vehicle (○,●) for 60 min and then stimulated with 10 μM of PGE1 (●, ■) or vehicle (○, □) for the indicated periods. (B) Cultured cells were pretreated with various doses of VER-155008 for 60 min and then stimulated with 10 μM of PGE1 (●) or vehicle (○) for 48 h. IL-6 concentrations of the culture medium were determined by ELISA. Each value represents the mean ± SEM of triplicate determinations from three independent cell preparations. *p †p 1 alone.</p

Characteristics of the study subjects.

<p>F indicates female; M, male; BMI, body mass index; sBP, systolic blood pressure; dBP, diastolic blood pressure; HbA_1c, hemoglobin A_1c; Glu, plasma glucose; TC, total cholesterol; TG, triglyceride; HDL, high-density lipoprotein; Plt, platelet counts. The data are presented as the means ± SD.</p><p>Characteristics of the study subjects.</p

The relationship between individual levels of released phosphorylated-HSP27 and the area under the curve (AUC) of platelet aggregation induced by collagen in type 2 DM patients.

<p>The levels of released phosphorylated-HSP27 in the supernatant of the conditioned mixture after platelet aggregation stimulated by 0.3 μg of collagen for 30 min was determined using specific ELISA kits and data were collected with the platelet counts. AUC of platelet aggregation stimulated by 0.3 μg/ml of collagen for 4 min were determined by an aggregometer using LS system recorded individually by the size of aggregates, (A) AUC of small aggregates, (B) AUC of medium aggregates, (C) AUC of large aggregates, (D) AUC of total transmission. Each data were plotted and analyzed by linear regression analysis. (a) Whole subjects (n = 35) were plotted. (b) The residual subjects after excluding what concentration of phosphorylated-HSP27 could not be detected (n = 30) were plotted.</p

Representative patterns of platelet aggregation induced by various doses of collagen as detected by an aggregometer with laser-scattering system and representative data showing the collagen-induced HSP27 phosphorylation in platelets from type 2 DM patients.

<p>PRP from type 2 DM patients was stimulated by various doses of collagen (0, 0.1, 0.3 and 1.0 μg/ml) in an aggregometer at 37°C for 4 min with a stirring speed of 800 rpm. (A) Time-dependent changes in the platelet aggregation after stimulation by 0 μg/ml (<i>i</i>), 0.1 μg/ml (<i>ii</i>), 0.3 μg/ml (<i>iii</i>), and 1.0 μg/ml (<i>iv</i>) are shown. The black line indicates the percentage of transmittance of each samples (the isolated platelets were recorded as 0%, and platelet free plasma was recorded as 100%). The blue line indicates small aggregates (9–25 μm); green line, medium aggregates (25–50 μm); red line, large aggregates (50–70 μm). (B) The reaction was terminated by the addition of an ice-cold EDTA (10 mM) solution. The extracts of platelets were subjected to a Western blot analysis using antibodies against phospho-specific HSP27 (Ser-78 and Ser-82), total HSP27 and P2Y12. The bands were quantified using the ImageJ software program as the counts of pixels. The bands of phospho-HSP27 and the bands of total HSP27 were normalized to the total HSP27 bands and the P2Y12 bands, respectively. The ratio (phospho-HSP27/total HSP27 and total HSP27/P2Y12) is presented for each value. Upper panel indicates the results of a Western blot analysis. In the left-lower panel, the semi-black bars indicate the phosphorylation ratio of HSP27 (Ser-78), and the black bars indicate the phosphorylation ratio of HSP27 (Ser-82). In the right-lower panel, the black bars indicate the ratio of total HSP27/P2Y12.</p