Effects of geldanamycin or 17-AAG on the phosphorylation of MYPT-1, the amounts of MYPT-1, RhoA and Rho-kinase induced by PGF_2α in MC3T3-E1 cells.

<p>The cultured cells were pretreated with various doses of geldanamycin (A) or 17-AAG (B) for 60 min, and then stimulated by 10 μM of PGF_2α or vehicle for 2 min. The cell extracts were then subjected to SDS-PAGE with subsequent Western blot analysis with antibodies against phospho-specific MYPT-1, MYPT-1, RhoA or Rho-kinase. (a) The histogram shows the quantitative representations of the levels of phosphorylated MYPT-1 after normalization with respect to MYPT-1 obtained from laser densitometric analysis. The levels were expressed as the fold increase to the basal levels presented as lane 1. (b),(c),(d) The histogram shows the quantitative representations of the levels of MYPT-1 (b), RhoA (c) and Rho-kinase (d) after normalization with respect to GAPDH obtained from laser densitometric analysis, respectively. The levels were expressed as the ratio to the levels presented as lane 1. Each value represents the mean ± S.E.M. of triplicate determinations from three independent cell preparations. ^*<i>p</i><0.05 compared to the value of control. N.S. designates no significant difference between the indicated pairs.</p

Schematic illustration of the regulatory mechanism underlying the amplifying effect of HSP90 inhibitors on the PGF_2α-induced IL-6 synthesis in osteoblast-like MC3T3-E1 cells.

<p>PGF_2α, prostaglandin F_2α; MAPK, mitogen-activated protein kinase; HSP90, heat shock protein 90; IL-6, interleukin-6.</p

Effects of geldanamycin or onalespib on the PGF_2α-induced expression levels of IL-6 mRNA in MC3T3-E1 cells.

<p>The cultured cells were pretreated with 0.3 μM of geldanamycin (A), onalespib (B) or vehicle for 60 min, and then stimulated by 10 μM of PGF_2α or vehicle for 3 h. The respective total RNA was then isolated and transcribed into cDNA. The expressions of IL-6 mRNA and GAPDH mRNA were quantified by RT-qPCR. The IL-6 mRNA levels were normalized to those of GAPDH mRNA. Each value represents the mean ± S.E.M. of triplicate determinations from three independent cell preparations. ^*<i>p</i><0.05 compared to the value of control. ^**<i>p</i><0.05 compared to the value of PGF_2α alone.</p

Effects of geldanamycin, 17-AAG or onalespib on the phosphorylation of p38 MAP kinase induced by PGF_2α in MC3T3-E1 cells.

<p>The cultured cells were pretreated with the indicated doses of geldanamycin (A) or 17-AAG (B) or onalespib (C) for 60 min, and then stimulated by 10 μM of PGF_2α or vehicle for 10 min. The cell extracts were then subjected to SDS-PAGE with subsequent Western blot analysis with antibodies against phospho-specific p38 MAP kinase or p38 MAP kinase. The histogram shows the quantitative representations of the levels of phosphorylated p38 MAP kinase after normalization with respect to p38 MAP kinase obtained from laser densitometric analysis. The levels were expressed as the fold increase to the basal levels presented as lane 1. Each value represents the mean ± S.E.M. of triplicate determinations from three independent cell preparations. ^*<i>p</i><0.05 compared to the value of control. ^**<i>p</i><0.05 compared to the value of PGF_2α alone.</p

Effects of 17-AAG, 17-DMAG or onalespib on the PGF_2α-stimulated IL-6 release in MC3T3-E1 cells.

<p>The cultured cells were pretreated with 1 μM of 17-AAG (A), 1 μM of 17-DMAG (B) or vehicle for 60 min, and then stimulated by 10 μM of PGF_2α or vehicle for 48 h. (C) The cultured cells were pretreated with various doses of onalespib for 60 min, and then stimulated by 10 μM of PGF_2α (●) or vehicle (○) for 48 h. IL-6 concentrations in the conditioned medium were determined by ELISA. Each value represents the mean ± S.E.M. of triplicate determinations from three independent cell preparations. (A,B) ^*<i>p</i><0.05 compared to the value of control. (A,B) ^**<i>p</i><0.05 compared to the value of PGF_2α alone. (C) ^*<i>p</i><0.05 compared to the value of PGF_2α alone.</p

Effect of PGF_2α on the phosphorylation of p38 MAP kinase in HSP90 knockdown MC3T3-E1 cells.

<p>(A) The cultured cells were transfected with 10 nM negative control siRNA (Neg) or 10 nM HSP90-siRNA (#1). (B) The cultured cells were transfected with 30 nM negative control siRNA (Neg) or 30 nM HSP90-siRNA (#2). Twenty-four hours after transfection, the cells were stimulated by 10 μM PGF_2α or vehicle for 10 min. The cell extracts were then subjected to SDS-PAGE with subsequent Western blot analysis with antibodies against phospho-specific p38 MAP kinase, p38 MAP kinase, HSP90 or GAPDH. (a) The histogram shows the quantitative representations of the levels of phosphorylated p38 MAP kinase after normalization with respect to GAPDH obtained from laser densitometric analysis. The levels were expressed as the fold increase to the basal levels presented as lane 1. (b),(c) The histogram shows the quantitative representations of the levels of (b) total p38 MAP kinase and (c) HSP90αβ after normalization with respect to GAPDH obtained from laser densitometric analysis, respectively. The levels were expressed as the ratio to the levels presented as lane 1. Each value represents the mean ± S.E.M. of triplicate determinations from three independent cell preparations. ^*<i>p</i><0.05 compared to the value of control. ^**<i>p</i><0.05 compared to the value of PGF_2α alone.</p

Effect of SB203580 on the enhancement by geldanamycin, 17-AAG or 17-DMAG of the PGF_2α-induced IL-6 release in MC3T3-E1 cells.

<p>The cultured cells were preincubated with 30 μM of SB203580 or vehicle for 60 min, subsequently pretreated with 1 μM of geldanamycin, 1 μM of 17-AAG, 1 μM of 17-DMAG or vehicle for 60 min, and then stimulated by 10 μM of PGF_2α or vehicle for 48 h. IL-6 concentrations of the conditioned mediums were determined by ELISA. Each value represents the mean ± S.E.M. of triplicate determinations from three independent cell preparations. ^*<i>p</i><0.05 compared to the value of control. ^**<i>p</i><0.05 compared to the value of PGF_2α alone. ^***<i>p</i><0.05 compared to the value of PGF_2α with the pretreatment of each HSP90 inhibitor.</p

Effect of geldanamycin on the PGF_2α-stimulated IL-6 release in MC3T3-E1 cells.

<p>(A) The cultured cells were pretreated with 1 μM of geldanamycin (●,○) or vehicle (■,□) for 60 min, and then stimulated by 10 μM of PGF_2α (●,■) or vehicle (○,□) for the indicated periods. (B) The cultured cells were pretreated with various doses of geldanamycin for 60 min, and then stimulated by 10 μM of PGF_2α (●) or vehicle (○) for 48 h. IL-6 concentrations in the conditioned medium were determined by ELISA. Each value represents the mean ± S.E.M. of triplicate determinations from three independent cell preparations. (A) ^*<i>p</i><0.05 compared to the value of control. ^**<i>p</i><0.05 compared to the value of PGF_2α alone. (B) ^*<i>p</i><0.05 compared to the value of PGF_2α alone.</p

Effects of rivaroxaban and edoxaban on the collagen-induced phosphorylation of HSP27 in human platelets.

<p>PRP was pretreated with various doses of rivaroxaban (A) or edoxaban (B) for 15 min, and then stimulated by 1.0 μg/ml collagen or vehicle for 5 min. The reaction was terminated by the addition of ice-cold EDTA (10 mM) solution. The lysate of platelets was subjected to Western blot analysis using antibodies against phospho-specific HSP27 (Ser-78 and Ser-82), total HSP27 or GAPDH. Representative results of rivaroxaban from ten healthy donors (A) and results of edoxaban from five healthy donors (B) are presented. The histogram shows quantitative representation of the collagen-induced levels obtained from laser densitometric analysis. The phosphorylation levels are expressed as the fold increase to the basal levels presented as lane 1. Each value represents the mean ± SEM. *p<0.05, compared to the value of the vehicle alone, and **p<0.05, compared to the value of collagen alone.</p

Effects of rivaroxaban and edoxaban on the collagen-induced phosphorylation of p44/p42 MAP kinase in human platelets.

<p>PRP was pretreated with various doses of rivaroxaban (A) or edoxaban (B) for 15 min, and then stimulated by 1.0 μg/ml collagen or vehicle for 5 min. The reaction was terminated by the addition of ice-cold EDTA (10 mM) solution. The lysate of platelets was subjected to Western blot analysis using antibodies against phospho-specific p44/p42 MAP kinase, p44/p42 MAP kinase or GAPDH. Representative results of rivaroxaban from ten healthy donors (A) and results of edoxaban from five healthy donors (B) are presented. The histogram shows quantitative representation of the collagen-induced levels obtained from laser densitometric analysis. The phosphorylation levels are expressed as the fold increase to the basal levels presented as lane 1. Each value represents the mean ± SEM. *p<0.05, compared to the value of the vehicle alone, and **p<0.05, compared to the value of collagen alone.</p