HSP20 represses the PI3K activities in the HCC cells.

<p>The HSP20-overexpressing HuH7 cells were stimulated with (<b>B</b>) or without (<b>A</b>) 20 ng/ml TGFα for 10 min. After stimulation, PI3K in the cells were immunoprecipitated followed by determination of the PIP3 producing activities. Values represent the means ± SD (n = 8). *: P<0.05.</p

Phospho-mimic HSP20 attenuated the TGF-α-induced JNK signaling in HuH7 cells.

<p>The empty vector-, WT-, SA- or SD-HSP20 transfected HuH7 cells were stimulated by 30 ng/ml of TGF-α for the indicated times. The cells were then harvested, and Western blot analysis was performed to determine the levels of phospho-p46 JNK (A and B) and phospho-EGFR (C). The lower bar graph (A and B) shows the quantification data for the relative levels of phospho-p46 JNK after normalization with GAPDH levels as determined by densitometry analysis. Each value represents the means ± SD (n = 3). *<i>P</i><0.05, compared to the value of lane 1. **<i>P</i><0.05, lanes 8 and 10, compared to the values of lanes 7 and 9, respectively.</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

Dibutyryl cAMP suppressed the TGF-α-induced cell migration of WT-HSP20 overexpressed HuH7 cells.

<p>The empty vector- (A) or the WT-HSP20- (B) transfected HuH7 cells were pretreated with the indicated concentrations of dibutyryl cAMP for 1 h, and then stimulated by 3 ng/ml of TGF-α or vehicle for 24 h. The migrated cells were fixed with paraformaldehyde and stained with DAPI for the nucleus (blue signal). The cells were photographed by fluorescent microscopy at a magnification of 20× (upper panel). The migrated cell numbers were counted and plotted as the ratio to the mean of migrated numbers of the cells stimulated by vehicle without dibutyryl cAMP treatment (lower panel). Each value represents the means ± SD (n = 3). *<i>P</i><0.05, lanes 2, 4 and 6 compared to the values of lanes 1, 3, and 5, respectively. **<i>P</i><0.05. lanes 4 and 6 compared to the value of lane 2.</p

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

Relationship between the protein level of phospho-HSP20 in HCC and tumor invasion.

<p>Relationship between the protein level of phospho-HSP20 in HCC and tumor invasion.</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

HSP20 interacts with PI3K in human HCC tissues.

<p>(<b>A</b>) The protein levels of HSP20 and PI3K p85, PI3K p110α and PI3K p110β subunits in stages I, II and III human HCC tissues were compared by a Western blot analysis. (<b>B</b>) The lysates from a stage II HCC tissue were immunoprecipitated (IP) with antibodies for PI3K p85, PI3K p110α PI3K p110β or normal rabbit IgG followed by Western blotting (WB) using HSP20 antibodies. Immunoprecipitation of the PI3K p85, PI3K p110α and PI3K p110β subunits in the stage II HCC tissue lysates was confirmed by WB using PI3K p85 antibodies, PI3K p110α antibodies and PI3K p110β antibodies, respectively.</p

The clinical and pathological characteristics of patients with HCC.

<p>The clinical and pathological characteristics of patients with HCC.</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