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Results: 9

1.
Figure 6

Figure 6. The effects of siRNA on PAK1 expression and vimentin reorganization are reversible. From: Silencing of p21-activated kinase attenuates vimentin phosphorylation on Ser-56 and reorientation of the vimentin network during stimulation of smooth muscle cells by 5-hydroxytryptamine.

(A) Smooth muscle cells that had been treated with control RNA, siRNA or no RNA were incubated in the absence of control RNA or siRNA for an additional 4 days. The representative immunoblot shows that PAK1 protein expression in siRNA-removed cells was similar to that in control RNA-removed cells or in cells not treated with RNA. (B) PAK1/actin ratios in cells after removal of siRNA or control RNA are normalized to the ratio obtained from cells not treated with RNA. The values represent means±S.E.M. (n=4). (C) After an additional 4-day incubation without siRNA or control RNA, vimentin filament reorientation in response to stimulation with 5-HT was also recovered. The values represent mean±S.E.M. (n=4).

Dale D. Tang, et al. Biochem J. 2005 June 15;388(Pt 3):773-783.
2.
Figure 9

Figure 9. Treatment with genistein inhibits protein tyrosine phosphorylation stimulated by 5-HT. From: Silencing of p21-activated kinase attenuates vimentin phosphorylation on Ser-56 and reorientation of the vimentin network during stimulation of smooth muscle cells by 5-hydroxytryptamine.

Smooth muscle cells were stimulated with 10 μM 5-HT for 15 min or unstimulated in the absence or presence of treatment with 100 μM genistein for 30 min. Blots of protein extracts (A) or CAS immunoprecipitates (B) obtained from these cells were probed with antiphosphotyrosine antibody (PY20 Ab), stripped and reprobed with antibodies against paxillin (A) or CAS (B) to ensure even protein loading. Molecular mass markers are indicated on the left. CAS and paxillin are indicated on the right. The blots are representative of three identical experiments. IB, immunoblot; IP, immunoprecipitate.

Dale D. Tang, et al. Biochem J. 2005 June 15;388(Pt 3):773-783.
3.
Figure 3

Figure 3. Treatment with siRNA depresses the expression of PAK1 in cultured smooth muscle cells. From: Silencing of p21-activated kinase attenuates vimentin phosphorylation on Ser-56 and reorientation of the vimentin network during stimulation of smooth muscle cells by 5-hydroxytryptamine.

(A) Representative immunoblots illustrating PAK1 silencing by siRNA in smooth muscle cells. Blots of protein extracts from tracheal smooth muscle cells that had been treated with siRNA or control RNA (C RNA), or with no RNA, were detected with antibodies against PAK1 and smooth muscle α-actin. The amount of PAK1 was lower in siRNA-treated cells than in cells treated with no RNA or control RNA. Similar amounts of α-actin were detected in all three groups of cells. Molecular mass markers are indicated on the left. (B) Ratios in siRNA-treated and control-RNA-treated cells are normalized to the ratio obtained from cells not treated with RNA. The values represent means±S.E.M. (n=4); *significantly lower ratio in siRNA-treated cells relative to cells treated with no RNA or control RNA (P<0.05).

Dale D. Tang, et al. Biochem J. 2005 June 15;388(Pt 3):773-783.
4.
Figure 4

Figure 4. PAK1 down-regulation inhibits vimentin phosphorylation at Ser-56 in response to stimulation with 5-HT. From: Silencing of p21-activated kinase attenuates vimentin phosphorylation on Ser-56 and reorientation of the vimentin network during stimulation of smooth muscle cells by 5-hydroxytryptamine.

Smooth muscle cells that had been treated with no RNA, control RNA (C RNA) or PAK1 siRNA were stimulated with 10 μM 5-HT for 15 min, or left unstimulated. The site-specific phosphorylation of vimentin was determined by immunoblot analysis of vimentin immunoprecipitates obtained from these cells. (A) Representative immunoblots illustrating the effects of PAK1 silencing on Ser-56 phosphorylation on vimentin in cells stimulated with 5-HT. Treatment with siRNA, but not with control RNA, inhibited vimentin phosphorylation on Ser-56 after stimulation with 5-HT. pS56 antibody, antibody against phospho-vimentin (Ser-56); IB, immunoblot; Ab, antibody. (B) Vimentin phosphorylation is quantified as multiples of phosphorylation levels obtained in unstimulated cells not treated with RNA. *Significantly higher phosphorylation levels in stimulated cells than in corresponding unstimulated cells (n=4, P<0.05); **significantly lower vimentin phosphorylation upon stimulation with 5-HT in cells treated with siRNA than in cells not treated with RNA or cells treated with control RNA (n=4, P<0.05).

Dale D. Tang, et al. Biochem J. 2005 June 15;388(Pt 3):773-783.
5.
Figure 7

Figure 7. PAK1 down-regulation inhibits the increase in the ratio of soluble vimentin to insoluble vimentin in smooth muscle cells stimulated with 5-HT. From: Silencing of p21-activated kinase attenuates vimentin phosphorylation on Ser-56 and reorientation of the vimentin network during stimulation of smooth muscle cells by 5-hydroxytryptamine.

(A) Immunoblot illustrating the effects of 5-HT on the amount of soluble (supernatant) and insoluble (pellet) vimentin in cells. Smooth muscle cells were stimulated with 10 μM 5-HT for 5 min or 15 min, or unstimulated. Supernatant (S) and pellet (P) fractions were assessed by fractionation. The stimulation of cells with 5-HT increased soluble vimentin and decreased insoluble vimentin. (B) The ratios of soluble to insoluble vimentin were determined by scanning densitometry of immunoblots for the fractions; *significantly greater ratios of soluble vimentin/insoluble vimentin in 5-HT-stimulated cells than in unstimulated cells (P<0.05, n=5–7). (C) Smooth muscle cells that had been treated with no RNA, control RNA (C RNA) or PAK1 siRNA were activated with 10 μM 5-HT for 5 min. The ratios of soluble vimentin/insoluble vimentin were then determined; *significantly greater ratios of soluble vimentin/insoluble vimentin in stimulated cells than in corresponding unstimulated cells (P<0.05, n=4); **significantly lower ratios of soluble vimentin/insoluble vimentin upon stimulation with 5-HT in cells treated with siRNA than in cells not treated with RNA or treated with control RNA (n=4, P<0.05).

Dale D. Tang, et al. Biochem J. 2005 June 15;388(Pt 3):773-783.
6.
Figure 5

Figure 5. PAK1 silencing attenuates the spatial rearrangement of vimentin filaments in response to stimulation with 5-HT. From: Silencing of p21-activated kinase attenuates vimentin phosphorylation on Ser-56 and reorientation of the vimentin network during stimulation of smooth muscle cells by 5-hydroxytryptamine.

(A) Smooth muscle cells that had been treated with control RNA or PAK1 siRNA were stimulated with 10 μM 5-HT for 15 min, or unstimulated. These cells were stained for vimentin (green) and F-actin (red). In the unstimulated cells treated with control RNA (a) or siRNA (c), the vimentin network displayed a curved filamentous structure. Stimulation with 5-HT induced the spatial reorganization of the vimentin network in cells treated with control RNA (b). However, vimentin filaments remained curved structures after stimulation with 5-HT of cells that had been treated with PAK1 siRNA (d). The increase in F-actin formation upon agonist stimulation was not affected in cells treated with control RNA or siRNA (ad). The bar (10 μm) indicates the magnification of the main figures; the insets are 1.5× the magnification of the main figures. (B) Quantitative analysis of the effects of PAK1 silencing on the spatial reorganization of vimentin filaments. *Significantly higher percentage of cells with rearranged vimentin filaments in stimulated cells relative to unstimulated cells treated with control RNA (P<0.05); **significantly lower percentage of cells with rearranged vimentin in stimulated cells treated with siRNA compared with stimulated cells treated with control RNA. The values represent means±S.E.M. (n=4).

Dale D. Tang, et al. Biochem J. 2005 June 15;388(Pt 3):773-783.
7.
Figure 8

Figure 8. Disruption of the actin cytoskeleton and inhibition of protein tyrosine phosphorylation do not inhibit the spatial reorganization of vimentin filaments in response to stimulation with 5-HT. From: Silencing of p21-activated kinase attenuates vimentin phosphorylation on Ser-56 and reorientation of the vimentin network during stimulation of smooth muscle cells by 5-hydroxytryptamine.

(A) Smooth muscle cells were pretreated with or without 5 μM cytochalasin D or 100 μM genistein for 30 min, and then activated with 10 μM 5-HT for 15 min, or unstimulated. These cells were stained for vimentin (green) and F-actin (red). Treatment with cytochalasin D decreased F-actin and disrupted actin stress fibres (c and d). Genistein inhibited the increase in F-actin formation in response to 5-HT stimulation (e and f). Curved vimentin filaments were observed in unstimulated control cells (a) and in cells treated with cytochalasin D (c) or genistein (e). Stimulation with 5-HT induced the reorganization of vimentin filaments in control cells (b) and cells preincubated with cytochalasin D (d) or genistein (f). The bar (10 μm) indicates the magnification of the main figures; the insets are 1.5× the magnification of the main figures. (B) Quantitative analysis of the effects of cytochalasin D and genistein on the spatial reorganization of vimentin filaments. The values represent means±S.E.M. (n=4).

Dale D. Tang, et al. Biochem J. 2005 June 15;388(Pt 3):773-783.
8.
Figure 1

Figure 1. Stimulation with 5-HT induces the reorientation of vimentin filaments in smooth muscle cells. From: Silencing of p21-activated kinase attenuates vimentin phosphorylation on Ser-56 and reorientation of the vimentin network during stimulation of smooth muscle cells by 5-hydroxytryptamine.

(A) Cultured tracheal smooth muscle cells were stimulated with 10 μM 5-HT for 5 or 15 min, or they were not stimulated. These cells were stained with anti-vimentin antibody (green) and rhodamine/phalloidin for F-actin (red). In unstimulated cells, vimentin exhibited curved filamentous structures (a). During stimulation with 5-HT for 5 min (b) or 15 min (c), the vimentin filaments became straight and were arranged along the long axis of cells. In addition, more F-actin was found in stimulated cells (b and c) than in unstimulated cells (a). The bar (10 μm) indicates the magnification of the main figures; the insets are 1.5× the magnification of the main figures. (B) Vimentin filaments displaying straightness longer than one-quarter of cell length are considered as straight filaments. Cells with more than four straight vimentin filaments are counted as cells with rearranged filaments. Quantitative analysis is expressed as (numbers of cells with rearranged vimentin filaments)/(numbers of total cells observed)×100. The asterisk (*) denotes a significantly higher percentage of cells with rearranged vimentin filaments in stimulated cells relative to unstimulated cells (P<0.05). The values represent means±S.E.M. (n=5). (C) An immunoblot of whole-cell extracts was obtained with monoclonal anti-vimentin antibody.

Dale D. Tang, et al. Biochem J. 2005 June 15;388(Pt 3):773-783.
9.
Figure 2

Figure 2. Vimentin undergoes phosphorylation on Ser-56 in smooth muscle cells stimulated with 5-HT. From: Silencing of p21-activated kinase attenuates vimentin phosphorylation on Ser-56 and reorientation of the vimentin network during stimulation of smooth muscle cells by 5-hydroxytryptamine.

(A) Characterization of the antibody (Ab) against phospho-vimentin (Ser-56). Wild-type vimentin or the S56A vimentin mutant was phosphorylated by PAK1 in vitro. Blots of the proteins (200 ng) were probed using the anti-phospho-vimentin (Ser-56) antibody (pS56 antibody), stripped and reprobed by use of anti-vimentin antibody in the presence or absence of synthetic peptides. The phospho-vimentin antibody recognizes the wild-type vimentin phosphorylated by PAK, but not unphosphorylated wild-type protein. Preincubation with phosphopeptide (pS56), but not non-phosphopeptide (S56), inhibits the immunoreactivity of pS56 antibody towards phosphorylated wild-type vimentin. The pS56 antibody does not recognize the S56A vimentin mutant in the absence or presence of PAK. (B) Cultured smooth muscle cells were stimulated with 10 μM 5-HT for 1, 5, 15 or 30 min, or they were unstimulated. Blots of vimentin immunoprecipitates obtained from these cells were probed using the pS56 antibody, stripped and reprobed by use of the vimentin antibody. Representative immunoblots show that stimulation of cells with 5-HT induces vimentin phosphorylation at Ser-56 at different time points. C, unstimulated cells; IB, immunoblot; IP, immunoprecipitation. (C) Vimentin phosphorylation is quantified as multiples of phosphorylation levels obtained in unstimulated cells. The values represent means±S.E.M. (n=4); *significantly higher phosphorylation levels in the stimulated cells compared with the level in unstimulated cells (P<0.05). (D) To determine the extent of vimentin phosphorylation at Ser-56, half-portions of protein extracts of unstimulated or stimulated cells was precleared with pS56 antibody, and then immunoprecipitated with vimentin antibody (P fraction). The other halves of the same protein extracts were immunoprecipitated with vimentin antibody (N fraction). Both fractions were subjected to immunoblot analysis using vimentin antibody. In response to stimulation with 5-HT, the level of the P fraction (normalized to the N fraction) was decreased to 0.35±0.07 (n=3), suggesting that 65% of total vimentin was phosphorylated in 5-HT-stimulated cells.

Dale D. Tang, et al. Biochem J. 2005 June 15;388(Pt 3):773-783.

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