Polo-like Kinase 1 Regulates Vimentin Phosphorylation at Ser-56 and Contraction in Smooth Muscle*

Polo-like kinase 1 (Plk1) is a serine/threonine-protein kinase that has been implicated in mitosis, cytokinesis, and smooth muscle cell proliferation. The role of Plk1 in smooth muscle contraction has not been investigated. Here, stimulation with acetylcholine induced Plk1 phosphorylation at Thr-210 (an indication of Plk1 activation) in smooth muscle. Contractile stimulation also activated Plk1 in live smooth muscle cells as evidenced by changes in fluorescence resonance energy transfer signal of a Plk1 sensor. Moreover, knockdown of Plk1 in smooth muscle attenuated force development. Smooth muscle conditional knock-out of Plk1 also diminished contraction of mouse tracheal rings. Plk1 knockdown inhibited acetylcholine-induced vimentin phosphorylation at Ser-56 without affecting myosin light chain phosphorylation. Expression of T210A Plk1 inhibited the agonist-induced vimentin phosphorylation at Ser-56 and contraction in smooth muscle. However, myosin light chain phosphorylation was not affected by T210A Plk1. Ste20-like kinase (SLK) is a serine/threonine-protein kinase that has been implicated in spindle orientation and microtubule organization during mitosis. In this study knockdown of SLK inhibited Plk1 phosphorylation at Thr-210 and activation. Finally, asthma is characterized by airway hyperresponsiveness, which largely stems from airway smooth muscle hyperreactivity. Here, smooth muscle conditional knock-out of Plk1 attenuated airway resistance and airway smooth muscle hyperreactivity in a murine model of asthma. Taken together, these findings suggest that Plk1 regulates smooth muscle contraction by modulating vimentin phosphorylation at Ser-56. Plk1 activation is regulated by SLK during contractile activation. Plk1 contributes to the pathogenesis of asthma.

Thr-210 and activation in smooth muscle. SLK is an upstream regulator of Plk1 in smooth muscle.

Contractile Activation Induces Plk1 Phosphorylation at Thr-210 in Smooth Muscle-In vitro biochemical studies suggest that
Plk1 is activated by phosphorylation at Thr-210 (18,21). We used immunoblot analysis to determine whether contractile stimula-tion induces Plk1 phosphorylation at this residue. In unstimulated smooth muscle cells, phosphorylation of Plk1 at Thr-210 was low. Stimulation with acetylcholine (ACh) increased the phosphorylation level of Plk1 at Thr-210 in smooth muscle (Fig. 1A).

Effects of ACh on Plk1 Activation in Live Smooth
Muscle Cells-To monitor Plk1 activation in live cells, we constructed a fluorescence resonance energy transfer (FRET)-based Plk1 biosensor. We fused DsRed to the N terminus and GFP to the C . E, the GFP and DsRed emissions of cells expressing GFP-tagged Plk1 were evaluated using a confocal microscope. GFP emission was very high, whereas DsRed signal was not detected. In addition, ACh stimulation did not affect GFP/DsRed emission. Because no DsRed (RED) emission was detected, we failed to calculate the ratio of GFP/DsRed (n ϭ 12 cells from 4 batches of cell culture). DIC, differential interference contrast. F, the emissions of GFP and DsRed of cells expressing DsRed-tagged construct were assessed. Neither GFP nor DsRed was found under the experimental conditions. ACh treatment did not affect the emission of GFP and DsRed. No GFP/DsRed ratios were calculated because of lack of fluorescent signals (n ϭ 14 cells from 4 batches of cell culture). Scale bar: 10 m.
terminus of Plk1 (Fig. 1B). We then evaluated the effects of contractile stimulation on Plk1 activation in live cells. Human airway smooth muscle (HASM) cells expressing the Plk1 sensor were treated with ACh, and emissions of DsRed and GFP were monitored live using a laser-scanning confocal microscope. Before stimulation, the emission ratios of GFP/DsRed were relatively lower. In response to ACh stimulation, the GFP signal was increased, whereas the DsRed signal was decreased (Fig.  1C). The GFP/DsRed ratios gradually increased during the course of contractile activation (Fig. 1D). These results suggest that contractile stimulation activates Plk1 in live smooth muscle cells.
To assess whether Thr-210 phosphorylation affects Plk1 activation in cells, we generated a T210A Plk1 biosensor (alanine substitution at Thr-210) and evaluated the effects of ACh stimulation on the activation of mutant Plk1. The emission ratios of GFP/dsRed for the T210A Plk1 biosensor were not increased in response to ACh stimulation ( Fig. 1, C and D). The results indicate that Thr-210 phosphorylation regulates Plk1 activation in smooth muscle cells.
To further characterize the biosensor, cells expressing single-probe constructs (GFP-tagged construct or DsRed-tagged construct) were treated with ACh, and emission of GFP and DsRed was evaluated using the confocal microscope under the same experimental condition. For cells expressing the GFP construct, GFP emission was high, whereas DsRed emission was undetected. In cells expressing the DsRed construct, neither GFP nor DsRed emission was detected (Fig. 1, E and F). The results validate the selectivity and sensitivity of the biosensor.
Plk1 Is Required for Smooth Muscle Contraction-As described earlier, Plk1 is a serine/threonine-protein kinase that has a role in cytokinesis (17,18) and smooth muscle cell proliferation (19,20). The role of Plk1 in smooth muscle contraction is largely unknown. To assess its role, we utilized a lentivirusmediated RNAi approach (26 -28) to inhibit the expression of Plk1. Contractile response of human bronchial rings to ACh was evaluated. Bronchial rings were then transduced with lentiviruses encoding control shRNA or Plk1 shRNA. These tissues were incubated in the serum-free medium for 3 days. Contractile force of these tissues was then evaluated. Immunoblot analysis verified the lower expression of Plk1 in tissues transduced with viruses encoding Plk1 shRNA compared with uninfected rings and tissues transduced with viruses for control shRNA ( Fig. 2A). More importantly, contractile responses of human bronchial rings were lower in Plk1-deficient tissues than in control tissues (Fig. 2B).
To further evaluate the role of Plk1 in smooth muscle, we also generated smooth muscle conditional Plk1 knock-out mice. Contractile responses of mouse tracheal rings to ACh stimulation were compared between Plk1 smko (smooth muscle knockout of Plk1) mice and Plk1 Ϫlox (control) mice. As shown in Fig.  2C, contractile responses of mouse tracheal rings to ACh were lower in Plk1 smko mice than in Plk1 Ϫlox mice, which was dose-dependent.
We also evaluated acute effects of the Plk1 pharmacological inhibitors BI6727 (29) on airway smooth muscle contraction. Treatment of mouse tracheal rings with BI6727 significantly induced relaxation of tracheal rings precontracted by ACh (Fig.  2D).

Plk1 Regulates Vimentin Phosphorylation at Ser-56 in Smooth Muscle and In
Vitro-Recent studies suggest that vimentin undergoes phosphorylation at Ser-56, which has a role in regulating various cellular functions including smooth muscle contraction (3,(5)(6)(7)(8)(9). To determine whether Plk1 mediates vimentin phosphorylation, we evaluated the effects of Plk1 knockdown on vimentin phosphorylation in smooth muscle cells. In cells treated with control shRNA, acetylcholine stimulation induced vimentin phosphorylation at Ser-56. In contrast, the ACh-induced vimentin phosphorylation was reduced in smooth muscle treated with Plk1 shRNA (Fig. 3A). However, ACh-induced vimentin phosphorylation in Plk1 knockdown cells was higher compared with unstimulated Plk1 knockdown cells (Fig. 3A). The results suggest that Plk1 partially regulates vimentin phosphorylation at this residue in smooth muscle.
We also used the in vitro kinase assay to assess whether Plk1 directly mediates vimentin phosphorylation. Phosphorylation of vimentin at Ser-56 was increased in the presence of Plk1. However, Plk1 did not enhance phosphorylation of S56A mutant vimentin (alanine substitution at Ser-56) ( Fig. 3B) (9,30). The results suggest that Plk1 mediates vimentin phosphorylation at Ser-56 in smooth muscle.
Plk1 Knockdown Did Not Affect Myosin Light Chain Phosphorylation at Ser-19 -Because myosin light chain phosphorylation at Ser-19 is known to regulate smooth muscle contraction (12,31,32), we evaluated the effects of Plk1 knockdown on myosin light chain phosphorylation at Ser-19. The knockdown of Plk1 did not affect myosin light chain phosphorylation in smooth muscle (Fig. 3C).

Expression of the Plk1 Nonphosphorylatable Mutant (T210A Plk1) Inhibits Smooth Muscle Contraction and Vimentin
Phosphorylation without Affecting Myosin Light Chain Phosphorylation-To determine the role of Plk1 phosphorylation at this residue, human bronchial rings were transfected with plasmids encoding T210A Plk1 using the method of reversible permeabilization (26,28,(33)(34)(35). The tissues were cultured in the serum-free medium for 2 days to allow for protein expression. Immunoblot analysis confirmed the expression of the recombinant proteins (Fig. 4A). It was noticed that endogenous Plk1 was reduced in tissues expressing wild type (WT) or T210A Plk1. This could be because exogenous Plk1 mRNA competes with endogenous Plk1 mRNA for the same translational machine (9,33). Contractile force of these tissues was compared before and after incubation. Contractile force was reduced in tissues expressing mutant T210A Plk1compared with tissues transfected with WT Plk1 (Fig. 4B).
We then determined the effects of mutant T210A Plk1 on vimentin phosphorylation at Ser-56 and myosin light chain phosphorylation. The ACh-induced vimentin phosphorylation was reduced in smooth muscle cells expressing Plk1T210A (Fig.  4C). In addition, ACh-induced vimentin phosphorylation in cells expressing T210A Plk1 was higher compared with basal vimentin Ser-56 phosphorylation in cells expressing T210A Plk1 (Fig. 4C). This indicates that vimentin phosphorylation at Ser-56 is regulated in part by Plk1 phosphorylation (activation).
However, myosin light chain phosphorylation at Ser-19 was comparable in cells expressing WT or mutant Plk1 (Fig. 4D).
To determine the role of vimentin phosphorylation at Ser-56 in contraction, we introduced WT vimentin or S56A vimentin (alanine substitution at Ser-56) (9, 30) into human bronchial rings by reversible permeabilization. Immunoblot analysis verified the expression of the recombinant proteins (Fig. 4E). Moreover, endogenous vimentin was reduced in tissues treated with WT or S56A vimentin. This could be because exogenous vimentin mRNA competes with endogenous vimentin mRNA for the same translational machine (9,33). More importantly, contractile force of tissues treated with S56A vimentin was reduced as compared with tissues expressing WT vimentin (Fig. 4F).
Knockdown of SLK Inhibits Plk1 Phosphorylation and Activation-SLK is a serine/threonine-protein kinase that has been implicated in spindle orientation and microtubule organization during mitosis (24,25). To determine whether SLK has a role in regulating Plk1, smooth muscle cells were transfected with siRNA against SLK. Immunoblot analysis verified SLK knockdown in cells (Fig. 5A). SLK knockdown attenuated Plk1 phosphorylation at Thr-210 upon ACh stimulation (Fig. 5B). The emission ratios of GFP/DsRed for Plk1 biosensor was also reduced in SLK knockdown cells (Fig. 5C). The results suggest that SLK mediates Plk1 activation in smooth muscle during contractile activation.

Conditional Knock-out of Plk1in Smooth Muscle Attenuates HDM-induced Airway Resistance and Contraction of Tracheal
Rings-Asthma is characterized by airway hyperresponsiveness, which is largely attributed to hyperreactivity of airway smooth muscle (36,37). We used the HDM-induced animal model of asthma to determine whether Plk1 in smooth muscle has a role in airway hyperresponsiveness. Briefly, Plk1 Ϫlox and Plk1 smko mice were exposed to HDM for 6 weeks. Airway resistance in response to methacholine (MCh) inhalation was measured using the FlexiVent system (27,37). HDM exposure induced a higher response to MCh inhalation in Plk1 Ϫlox mice as compared with Plk1 Ϫlox mice treated with PBS. However, FIGURE 2. Plk1 is necessary for smooth muscle contraction. A, human bronchial rings were transduced with lentiviruses encoding control shRNA or Plk1 shRNA. These tissues were then incubated in the serum-free medium for 3 days. Immunoblot analysis was used to assess protein expression in tissues. UI, uninfected. *, significantly lower protein ratios of Plk1/GAPDH in tissues transduced with virus encoding Plk1 shRNA than in uninfected tissues and tissues expressing control shRNA (p Ͻ 0.05). Data are the mean values of experiments from three donors. Error bars indicate S.D. B, contraction of human bronchial rings to ACh was evaluated, after which they were transduced with lentiviruses as described above. Contractile responses were compared before and after incubation. *, significantly lower contractile force in bronchial rings treated with Plk1 shRNA as compared with uninfected tissues or tissues infected with airway resistance induced by MCh was lower in Plk1 smko mice exposed to HDM than in Plk1 Ϫlox mice treated with HDM (Fig.  6A). In addition, airway resistance in Plk1 smko mice treated with PBS was also lower than that in Plk1 Ϫlox mice treated with PBS (Fig. 6A).
We also assessed the effects of Plk1 knock-out on airway smooth muscle hyperreactivity in vitro. Contractile force in isolated tracheal rings from HDM-treated Plk1 Ϫlox mice was greater compared with naïve Plk1 Ϫlox mice. However, active force of isolated tracheal rings from HDM-treated Plk1 smko mice was reduced compared with HDM-treated Plk1 Ϫlox mice (Fig. 6B). Moreover, tracheal contraction of Plk1 smko mice treated with PBS was reduced as compared with Plk1 Ϫlox mice treated with PBS (Fig. 6B).
Furthermore, we evaluated the effects of the Plk1 inhibitor BI6727 on airway resistance and contraction of isolated tracheal rings. Treatment with BI6727 attenuated the HDM-induced increase in airway resistance and airway smooth muscle hyperreactivity (Fig. 6, C and D).
Conditional Knock-out of Plk1 Does Not Affect the Size of Animal Lungs and Expression of ␣-Actin and Myosin Heavy Chain-Because Plk1 conditional knock-out attenuates airway resistance without allergen exposure (Fig. 6A), this raises a possibility that Plk1 knock-out could impair lung development, which results in smaller lungs. To test this, we compared the lung size of Plk1 Ϫlox mice and Plk1 smko mice. The lung size of Plk1 smko mice was similar to that of Plk1 Ϫlox mice (Fig. 6E). Furthermore, the expression of ␣-actin and myosin heavy chain in tracheal smooth muscle was comparable between Plk1 Ϫlox mice and Plk1 smko mice (Fig. 6F). These results suggest that Plk1 conditional knock-out does not affect lung development and contractile protein expression in smooth muscle.

Discussion
Plk1 is a serine/threonine-protein kinase that has been implicated in cytokinesis, mitosis (17,18), and smooth muscle cell proliferation (19,20). The role of Plk1 in smooth muscle contraction has not been previously investigated. In this study, knockdown of human airway smooth muscle inhibited the agonist-induced contraction. Furthermore, smooth muscle conditional knock-out of Plk1 in mice also attenuated tracheal ring contraction. To the best of our knowledge, this is the first evidence to suggest that Plk1 is necessary for smooth muscle contraction.
Contractile activation induces myosin light chain phosphorylation at Ser-19, initiating cross-bridge cycling (31,32). In addition, actin filament polymerization transpires in smooth muscle upon agonist stimulation, which promotes the force transmission between the contractile units and the extracellular matrix (12, 19, 33, 38 -41). Furthermore, vimentin phosphorylation at Ser-56 occurs in response to contractile stimulation, facilitating reorganization of the vimentin network and intercellular/intercellular force transmission (1,2,4,5). Thus, myosin may serve as an "engine" for smooth muscle contraction, whereas the actin cytoskeleton and the vimentin network may function as a "transmission system" in smooth muscle (1,5,26,27). Actin dynamics in smooth muscle is largely regulated by protein tyrosine phosphorylation. Because Plk1 is a serine/threonine kinase (17,21), we have assessed the role of Plk1 in vimentin phosphorylation and myosin light chain phosphorylation. Knockdown of Plk1 diminished the agonist-induced vimentin phosphorylation at Ser-56 but not myosin light chain phosphorylation at Ser-19. The results suggest that Plk1 regulates smooth muscle contraction by controlling vimentin phosphorylation.
Plk1 undergoes phosphorylation at Thr-210 during cell division and stimulation of smooth muscle cells with growth factors (17,20,23). In the present study, ACh stimulation induced Plk1 phosphorylation at Thr-210, suggesting Plk1 activation during contractile activation. Furthermore, we provide the first evidence that contractile stimulation activated Plk1 in live smooth muscle cells by using the Plk1 biosensor. The results also provide direct evidence that contractile stimulation is able to induce conformational changes of Plk1 from a "closed" structure to an "open" conformation. When in closed state, PBD binds to the catalytic domain, which inhibits the access of substrates to the catalytic domain. Plk1 phosphorylation at Thr-210 may induce the dissociation of PBD from the catalytic domain, increasing kinase activity (17,18,21,22). More importantly, the introduction of T210A Plk1 inhibited vimentin phosphorylation and contraction without affecting myosin light chain phosphorylation. Therefore, Plk1 phosphorylation at Thr-210 is critical for vimentin phosphorylation at Ser-56 and force development in smooth muscle.
The serine/threonine kinase SLK has been implicated in spindle orientation and microtubule organization during mitosis (24,25). SLK may directly catalyze Plk1 phosphorylation at Thr-210 as evidenced by mass spectrometry analysis (43). Here, we showed that knockdown of SLK inhibited Plk1 phosphorylation at Thr-210 and activation in response to contractile activation. Thus, SLK is an upstream regulator of Plk1 in smooth muscle.
Abnormal airway smooth muscle contraction contributes to the pathogenesis of airway hyperresponsiveness, a cardinal characteristic of asthma (36,37). IL-13 has been implicated in the hyperactivity of airway smooth muscle (44). In addition, c-Abl tyrosine kinase has been implicated in the pathogenesis of airway hyperresponsiveness (19,37). Here, conditional knock-out of Plk1 or inhibition of Plk1 attenuated the allergeninduced airway hyperresponsiveness and airway smooth muscle hyperreactivity. These results suggest that Plk1 plays a critical role in the pathogenesis of airway hyperresponsiveness.
In summary, we unveiled a novel mechanism that is indispensable for smooth muscle contraction. In response to con-tractile activation, Plk1 is phosphorylated at Thr-210 and activated, which subsequently mediates vimentin phosphorylation at Ser-56. The phosphorylation of vimentin may facilitate intracellular and intercellular force transduction and smooth muscle contraction. Plk1 phosphorylation is regulated by SLK (Fig. 7).

Experimental Procedures
Cell Culture-HASM cells were prepared from human bronchi and adjacent tracheas obtained from the International Institute for Advanced Medicine (26 -28, 33, 45). Human tissues were non-transplantable and consented for research. This study was approved by the Albany Medical College Committee on Research Involving Human Subjects. Briefly, muscle tissues were incubated for 20 min with dissociation solution (130 mM NaCl, 5 mM KCl, 1.0 mM CaCl 2 , 1.0 mM MgCl 2 , 10 mM Hepes, 0.25 mM EDTA, 10 mM D-glucose, 10 mM taurine, pH 7, 4.5 mg of collagenase (type I), 10 mg of papain (type IV), 1 mg/ml BSA, and 1 mM dithiothreitol). All enzymes were purchased from Sigma. The tissues were then washed with Hepes-buffered saline solution (10 mM Hepes, mM 130 NaCl, 5 mM KCl, 10 mM glucose, 1 mM CaCl 2 , 1 mM MgCl 2 , 0.25 mM EDTA, 10 mM taurine, pH 7). The cell suspension was mixed with Ham's F-12 medium supplemented with 10% (v/v) fetal bovine serum (FBS) and antibiotics (100 units/ml penicillin, 100 g/ml streptomycin). Cells were cultured at 37°C in the presence of 5% CO 2 in the same medium. The medium was changed every 3-4 days until cells reached confluence, and confluent cells were passaged with trypsin/EDTA solution (9,10,45,46). Smooth muscle cells within passage 5 were used for the studies.
Immunoblot Analysis-Cells were lysed in SDS sample buffer composed of 1.5% dithiothreitol, 2% SDS, 80 mM Tris-HCl, pH 6.8, 10% glycerol, and 0.01% bromphenol blue. The lysates were boiled in the buffer for 5 min and separated by SDS-PAGE. Proteins were transferred to nitrocellulose membranes. The membranes were blocked with bovine serum albumin or milk for 1 h and probed with the use of primary antibodies followed by horseradish peroxidase-conjugated secondary antibodies (Thermo Fisher Scientific). Proteins were visualized by enhanced chemiluminescence (Thermo Fisher Scientific) using the LAS-4000 Fuji Image System or GE Amersham Biosciences Imager 600 system. Total Plk1 antibody (1:1000) was purchased from EMD Millipore (#05-844, L/N 2477015). Anti-SLK (1:250) was purchased from Santa Cruz Biotechnology (#sc-79068, L/N B2410). Anti-Plk1 and anti-SLK were validated by using corresponding knockdown cells. Phospho-vimentin (S56) antibody (1:500) was produced as previously described (9,30) and validated by examining molecular weight of detected bands and higher intensity in stimulated cells. Vimentin antibody (1:2000) was purchased from BD Biosciences (#550515, L/N 3214517) and validated by examining molecular weight of detected bands. Phospho-Plk1 (T210) antibody (1:500) was purchased from Cell Signaling (#9062S, L/N 1) and validated by assessing molecular weight of detected bands and higher intensity in stimulated cells. Antibody against phospho-myosin light chain (Ser-19, 1:250) was purchased from Santa Cruz Biotechnology (sc-19849-R, L/N B2411) and validated by assessing molecular weight of detected bands and higher intensity in stimulated FIGURE 6. Knock-out or inhibition of Plk1 diminishes airway resistance and contractile response of tracheal rings from HDM-exposed mice. A, Plk1 Ϫlox and Plk1 smko mice were exposed to HDM for 6 weeks. Airway resistance (R AW ) in response to inhalation of 80 mg/kg MCh was then measured. The data represent the means Ϯ S.D. (*, p Ͻ 0.05). **, significantly lower airway resistance in Plk1 smko mice treated with PBS than in Plk1 Ϫlox mice treated with PBS (p Ͻ 0.05). n ϭ 6 -8 mice/each group. B, the contractile response of tracheal rings from these mice was determined by using an in vitro organ bath system. Contractile force is normalized to the mean maximal force of rings from HDM-treated Plk1 Ϫlox mice. The data represent the means Ϯ S.D. (*, p Ͻ 0.05). **, significantly lower tracheal contraction of Plk1 smko mice treated with PBS than Plk1 Ϫlox mice treated with PBS (p Ͻ 0.05). n ϭ 6 -8 mice/each group. C, C57BL/6J mice were exposed to HDM in the presence or absence of the Plk1 inhibitor BI6727 as described under "Experimental Procedures." Intranasal instillation of BI6727 inhibits R AW in mice treated with HDM. The data represent the means Ϯ S.D. (*, p Ͻ 0.05, n ϭ 4 -6 mice/group). D, treatment with the Plk1 inhibitor BI6727 attenuates HDM-sensitized tracheal contraction ex vivo. Contractile force is normalized to maximal force of rings from HDM-and vehicle-treated mice. Error bars indicate S.D. (*, p Ͻ 0.05, n ϭ 4 -5 mice/group). E, the size of lungs from Plk1 smko mice is similar to that from Plk1 Ϫlox mice. Shown are images of lungs from 3 Plk1 Ϫlox mice and 3 Plk1 smko mice. Scale bar, 1 cm. F, blots of tracheal smooth muscle tissues from Plk1 Ϫlox mice and Plk1 smko mice were probed with antibodies against smooth muscle myosin heavy chain 11 (MYH11), ␣-actin. and GAPDH. Protein ratios of MYH11/GAPDH and ␣-actin/GAPDH from Plk1 smko mice are normalized to those from Plk1 Ϫlox mice. The data represent the means Ϯ S.D. (NS, not significant, n ϭ 3 mice/group). (1:2000) was a gift of Dr. Gunst (34,47) and validated by assessing molecular weight of detected bands. GFP antibody (1:3000) was purchased from Life Technologies (#G10362, L/N1696193) and validated by assessing molecular weight of detected bands. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (1:10,000) was purchased from Ambion (AM4300, L/N 1311029) and validated by assessing molecular weight of detected bands. MYH11 antibody (1:250) was purchased from Santa Cruz Biotechnology (#sc-6956, L/N C1815) and validated by examining molecular weight of detected bands. Finally, vendors have provided data sheet to show that antibodies were validated by positive controls. The levels of proteins were quantified by scanning densitometry of immunoblots (Fuji Multigauge Software or GE IQTL software). The luminescent signals from all immunoblots were within the linear range.

cells. Anti-myosin light chain
Virus-mediated RNAi-For Plk1 knockdown (KD), lentiviruses encoding Plk1 shRNA (sc-36277-V) or control shRNA (sc-108080) were purchased from Santa Cruz Biotechnology. HASM cells were infected with control shRNA lentivirus or Plk1 shRNA lentivirus for 12 h. They were then cultured for 3-4 days. Positive clones expressing shRNAs were selected by puromycin. Immunoblot analysis was used to determine the expression levels of Plk1 in these cells. Plk1 KD cells and cells expressing control shRNA were stable at least five passages after initial infection.
In Vitro Kinase Assay-Active Plk1 were purchased from EMD Millipore. Recombinant vimentin was produced by our laboratory as previously described (9). Active Plk1 (40 ng) and 1 g of vimentin were placed in 20 l of kinase buffer containing 20 mM HEPES, pH 7.5, 60 mM NaCl, 2 mM MgCl 2 , 5 mM EGTA, and 100 M ATP. The kinase reaction mix was incubated at 30°C for 30 min and stopped by the addition of the SDS sample buffer (9,35). The samples were boiled for 5 min and separated by SDS-PAGE followed by membrane transfer. The membrane was probed with phospho-vimentin antibody, stripped, and reprobed with vimentin antibody.
FRET Analysis-HASM cells cultured in glass-bottom dishes were transfected with plasmids encoding the Plk1 sensor using the FuGENE HD transfection reagent kit (Promega) and analyzed 2 days after transfection by using a laser scanning confocal microscope (Zeiss 510 Meta, C-Apochromat 40ϫ/1.2 watt corr, objective). Briefly, cells were excited at wavelength of 488 nm, and emission of DsRed and GFP was simultaneously collected every 50 s. Exposure time was between 1 s and 3 s. Appropriate microscope setting (laser power level, detector gain, and amplifier gain) was used to minimize potential bleed-through. The same microscope setting was used for the experiments. For quantification of FRET efficiency, region of interest (ROI) of cells were positioned, and the fluorescent intensity of each channel was measured by Zeiss Analysis software. GFP/DsRed fluorescent ratios were used to assess the FRET efficiency.
Measurement of Human Bronchial Ring Contraction-The study was approved by Albany Medical College Institutional Review Board. Bronchial rings (diameter, 5 mm) were prepared from human lungs obtained from the International Institutes for Advanced Medicine (see above). Bronchial rings were placed in physiological saline solution (PSS) at 37°C in a 25-ml organ bath and attached to a Grass force transducer that had been connected to a computer with A/D converter (Grass). For lentivirus-mediated RNAi in tissues, the thin epithelium layer of human bronchial rings was removed by using forceps. They were then transduced with lentivirus encoding Plk1 shRNA or control shRNA for 3 days. Force development in response to ACh (100 M, 10 min) activation was compared before and after lentivirus transduction. For biochemical analysis, human tissues were frozen using liquid nitrogen and pulverized as previously described (3,4,26).
Animals and Measurement of Airway Resistance-All animal protocols were reviewed and approved by the Institutional Animal Care and Usage Committee of Albany Medical College. To generate smooth muscle conditional Plk1 knock-out mice (Plk1 smko mice), Plk1 Ϫlox mice (from the Toronto Centre for Phenogenomics; genetic background, C57BL/6N) were crossed with B6.Cg-Tg (Myh11-cre,-EGFP) 2Mik/J mice (from The Jackson Laboratory, genetic background, C57BL/6J). Plk1 Ϫlox , Cre-, and Plk1 smko mice (6 -8 weeks old, male and female) were randomly exposed to HDM extracts (50 g of Dermatophagoides pteronyssinus, Greer) or PBS (control) intranasally for 5 days followed by every other day exposures weekly for 5 weeks. Airway resistance in these mice was then assessed on day 43.
To measure airway resistance, mice were anesthetized with intraperitoneal injection of ketamine/xylazine mixture (320 mg/kg), tracheotomized, and connected to the FlexiVent system (SCIREQ, Montreal, Canada). Mice were mechanically ventilated at 150 breaths/min with a tidal volume of 10 ml/kg and a positive end-expiratory pressure of 3.35 cm H 2 O. After baseline measurements, mice were challenged with MCh aerosol for 10 s at different doses. Airway resistance was measured for each mouse after inhalation of the aerosol. Contractile response was then determined. This study was approved by the Institutional Committee of Animal Care and Usage of Albany Medical College.
To assess the effects of inhibitors, C57BL/6J mice were exposed to HDM extracts or PBS intranasally for 5 days followed by every other day exposures weekly for 5 weeks. In addition, animals were intranasally instilled with 10 mg/kg BI6727 (Santa Cruz Biotechnology, #sc-364432, L/N H1513) or PBS for 1 h before HDM instillation and 5 h after HDM instillation for last 2 weeks. Airway resistance in these mice was then assessed on day 43.
Each animal experiment was performed on monthly basis. The results were substantiated by repetition by at least two researchers. Animals were randomly treated with PBS or HDM. Animal experiments associated with the inhibitor were blindly performed. The inhibitor and vehicle control were coded and blinded to researchers. Animal experimental data were excluded if anesthesia was not appropriate, which can be detected by the FlexiVent software.
Assessment of Tracheal Ring Contraction-Mice were euthanized by intraperitoneal injection of euthanasia solution (VEDCO, 0.1 ml/25g). All experimental protocols were approved by the Institutional Animal Care and Usage Committee. A segment of tracheas (4 -5 mm in length) was immediately removed and placed in PSS containing 110 mM NaCl, 3.4 mM KCl, 2.4 mM CaCl 2 , 0.8 mM MgSO 4 , 25.8 mM NaHCO 3 , 1.2 mM KH 2 PO 4 , and 5.6 mM glucose. The solution was aerated with 95 %O 2 , 5% CO 2 to maintain a pH of 7.4. Two stainless steel wires were passed through the lumen of tracheal rings. One of the wires was connected to the bottom of organ baths, and the other was attached to a Grass force transducer that had been connected to a computer with A/D converter (Grass). Tracheal segments were then placed in PSS at 37°C. At the beginning of each experiment, 0.5 g of passive tension was applied to tracheal rings. After 60 min of equilibrium they were stimulated with 80 mM KCl repeatedly until contractile responses and passive tension reached a steady state. Contractile force in response to acetylcholine was then measured.
Statistical Analysis-All statistical analysis was performed using Prism 6 software (GraphPad Software, San Diego, CA). Differences between pairs of groups were analyzed by Student-Newman-Keuls test or Dunn's method. Values of n refer to the number of experiments used to obtain each value. p Ͻ 0.05 was considered to be significant.