Biopsies of human myometrial tissue during pregnancy were obtained at elective (pregnant not in labour, PNL) and intrapartum (pregnant in labour, PL) and preterm emergency caesarean delivery (prior to 34 weeks, PT). The biopsies were excised from the upper lip of the lower uterine segment incision in the midline, i.e. the upper portion of the lower uterine segment. Biopsies of human non-pregnant myometrial tissue (non-pregnant, NP) were obtained from pre-menopausal hysterectomy specimens and women receiving therapy with progestagens, luteinising hormone-releasing hormone analogues, or steroid based medications were excluded from the study. Women who had received prostaglandins or oxytocin for either induction or augmentation of labor were excluded from the study. Women with gynaecologic malignancy were also excluded from the study. Myometrial samples were carefully dissected to minimize decidual inclusion. Ethical Committee approval for the tissue collection procedure was obtained from the Research Ethics Committee University College Hospital, Galway and recruitment was by written informed consent. Immediately upon collection, tissue was rinsed in normal saline for myometrial cell isolation or snap-frozen in liquid nitrogen, and stored at -80°C.

Myometrial biopsies were obtained at hysterectomy (n = 1), elective (n = 2) and intrapartum caesarean section (n = 2). The reason for hysterectomy was menorrhagia, aged 45. The reasons for elective caesarean section included previous caesarean section (n = 2). The reasons for emergency caesarean section were face presentation (n = 2). The mean age of the caesarean section women was 38.5 years (range, 36–41) and all the caesarean sections were multigravida and delivered between 37 and 39 weeks' gestation.

For real-time RT-PCR confirmation analysis, biopsies of myometrium were obtained at the time of elective (n = 7) and intrapartum (n = 7) caesarean section. The reasons for elective caesarean section included previous caesarean section (n = 6) and placenta praevia (n = 1). The reasons for emergency caesarean section were face presentation (n = 4), non-reassuring foetal testing (n = 1) and previous classical caesarean section (n = 2). The mean age of each group was PNL, 34.4 and PL, 34.3 and the overall mean age was 34.35 years (range, 29–41), 3 of whom were primagravida and 11 multigravida. All women were delivered between 37 and 41 weeks' gestation.

Biopsies of myometrium (for the RHOGEF GAP and GDI tissue westerns) during pregnancy were obtained at elective (n = 4) and intrapartum (n = 4) caesarean section and at hysterectomy (n = 4). The reasons for elective caesarean section were previous caesarean sections (n = 4). The reasons for emergency caesarean delivery were non-reassuring foetal testing (n = 2), face presentation (n = 1) and previous classical caesarean section (n = 1). The mean age of the women was 33 years (range 31–37), 4 were primagravida and 4 were multigravida. All women were delivered between 39 and 40 weeks' gestation. The hysterectomies were pre-menopausal (n = 4) with a mean age of 46.

Biopsies of myometrium during pregnancy for the first moesin western blots, were obtained at elective (n = 3) and intrapartum (n = 3) caesarean section. The reasons for elective caesarean section included maternal request (n = 1) and previous caesarean section (n = 2). The reasons for emergency caesarean delivery were non-reassuring foetal testing (n = 1), failed induction (n = 1) and failure to progress (n = 1). The mean age of the women was 35.5 years (range 30–41), 3 were primagravida and 3 were multigravida. All women were delivered between 39 and 40 weeks' gestation.

Biopsies of myometrium for the last set of moesin western blots, during pregnancy were obtained at intrapartum (n = 3) and preterm (n = 3) caesarean sections. The reasons for emergency caesarean delivery were non-reassuring foetal testing (n = 2) and undiagnosed breech birth (n = 1). The reasons for preterm birth were HELLP syndrome (n = 1), foetal abnormality (n = 1) and ulcerative colitis (n = 1). The mean age of the women was 35.5 years (range, 29–37) of which 2 were primagravida and 2 were multigravida. The preterm women were delivered at 32 weeks gestation. The elective and emergency caesarean sections were delivered between 39 and 41 weeks gestation.

Myometrial tissue samples were minced (finely with any fibrous tissue removed) and digested in sterile filtered Dulbecco's Modified Eagle Medium (DMEM) (minus calf serum) (Sigma-Aldrich, Ireland) containing collagenase type IA (Sigma-Aldrich, Ireland), collagenase type XI (Sigma-Aldrich, Ireland) and 0.1% bovine serum albumin (w/v) (BSA) (Sigma-Aldrich, Ireland) for 45 minutes. The resulting suspension was vortexed and the non-dispersed tissue fragments were separated by filtration of the mixture through sterile gauze layers. Individual cells were then collected by centrifugation at 400 g for 10 minutes. Cells were then washed and centrifuged 2 to 3 times in sterile 1X phosphate-buffered saline (PBS). After washing, cells were cultured in SGM-2 medium (Cambrex, Biowhittaker UK Ltd., Berkshire, UK) at 37°C and 5% CO₂. Cells were sub-cultivated with trypsin/ethylenediaminetetraacetic acid (EDTA) at a 1:2 or 1:3 split after reaching confluence. Myometrial cells used in experiments were used to passage 8.

Myometrial smooth muscle cells were characterised for mRNA expression of calponin, and estrogen receptor α and for SM α-actin mRNA and protein expression.

Total RNA was isolated from myometrial tissue using TRIzol reagent (Life Technologies Ltd., UK) 29 and from myometrial smooth muscle cells using the RNeasy mini RNA isolation kit (Qiagen, Crawley, West Sussex, UK). All RNA samples were DNase I treated using the DNA-free™ kit (Ambion, Spitfire Close, Huntingdon, Cambridgeshire, UK). RNA (500 ng – DNase I treated) was reverse transcribed into complementary DNA (cDNA) for use as a template for Polymerase Chain Reaction (PCR). The RNA samples were then denatured at 65°C for 10 minutes. Reverse transcription was performed at 42°C for 60 minutes in a reaction volume of 20 μl containing the following: oligo dT primer (500 ng), Moloney murine leukaemia virus (M-MLV) reverse transcription buffer (50 mol l^-3 Tris-HCl, pH 8.3, 75 mol l^-3 KCl, 3 mol l^-3 MgCl₂, 10 mol l^-3 dithiothreitol (DTT)) (Promega, Southampton Science Park, Southampton, UK), diethylpyrocarbonate (DEPC) treated water (BDH, UK), deoxyribonucleotide triphosphates (dNTPs) (0.2 mol l^-3) (Promega, UK) and 200U M-MLV reverse transcriptase (Promega, UK). Reverse transcriptase activity was stopped by heating samples at 65°C for 10 minutes. Control RNA samples, in which no reverse transcriptase was added, were included to confirm that no genomic DNA contamination was present.

0.5–1 μl of this 20 μl reaction was then used in the subsequent PCR. The PCR reaction was performed in a final volume of 50 μl containing 1.5 mol l^-3 MgCl₂, 20 mol l^-3 Tris-HCl, 50 mol l^-3, KCl pH 8.3 (Promega, UK), 1.25 U Taq DNA polymerase (Promega., UK), 40 mol l^-6 dNTPs and 10 mol l^-12 of each sense and antisense primer. cDNA amplification was carried out by an initial denaturation step of 5 minutes at 95°C followed by 34 cycles of denaturation at 94°C for 20 s, annealing at 57°C for 45 s and elongation at 72°C for 45 s, followed by a final extension step at 72°C for 10 minutes. 10 μl of each PCR product was then separated by gel electrophoresis on 1–1.5% (w/v) agarose gels. Products were separated alongside a 100 bp DNA molecular weight ladder (Promega, UK) for sizing. The oligonucleotides synthesised (MWG, Ebersberg, Germany) to PCR amplify the genes of interest were:

ARHGEF1 Sense 5'-AGCGAGTTCAAGAACCTGGA-3'

Antisense 5'-TCGTATATCTGGGCCTCCTG-3' [Genbank: NM_198977]

ARHGEF11 Sense 5'-GTCTCGAAAGGCAGAGAACG-3'

Antisense 5'-ATCCTTGCCCACTGTATGCT-3' [Genbank: NM_198236]

ARHGEF12 Sense 5'-GGAGCATCTGGGAATATGGA-3'

Antisense 5'-TCTTGCAGCTGAGGAATGTG-3' [Genbank: NM_015313]

ARHGAP5 Sense 5'-TCCCCCATCCTATACCATCA-3'

Antisense 5'-CAGGCATTTGCTTCTGTTCA-3' [Genbank: NM_001173]

ARHGAP24 Sense 5'-GGGCAACAGCAGCAACCACA-3'

Antisense 5'-TCGCTCGGCATTTCGCATTTTTAT-3' 18

ARHGDIA Sense 5'-CATCCAGATCCAGGAGC-3'

Antisense 5'-GACTTGATGCTGTAGCTGCC-3' 30

MSN Sense 5'-CTGATGGAGAGGCTGAAGCA-3'

Antisense 5'-TCTTGGACTCATCTCTGGCA-3' 31

ACTB Sense: 5'-CAACTCCATCATGAAGTGTGAC-3',

Antisense 5'-GCCATGCCAATCTCTCATCTTG-3' [Genbank: M10277]

Real-time PCR was performed on a 1/125 dilution of each the 7 PNL and 7 PL myometrial cDNA in triplicate for each transcript, using the Applied Biosystems ABI Prism 7000 sequence Detection System (ABI, USA). The PCR reactions were performed in a final volume of 25 μl containing 12.5 μl Sybr Green PCR Master Mix (ABI, USA), 5 μl diluted cDNA and 0.4 μM of each sense and antisense primer. The final volume of 25 μl was achieved using PCR grade water (Sigma-Aldrich, Ireland). cDNA amplification was performed by an initial step of 50°C for 2 minutes an initial denaturation step at 95°C for 10 minutes, followed by 40 cycles of denaturation at 95°C for 15 seconds, annealing at 60°C and elongation at 72°C for 30 seconds each. The sequences of the real time fluorescence RT-PCR oligonucleotide primers for the genes are as above, except ARHGEF12 and ARHGDIA, as alternative primers for these genes were utilised for the real time assay:

ARHGEF12 Sense 5'-TGTTGGTGACTCTCGGTTCA-3'

Antisense 5'-CCAATGAGTGGCACACAGTCT-3' 32

ARHGDIA Sense 5'-CAGGAAAGGCGTCAAGATTG-3'

Antisense 5'-GTCAGGAACTCGTACTCCTC-3' 33

Fluorescence data was acquired at the end of each PCR cycle. Melting curve analysis was performed by an initial denaturation step of 95°C for 15 seconds, cooling to 60°C for 10 seconds, and 72°C for 15 seconds. Fluorescence was measured continually during the melting curve cycle. The mean Cycle Threshold (Ct) of each gene for every patient (performed in triplicate) from their standard curves was normalised to the corresponding mean Ct value of β-Actin (ACTB), Gene Ct values/ACTB Ct values). β-Actin is a housekeeping gene, it is constitutively expressed and is used to normalise mRNA levels between different samples. The normalised Ct values of the 7 PL and the 7 PNL myometrial tissue types (PL v PNL) were analysed using the independent samples t test. Results were expressed as mean normalised Ct units ± the standard error of the mean (SEM). A P value of < 0.05 was considered to be statistically significant. Relative fold changes were then calculated using the difference in the mean normalised Ct values (x) between the pregnant at-term and the labouring myometrium for each transcript, Relative fold change = 2^x. All statistical analysis was performed using the SPSS statistical package using one way ANOVA with Tukey's post hoc analysis (Statistical Package for the Social Sciences, v.11, SPSS Inc., Chicago, IL, USA) and GraphPad Prism version 4 (GraphPad Software Inc., San Diego, CA, USA). Real time fluorescence RT-PCR products were gel electrophoresed, bands extracted, purified with the Qiaquick Gel Extraction kit (Qiagen, UK) and DNA sequence verified (MWG, Germany).

Human myometrial tissue was homogenised in ice-cold buffer containing 1% Triton-X (Sigma-Aldrich, Dublin, Ireland) and 0.1% sodium dodecyl sulphate (w/v) (SDS) (Sigma-Aldrich, Ireland). Cellular debris was removed by centrifugation at 10,000 × g, 4C for 15 minutes. The resultant supernatant was used for Western blot analysis. Human myometrial tissue or myometrial smooth muscle cells or Hela cells were homogenised in Protein lysis buffer: 50 mM Tris pH 7.4, 100 mM NaCl, 5 mM MgCl2, 0.1% Triton X-100, 10% glycerol with inhibitors (10 μg/ml leupeptin, 10 μg/ml aprotinin, 1 mM PMSF) ice-cold buffer (Sigma-Aldrich, Ireland). Cellular debris was removed by centrifugation at 10,000 × g, 4°C for 15 minutes. The resultant supernatant was used for Western blot analysis. Protein concentrations were determined using the BCA protein assay reagent kit (Pierce Technology, Rockford, Illinois, USA) as per the manufacturer's protocol, with bovine serum albumin as a standard.

Protein samples (30 μg) were heated at 95°C for 5 minutes after addition of an equal volume of 2X sample buffer containing: 62.5 mol l^-3 Tris pH 6.8, 2% SDS (w/v) (Sigma-Aldrich, Ireland), 10% glycerol (v/v) (BDH, Alkem Chemical Ltd, Dublin, Ireland), 5% 2-β mercaptoethanol w/v (Sigma-Aldrich, Ireland) or 5X Laemmli containing 50 mM DTT. This preparation was resolved by electrophoresis on 4–20% (Pierce Technology, USA) or 12% SDS (w/v) polyacrylamide gel electrophoresis (SDS-PAGE) gel at 100 V, room temperature for 60 minutes (BioRad, Hercules, CA, USA) in a buffer containing 25 mol l^-3 Tris base, pH 8.3, 19.2 mol l^-2 glycine, and 0.1% (w/v) SDS. The separated proteins were transferred to 0.2 μm nitrocellulose or PVDF membranes at a constant voltage of 100 V at 4C for 60 minutes in transfer buffer containing 25 mol l^-3 Tris base, 192 mol l^-3 glycine, and 20% methanol (v/v), high-performance liquid chromatography (HPLC) grade (Sigma-Aldrich, Ireland). To ensure transfer and equal loading of proteins, blots were stained with Ponceau S solution (Sigma-Aldrich, Ireland) for 5 minutes, followed by washing with de-ionised water, rapid immersion with agitation in 0.1 mol l sodium hydroxide (Sigma-Aldrich, Ireland), and finally washing under flowing de-ionised water for 3 minutes. Membranes were incubated for 1 hour at room temperature with phosphate-buffered saline (PBS; 1 mol l^-2 phosphate buffer, 2.7 mol l^-3 potassium chloride, and 1.37 mol l^-1 sodium chloride, pH 7.4) containing 0.05% Tween 20 (v/v) (Sigma-Aldrich, Ireland) and 5% low-fat milk powder (w/v) (Dawn Dairies, Westmeath, Ireland) to block nonspecific binding. Blots were either incubated for 60 minutes at room temperature or overnight at 4°C, with 1:200 dilution FilGAP antibody or a 1:100 dilution of p115RhoGEF (H-165) sc-20804, 1:100 dilution LARG (H-70) sc-25638, 1:100 dilution p190B RhoGAP (H-160) sc-30206) or 1:750–1000 dilution of RHOGDI (ARHGDIA) (A20 sc360 Santa Cruz Biotechnology, Inc, Heidelberg, Germany) rabbit polyclonal IgG anti-human primary antibodies or 1:100 dilution PDZ-RhoGEF (N-14 sc-46234), 1:1000 dilution of MSN (C-15 sc 6410), or 1:1000 dilution of p-MSN (Thr 558 sc 12895) goat polyclonal IgG anti-human primary antibodies (Santa Cruz Biotechnology, Germany), or with a 1:5000–15,000 dilution of β-actin (ACTB) mouse monoclonal antibody clone number AC-15 (Sigma-Aldrich, Ireland), diluted in PBS containing 3% bovine serum albumin (w/v) or 5% low-fat milk powder (w/v) (Dawn Dairies, Ireland), 0.03% Tween 20 (v/v) and 0.1% NaN₃ (v/v). Blots were then washed three times with PBS-0.03%T for 10 minutes each and incubated in a 1:4,000 dilution of a rabbit anti-goat horseradish peroxidase-conjugated antibody (DakoCytomation Ltd, Cambridgeshire, UK) or 1:1000 dilution anti-rabbit secondary (Pierce Technology, USA) or 1:4000 swine anti-rabbit IgG horseradish peroxidase-conjugated antibody (P-0217 DakoCytomation Ltd, Cambridgeshire, UK) or in 1:4000 dilution of a goat anti-mouse horseradish peroxidase-conjugated antibody (sc2005 Santa Cruz Biotechnology, Germany) or 1:1000 anti-mouse secondary (Pierce Technology, USA) in 1X PBS, 3%BSA (w/v), 0.05% Tween 20 (v/v) or in 1X PBS, 5% low-fat milk powder (w/v) (Dawn Dairies, Ireland) with 0.05% Tween 20 for 1 hour at room temperature. Blots were then washed with PBS-0.05%T. Bound secondary antibody was detected using the West-Pico chemiluminescent detection kit as per the manufacturer's protocol (Pierce Technology, USA) or Immobilon Western chemiluminescent HRP substrate (Millipore, USA). The membranes were scanned with the fluorescence imager (FluorchemTM 8900, Alpha Innotech Corporation, San Leandro, California, USA) and AlphaEaseFC software was used to detect the signal, the image was processed and protein expression levels where possible, were determined by densitometric analysis compared to corresponding levels of the housekeeping protein, β-Actin (ACTB). Statistical analysis of the densitometric data (one way ANOVA and Tukey's post hoc analysis) and graph construction were performed using GraphPad Prism version 4 (GraphPad Software Inc., USA).

Primary myometrial cells (to passage 8) were cultivated on LabTekII 8 well chamber slides (Nalge Nunc Int., Naperville, IL, USA) overnight. The samples were fixed in 4% paraformaldehyde (w/v) for 30 minutes at room temperature and blocked in 1X PBS, 0.01% Triton X-100 (v/v) and 5% serum (v/v) (donkey or goat). Cells were subsequently incubated with primary antibody in blocking solution, either a 1:50 dilution of MSN (C-15 sc 6410) goat polyclonal IgG anti-human primary antibody, p-MSN (Thr 558 sc12895) goat polyclonal IgG anti-human primary antibody or RHOGDI (ARHGDIA) (A20 sc360) rabbit polyclonal IgG anti-human primary antibody (Santa Cruz Biotechnology, Germany) in PBS/1%BSA overnight at 4°C. Samples were rinsed in 1XPBS 3 times and incubated with Alexa Fluor 488 donkey anti-goat IgG (A11055) or Alexa Fluor 488 goat anti-rabbit IgG (A11070) (Molecular Probes, Eugene, OR, USA) for 1 hour at room temperature and then rinsed in PBS. Control cells were incubated with the secondary antibody alone. After washing the coverslips were mounted on glass slides with Vectashield mounting medium with DAPI (Vector Laboratories, Burlingame CA, USA). Fluorescent images were obtained using the Laser Scanning Microscope LSM 510 (confocal microscope) (Carl Zeiss AG, Strasse 22, Oberkocken, Germany) and the DP70 fluorescence microscope (Olympus, Tokyo, Japan).

RT-PCR analysis demonstrated expression of ARHGEF1, ARHGEF11, ARHGEF12, ARHGAP5, ARHGAP24 and MSN mRNA in human myometrial smooth muscle cells, that were sub-passaged from primary cells in culture (Figure 4a(i–vi)).

Western Blot analysis demonstrated expression of pMSN and ARHGDIA proteins in the human myometrial smooth muscle cells (Figure 4b(i–ii)).

Immunolabelling confocal microscopy localised ARHGDIA protein to the cytoplasm of human myometrial smooth muscle cells (Figure 11a (i) and (ii)). MSN and pMSN immunofluorescence studies localised the proteins to vesicle structures in the cytoplasm of the myometrial smooth muscle cells (Figure 11c and 11d).