2.3. Histological and Immunostaining Analysis
Uteri were fixed in 4% paraformaldehyde for histology and immunostaining. After embedded in paraffin block, uterine blocks were sectioned with 5 μm-thickness using a microtome and put on Superfrost Plus Stain slides (Thermo Fisher Scientific, Waltham, MA, USA). For immunofluorescent assay, the slides were deparaffinized and boiled in sodium citrate buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0) for 40 min using antigen retrieval steamer (IHC world, Gyeonggi-do, Korea). The antigen-retrieved slides were washed in PBS-T (PBS containing 0.05% Tween-20) and blocked by incubating in blocking buffer (PBS-T with 4% BSA) for 4 h (h) at room temperature. Then, the slides were treated with the indicated primary antibodies for overnight at 4 °C. After washing with PBS-T, the slides were incubated in blocking buffer containing the secondary antibody conjugated with Alexa-Fluor antibodies 488 or 546 (Thermo Fisher Scientific, Waltham, MA, USA) at room temperature for 1 h. The slides were washed with PBS and treated with DAPI (4′,6-diamidino-2-phenylindole) (Thermo Fisher Scientific, Waltham, MA, USA). After washing, the slides were mounted by treating with Mounting Medium (DAKO, Glostrup, Denmark) and covered with glass coverslip. The expression and localization of proteins were analyzed using confocal microscopy (Leica Microsystems, Wetzlar, Germany). Images were converted to JPEG format at the resolution of 1024 × 1024 pixels. The intensity of positive signals was calculated by the software LAS X (Leica Microsystems, Wetzlar, Germany) and Image J software.
To perform immunohistochemical staining, the deparaffinized slides were treated with 0.3% H2O2 in distilled water to quench endogenous peroxidase activity before antigen retrieval. After antigen retrieval, the sections were incubated in a blocking buffer (PBS-T with 5% normal serum and 1% BSA) and treated with the indicated antibody followed by HRP-conjugated secondary antibody (Thermo Fisher Scientific, Waltham, MA, USA). After washing, the signals from antibody were acquired by incubating with DAB solution (Vector Laboratories, Burlingame, CA, USA). The slides were counterstained with hematoxylin. The slides were dehydrated and mounted with Permount Mounting Medium (Thermo Fisher Scientific, Waltham, MA, USA).
The primary antibodies used in this study are as follows; anti-STK4 (ab51134, Abcam, Burlington, CA, USA), anti-STK4 phospho-Thr183 (FITC) (orb9297, Biorbyt, Cambridge, UK), anti-STK4 phospho-Thr183/STK3 phospho-Thr180 (#3681, Cell Signaling Technology, Seoul, Korea), anti-LATS1 phospho-Thr1079 (ABIN872292, Antibodies-online, Limerick, PA, USA), anti-YAP (sc-271134, Santa Cruz Biotechnology, Dallas, TX, USA), anti-YAP phospho-Ser127 (orb8478, Biorbyt, Cambridge, UK), and anti-β-Actin (sc-47778, Santa Cruz Biotechnology, Dallas, TX, USA)
2.4. Ovariectomy and Hormone Treatments
Mice were ovariectomized to investigate the effects of estrogen and progesterone on the expression of Hippo signaling-related genes in the uterus as previously described [
8]. After 14 days of recovery, β-estradiol (E
2, 200 ng/mouse, Sigma-Aldrich, St. Louis, MO, USA) or progesterone (P
4, 2 mg/mouse, Sigma-Aldrich, St. Louis, MO, USA) was subcutaneously injected into mice. Mice were sacrificed and the uteri were collected at 0, 1, 3, 16, 12, and 24 h after hormone treatment. Five mice were used in each group. To test estrogen receptor-mediated regulation of gene expression, ovariectomized mice were either injected with E
2 (200 ng/mouse) or pretreated with estrogen receptor antagonist, ICI 182,780 (ICI, 500 μg/mouse, Sigma-Aldrich, St. Louis, MO, USA) 30 min before E
2 injection. Sesame oil (100 μL/mouse, Sigma-Aldrich, St. Louis, MO, USA) was used as control.
2.5. RNA Preparation, RT-PCR, and qRT-PCR
Total RNAs were isolated from uteri using RNeasy total RNA isolation kit (Qiagen, Hilden, Germany) according to the manufacturer’s instruction. The total RNAs (2 μg) were reverse-transcribed using SuperScript
® III First-Strand Synthesis System (Thermo Fisher Scientific, Waltham, MA, USA). The reverse-transcribed cDNAs were used for RT-PCR and quantitative RT-PCR analyses. RT-PCR was performed using C1000 Touch™ Thermal Cycler (Bio-Rad Life Sciences, Hercules, CA, USA). The annealing temperatures used in the thermal cycling were indicated in
Table 1. The products were analyzed in 2% agarose gel.
Semi-quantitative RT-PCR analysis was performed using The iQ™ SYBR
® Green Supermix (Bio-Rad Life Sciences, Hercules, CA, USA) and the CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad Life Sciences, Hercules, CA, USA). The results were analyzed with the iQ5™ Optical system software (Bio-Rad Life Sciences, Hercules, CA, USA). The relative gene expression was calculated by the comparative CT (ΔΔCT) method [
11]. The relative gene expression was compared with the amounts of either ribosomal protein L-7 (
Rpl7) or
Gapdh as reference genes.
2.6. Knockdown of STK4 Expression in Human Uterine Endometrial Cells
To examine the effect of STK4 knockdown on gene expression in human endometrial cells, Ishikawa cell line was used. Ishikawa cells were transfected with STK4 siRNAs (SR415716, Dharmacon, Lafayette, CO, USA) or universal scrambled negative control siRNA (SR30004, Dharmacon, Lafayette, CO, USA) using Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA).
2.7. Western Blot and Statistics
Total proteins from cells or uteri were separated on 10% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membrane. After blocking with ProNATM phospho-block solution (TransLab, Seoul, Korea), the membrane was incubated with the indicated primary antibody diluted in ProNATM phospho-block solution (TransLab, Seoul, Korea) at 4 °C for overnight. The membrane was treated with HRP-conjugated secondary antibody (OriGene Technologies, Rockville, MD, USA) in ProNATM phospho-block solution to detect protein expression. The immunoreactive bands were detected by chemiluminescence using ECL Western Blotting substrate kit (GenDEPOT, Barker, TX, USA). The relative expression was imaged by ChemiDoc XRS system (Bio-Rad Life Sciences, Hercules, CA, USA) and analyzed by One-way ANOVA analysis.