Fig 2: Expression of STK3 and STK4 in the mouse uteri during the estrous cycle. (A,B) The total RNA was isolated from the tissues of 7-week-old mice. RT-PCR and qRT-PCR analysis for STK3 and STK4 transcripts in the mouse uteri at four stages of the estrous cycle (P, proestrus; E, estrus; M, metestrus; D, diestrus). Relative expression level of Stk4 was normalized with Rpl7 transcript. Data were shown with mean ± SEM. p-value; ** p < 0.05. (C) Western blot analysis of STK3/4 protein was performed using whole cell lysate from mouse uteri during estrous cycle. (D) Vaginal smear assays confirming each stage of the estrous cycle. LK, leukocyte; NE, nucleated epithelial cells; CE, cornified epithelial cells. (E) Immunohistochemical analysis of STK3/4 and phosphorylated STK3/4 (pSTK3/4) in the 7-week-old mouse uteri at different stages during the estrous cycle. Negative control image is a proestrous uteri stained using normal rabbit IgG (IgG control). LE, luminal epithelium; GE, glandular epithelium; S, stroma. Images were analyzed using a confocal microscope. White bar represents scale bar (scale bar; 25 µm).

Fig 3: Regulation of STK3/4 expression through estrogen receptor. (A,B) Ovariectomized mice were treated with an estrogen receptor antagonist, ICI 182,780 (ICI, 500 µg), for 30 minitues (min) before estrogen (E2, 200 ng) injection and sacrificed at 6 h after E2 administration. STK4 expression was analyzed by RT-PCR and western blot analysis. Data were shown with mean ± SEM. p-value; ** p < 0.01 (C) Immunostaining images showed localization of STK3/4 (red) and pSTK3/4 (green) in uteri of ovariectomized mice treated with oil, E2, or a combination of E2 and ICI. LE, luminal epithelium; GE, glandular epithelium. Nuclear DNA was stained with DAPI. The scale bars indicate 25 µm.