Effects of icaritin on MAPK pathways.

<p>Hec1A cells were treated with the indicated icaritin concentration or the indicated interval, total cellular extracts were prepared and subjected to Western blot assay to measure levels of phosphorylated forms of ERK1/2 (A), JNK (B) and p38 (C). Membranes were reprobed with antibodies against total ERK1/2, JNK and p38 for normalization.</p

Icaritin inhibits Hec1A cells growth.

<p>(A) The chemical structure of icaritin. (B) Effects of icaritin on the growth inhibition of Hec1A cells. Cells were maintained in phenol red-free media with 2.5% charcoal-stripped fetal serum for 24 h, and then treated with the indicated concentration of icaritin. Cells were harvested at different time points as the indicated and proliferation potential was assessed by MTT assay. Results of eight independent experiments were averaged and mean ± SEM. *, <i>P</i><0.05 compared to control cells.</p

Icaritin induced Hec1A cells apoptosis.

<p>(A) Visualization of apoptotic cells by the TUNEL assay on Hec1A cells. (B) DNA fragmentation was evaluated using a Cell Death Detection ELISA kit. The data are expressed as mean ± SEM of three separate experiments. *, <i>P</i><0.05 compared to control cells. (C) The apoptotic status was determined by Annexin V/PI staining method. Percentages of negative (viable) cells, annexin V-positive (early apoptotic) cells, PI-positive (necrotic) cells, or annexin V and PI double-positive (late apoptotic) cells were shown (mean of three independent experiments) by a flow cytometry analysis.</p

Effects of icaritin on cell cycle regulators in Hec1A cells.

<p>(A) Hec1A cells were treated with the indicated concentration of icaritin for 24 h. The levels of cyclin D1 and cdk4 were determined by western blot. Protein levels of β-actin were also measured as controls. (B) Hec1A cells were treated with the indicated concentration of icaritin for 24 h. The levels of p21 and p27 were determined by western blot. Protein levels of β-actin were also measured as controls.</p

Icaritin induced Hec1A cells apoptosis was mediated through ERK1/2 activation.

<p>(A) Hec1A cells were treated with icaritin in the presence or absence of 10 µM U0126, 10 µM SP600125, or 40 µM SB203580 for 24 h, Cell growth was determined by MTT. The bars represent the mean ± SEM of three separate experiments. *, <i>P</i><0.05 compared for the icaritin-treated group. (B) Hec1A cells were treated with icaritin in the presence or absence of 10 µM U0126, 10 µM SP600125, or 40 µM SB203580 for 24 h, and DNA fragmentation was determined. The data are expressed as mean ± SEM of three separate experiments. *, <i>P</i><0.05 compared to icaritin-treated cells. (C) Hec1A cells were treated with icaritin in the presence or absence of 10 µM U0126, 10 µM SP600125, or 40 µM SB203580 for 24 h, and caspase-3 activity was determined by caspase-Glo assay. The data are expressed as mean ± SEM of three separate experiments. *, <i>P</i><0.05 compared to icaritin-treated cells. (D) Hec1A cells were pretreated or not pretreated with 10 µM U0126 then added with DMSO (vehicle) or 10 µM icaritin for 12 h, 24 h respectively. Protein extracts were prepared and subjected to Western blot assay to measure levels of phosphorylated ERK1/2. Protein levels of ERK1/2 were also measured as controls. (E) Hec1A cells were pretreated or not pretreated with 10 µM U0126 then added with DMSO (vehicle) or 10 µM icaritin for 12 h, 24 h respectively. Protein extracts were prepared and the cleavage of PARP was analyzed with Western bolt. Protein levels of β-actin were also measured as controls. (F) Hec1A Cells were pretreated with or without z-VAD-fmk (10 µM) were further incubated with 10 µM icaritin for 24 h. Protein extracts were prepared and subjected to Western blot assay to measure levels of phosphorylated ERK1/2. Protein levels of ERK1/2 were also measured as controls.</p

Induction of caspases activities by icaritin.

<p>(A) Hec1A cells were treated with the indicated icaritin concentration or the indicated time points, total cellular extracts were prepared and subjected to Western blot assay to measure levels of cleaved-caspase-3 and cleaved-caspase-9. Protein levels of β-actin were also measured as controls. (B) Hec1A cells were treated with the indicated icaritin concentration or the indicated time points, total cellular extracts were prepared and subjected to Western blot assay using antibodies against PARP. Protein levels of β-actin were also measured as controls. (C) Hec1A cells were fixed and labeled for cytochrome c (red) and DNA (blue). (D) Hec1A cells were pretreated with 10 µM of z-VAD-fmk for 1 h, followed with 10 µM icaritin for 24 h, and DNA fragmentation and caspase-3 activity were determined. The data are expressed as mean ± SEM of three separate experiments. *, <i>P</i><0.05 compared to control cells. (E) Hec1A cells were pretreated with 10 µM of z-VAD-fmk for 1 h, followed with 10 µM icaritin for 24 h. Protein extracts were prepared and subjected to Western blot assay using antibody against PARP. Protein levels of β-actin were also measured as controls.</p

Effects of icaritin on the expression of Bcl-2 family.

<p>(A) Hec1A cells were treated with the indicated concentration of icaritin, total cellular extracts were prepared and subjected to Western blot assay to measure levels of Bcl-2 and Bax. Protein levels of β-actin were also measured as controls. (B) Hec1A cells were treated with the indicated icaritin time points of icaritin, total cellular extracts were prepared and subjected to Western blot assay to measure levels of Bcl-2 and Bax. Protein levels of β-actin were also measured as controls.</p

The Roles of Parathyroid Hormone-Like Hormone during Mouse Preimplantation Embryonic Development

<div><p>Parathyroid hormone-like hormone (PTHLH) was first identified as a parathyroid hormone (PTH)-like factor responsible for humoral hypercalcemia in malignancies in the 1980s. Previous studies demonstrated that PTHLH is expressed in multiple tissues and is an important regulator of cellular and organ growth, development, migration, differentiation, and survival. However, there is a lack of data on the expression and function of PTHLH during preimplantation embryonic development. In this study, we investigated the expression characteristics and functions of PTHLH during mouse preimplantation embryonic development. The results show that <em>Pthlh</em> is expressed in mouse oocytes and preimplantation embryos at all developmental stages, with the highest expression at the MII stage of the oocytes and the lowest expression at the blastocyst stage of the preimplantation embryos. The siRNA-mediated depletion of <em>Pthlh</em> at the MII stage oocytes or the 1-cell stage embryos significantly decreased the blastocyst formation rate, while this effect could be corrected by culturing the <em>Pthlh</em> depleted embryos in the medium containing PTHLH protein. Moreover, expression of the pluripotency-related genes <em>Nanog</em> and <em>Pou5f1</em> was significantly reduced in <em>Pthlh</em>-depleted embryos at the morula stage. Additionally, histone acetylation patterns were altered by <em>Pthlh</em> depletion. These results suggest that PTHLH plays important roles during mouse preimplantation embryonic development.</p> </div

Primer sequences used for real-time PCR.

<p>Primer sequences used for real-time PCR.</p

Effect of PTHLH (1–141) on development of mouse embryos injected with <i>Pthlh</i> siRNA.

*<p>p<0.01 compared with the control group.</p