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 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

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

ER-a36 mediates E2 stimulated cell proliferation.

<p>A. Ishikawa/V and Ishikawa/RNAiER36 cells were treated with 10 nM E2-BSA for 24 h, 48 h, 72 h and 96 h. MTT assay was performed as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015408#s4" target="_blank">materials and methods</a>. Results of three independent experiments were averaged and mean value ± SEM are shown. *, P<0.05 compared to E2-BSA treated Ishikawa/V cells respectively. B. Ishikawa cells were treated with 10 nM E2-BSA alone or together with 5 µM rottlerin, a PKCδ specific inhibitor, or 5 µM bisindolylmaleimide, a pan-PKC inhibitor, or HBDDE, a PKCα specific inhibitor, or 5 µM H89, a PKA specific inhibitor for 72 h, and MTT assays were then performed. Results of three independent experiments were averaged and mean value ± SEM are shown. *, P<0.05 compared to control cells.</p

The GVBD inhibition after preservation for different times in different media at 27.5°C.

<p>Data are presented as means ± SEM from three replicated experiments.</p>1<p>Means oocytes treated in every group.</p>2<p>Percentages are based on the total number of oocytes examined in every group.</p>A, B<p>Values with different superscripts are significantly different in each column (P<0.05).</p>a, b<p>Values with different superscripts are significantly different in each line (P<0.05).</p

Overexpression of GPR3 inhibits meiotic resumption in porcine oocytes.

<p>(A) Oocytes cultured in normal medium without HX were injected with 2.5 mg/ml Myc₆-GPR3 or control Myc₆ and then cultured for 24 h or 30 h. The GVBD rates of the injected oocytes are shown along with non-injected oocytes. (B) Cyclin B (upper panel) and CDC2 (middle panel) levels were detected by western blot using 150 oocytes in each sample. The results are shown along with those of non-injected oocytes cultured without HX. Samples were collected after culture in medium without HX at 0 h, 24 h and 30 h. (C) cAMP levels of porcine oocytes in the Myc₆-GPR3 injection group and control Myc₆ injection group after culture without HX at 24 h or 30 h. Dotted line represents cAMP level at time 0. (D) cGMP levels of porcine oocytes in the Myc₆-GPR3 injection group and control Myc₆ injection group after culture without HX at 24 h or 30 h. Dotted line represents cGMP level at time 0. The number “n” on top of the bars indicates the total number of treated oocytes in each group. Data are shown as mean ± SEM of at least three repeated experiments and letters ‘a’ and ‘b’ indicate statistically significant difference (p<0.05).</p