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

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

Effects of E2 and E2-BSA on the activation of PKCδ, PKCα and PKA in Ishikawa cells.

<p>A and B. Serum starved Ishikawa cell was treated with 10 nM E2 or 10 nM E2-BSA for the indicated time points. Protein extracts were prepared and used for Western blot analysis to measure levels of PKCδ phosphorylation. Protein levels of total PKCδ were also examined as controls. Each bar represents mean value ± SEM (n = 3). *, P<0.05 compared to untreated cells. C. Serum starved Ishikawa cell was treated with 10 nm E2 for the indicated time points and cells were lysed for western blot analysis to measure levels of PKCα phosphorylation. Protein levels of total PKCα were measured as controls. D, Serum starved Ishikawa cell was treated with 0, 0.1,1, 10, 100 and 1,000 nM E2 for 20 min. Protein extract was prepared for Western blot analysis to measure levels of PKCα phosphorylation and total PKCα. E and F, Serum starved Ishikawa cells were treated with 10 nM E2 or 10 nM E2-BSA for indicated time points, 20 µM Forskolin (F) was added for 15 min as a positive control, after which the cells were lysed and tested in the PepTag PKA assay. Samples were separated on an agarose gel. The lower band represents phosphorylated peptide, and the upper band represents the remaining unphosphorylated peptide.</p

ER-α36 mediates E2-stimulated PKCδ activation.

<p>A and B. ER-α36 expression in Ishikawa/V and Ishikawa/RNAiER36 cells. Each bar represents mean value ± SEM (n = 3). *, P<0.05 compared to Ishikawa/V cells. C. Ishikawa/V and Ishikawa/RNAiER36 cells were treated with 10 nm E2 or 10 nm E2-BSA for 10 min, and PKCδ phosphorylation was analyzed by Western blot. Total levels of PKCδ were measured as controls, and each bar repents mean value ± SEM (n = 3). *, P<0.05 compared to untreated cells. #, P<0.05 compared to E2- or E2-BSA- treated Ishikawa/V cells. D and E. ER-α66 expression in Ishikawa/V and Ishikawa/RNAiER66 cells. Each bar represents mean value ± SEM (n = 3). *, P<0.05 compared to Ishikawa/V cells. F. Ishikawa/V and Ishikawa/RNAiER66 cells were treated with 10 nM E2 or 10 nM E2-BSA for 10 min, and then PKCδ phosphorylation was assessed with Western blot. Total PKCδ was measured as controls. Each bar represents mean value ± SEM (n = 3). *, P<0.05 compared to untreated cells. G, Hec1A/V and Hec1A/RNAiER36 cells were treated with 10 nM E2 or 10 nM E2-BSA for 10 min, and PKCδ phosphorylation was analyzed by Western blot. Total PKCδ was measured as controls. Each bar represents mean value ± SEM (n = 3).*, P<0.05 compared to untreated cells. #, P<0.05 compared to E2- or E2-BSA-treated Hec-1A/V cells.</p