OEOA was retained for a longer period in mouse blood plasma than OA.

<p>MS-MS product ion mass spectra of OEOA (A) and OA (B). (C) K562 cells were treated with OA or OEOA (1 µM) for 6 h. Culture media and cell lysates were collected at indicated time points for HPLC-MS/MS analysis. Data was represented as mean ± SEM in cell lysate compared to total exposure, n = 2 (* <i>p</i><0.05). (D) Concentrations of OA and OEOA in mouse plasma after intraperitoneal injection. Blood was collected from mice at different time points after single administration of OA and OEOA. Data was represented as mean ± SEM, n = 2 animals per time point (* <i>p</i><0.05).</p

OEOA did not induce cell death in K562 and HEL cells.

<p>K562 (A & C) and HEL (B & D) cells were treated with OEOA (1 µM) for 6 days. Cell viability was measured by trypan blue exclusion as described in Materials and Methods. Data are mean ± SEM of three independent experiments (* <i>p</i><0.05).</p

OEOA attenuated phosphorylation of Rb protein in K562 and Jurket cells.

<p>(A) K562 and Jurket cells were treated with OEOA (0.1–10 µM) for 2 days, and the cell lysates were subjected to Western blot analysis for p-Rb and Rb. Actin served as an equal loading control. Histograms in (B) show the relative expression of p-Rb (normalized to actin) as compared to the vehicle-treated cells. Results were representative blots from three separate experiments, (* <i>p</i><0.05).</p

OEOA induced G1 cell cycle arrest in K562 cells.

<p>(A) Cells were incubated with OEOA (1 or 10 µM) for 24 h. The distribution of cell cycle was examined by PI staining method. The table summarized the distribution of cells in OEOA-treated or control cells. Data represented mean ± SEM of three independent experiments (* <i>p</i><0.05). (B) K562 cells were cultured in the presence of OEOA (1 or 10 µM) for 2 days. Total proteins were collected for Western blot analysis to detect the expression of p27, Cdk4, Cdk6, Cyclin D1, Cyclin E and RAMP. Actin served as an equal loading control. Histograms on the right show the relative expression of various proteins (normalized to actin) as compared to the control cells. Results were representative blots from three separate experiments, (* <i>p</i><0.05).</p

OEOA promoted erythroid differentiation in K562 cells.

<p>K562 cells were treated with OEOA (0.1–10 µM) for 2 days. Total RNA was reverse transcribed and subjected to real time-PCR analysis with primers specific to <i>γ-globin</i> (A) and <i>cd41b</i> (B), respectively. <i>hprt1</i> served as an internal housekeeping gene control. Data were expressed as fold change to the control cells as mean ± SEM of three independent experiments (* <i>p</i><0.05). (C) K562 cells were treated with OEOA (0.1–10 µM) for 2 days. Western blot analysis of Bcr-Abl and Erk1/2 was performed. Actin served as an equal loading control. Histograms on the right show the relative expression of various proteins (normalized to actin) as compared to the control cells. Results were representative blots from three separate experiments, (* <i>p</i><0.05).</p

OEOA inhibited cell proliferation in leukemia cell lines.

<p>Different cell lines were treated with various concentrations of OEOA or OA for 2 days. Cell growth was measured by MTT assay: (A) K562, (B) HEL, (C) Jurket (D) HEKneo, and (E) HepG2, MCF<b>-</b>7 and HeLa cells. Data are mean ± SEM of three independent experiments (* <i>p</i><0.05).</p

Axin is expressed at neuronal synapses.

<p>(A) Axin co-localized with PSD-95 puncta in hippocampal neurons. Hippocampal neurons (20 DIV) were stained with antibodies against Axin and PSD-95. Upper panels: representative images. Scale bar: 25 μm. Lower panels: higher-magnification images showing Axin colocalization with PSD-95 at synapses. Scale bar: 10 μm. (B) Axin was readily detected in the P2′, SPM, and PSD fractions prepared from mouse brains. PSD-95 and synaptophysin are pre- and postsynaptic markers, respectively. Hom: homogenate; P1: nuclear fractions; P2′: crude synaptosomal fraction; SPM: synaptic plasma membrane; PSD: postsynaptic density. (C) Mass spectrometry analysis identified unique peptides representing CaMKIIα and CaMKIIβ in the mouse brain synaptosomal fraction pulled down by Axin antibody. (D) Co-immunoprecipitation assay demonstrated that Axin strongly associated with CaMKIIα and CaMKIIβ in HEK293T cells. (E) CaMKIIα was co-immunoprecipitated with Axin from the mouse brain synaptosomal fraction. (F) Schematic structure of Axin protein. (G) Amino acids 216–353 of Axin were important for Axin and CaMKIIα interaction.</p

Axin is required for dendritic spine morphogenesis.

<p>(A–D) Axin knockdown led to the simplification of dendritic trees and reduction of dendritic spine number. (A) Upper panels: representative images showing hippocampal neuron morphology. Scale bar: 20 μm. Lower panels: higher-magnification images showing dendritic spines morphology. Scale bar: 10 μm. (B) Axin knockdown significantly reduced protrusion density, which was partially rescued by re-expressing the RNAi-resistant form of Axin. One-way ANOVA, <i>n</i> = 45, **<i>p</i> < 0.01 shAxin vs shAxin+Axin, ***<i>p</i> < 0.001 shAxin vs Con. (C) The number and total length of dendrites in Axin-knockdown neurons were reduced. One-way ANOVA, <i>n</i> = 15, *<i>p</i> < 0.05 shAxin vs shAxin+Axin, ***<i>p</i> < 0.001 shAxin vs Con. (D) Sholl analysis showed that the complexity of dendritic trees was reduced in Axin-knockdown neurons. <i>n</i> = 15. (E) Lentiviral knockdown of Axin in the hippocampal CA1 region reduced dendritic spine density. Left panel: representative image showing virus-infected neurons in the hippocampal CA1 region. Scale bar: 50 μm. Right panels: higher-magnification images showing the dendritic spines along dendrites. Scale bar: 10 μm. (F) Silencing Axin significantly reduced dendritic spine density in the CA1 region. Student’s <i>t</i>-test; GFP, <i>n</i> = 56; shRNA, <i>n</i> = 24; ***<i>p</i> < 0.001. (G) Overexpression of Cdc42 but not Rac1 rescued the defective dendritic spine phenotype in Axin-knockdown neurons. Left panels: representative images showing the dendritic morphology. Right panels: quantitation of dendritic spine density. Scale bar: 10 μm; one-way ANOVA, <i>n</i> = 15, **<i>p</i> < 0.01 shRNA+vector vs shRNA+Cdc42; shRNA+Rac1 vs shRNA+Cdc42.</p

Axin stabilization increases dendritic spine density and synaptic transmission.

<p>(A) Treating hippocampal neurons (17 DIV) with the Axin stabilizer XAV939 for 3 days significantly increased the density of mature dendritic spines and total protrusions. Scale bar: upper panels = 20 μm, lower panels = 10 μm; Student’s <i>t</i>-test, <i>n</i> = 24, *<i>p</i> < 0.05. (B) XAV939 treatment increased PSD-95–positive puncta along dendrites. Left panels: representative images showing the dendritic morphology of control and XAV939-treated neurons. Right panels: quantitation of PSD-95 puncta density and intensity. Scale bar: 10 μm; Student’s <i>t</i>-test, <i>n</i> = 15, *<i>p</i> < 0.05, **<i>p</i> < 0.01. (C) Representative mEPSC traces of control and XAV939-treated neurons. (D) XAV939 treatment increased the frequency but not the amplitude of mEPSCs in hippocampal neurons. One-way ANOVA, <i>n</i> = 22, **<i>p</i> < 0.01 vs DMSO for 72 h. (E) Cortical neurons were treated with XAV939 for the indicated times. Enhanced GluA1 phosphorylation at Ser831 was observed from 0.5–72 h after treatment. (F) Live imaging demonstrated that XAV939 treatment did not induce an obvious change in the formation rate of dendritic spines but significantly reduced their elimination rate. Left panels: representative images showing spine morphology in cultured neurons. Right panels: quantitative results of spine formation/elimination rate. Scale bar: 10 μm; Student’s <i>t</i>-test, <i>n</i> = 21, ***<i>p</i> < 0.001.</p

The COG function annotaion of <i>G. lucidum</i>.

<p>Distribution of Genes in different COG function classification.</p