The lymphocytes exposed to BE <i>in vivo</i> underwent increased apoptosis upon subsequent culture <i>in vitro</i>.

<p>Murine hepatitis was induced by an intravenous injection of Con A at a dose of 15 mg/kg. BE (100 mg/kg) was administrated intraperitoneally immediately after Con A injection. The liver MNCs and splenocytes were prepared at 8 h after Con A injection. After 2 hours of in-vitro culture, the adherent macrophages were removed and the nonadherent cells were further cultured <i>in vitro</i> for 12 and 24 h. (A) The percentages of apoptosis in the <i>in vitro</i> cultured nonadherent cells at each time point were determined by flow cytometry using FITC-annexin V/PI staining. (B) After <i>in vitro</i> culture for 24 h, the absolute numbers of apoptotic cells in liver MNCs and splenocytes, as well as in CD3⁺ T cell and CD19⁺ B cell subsets, were determined by cell counting and flow cytometry using PE-anti-CD3/FITC-annexin V staining and PE-anti-CD19/FITC-annexin V staining, respectively. Data are mean ± SEM of 6–8 mice/group. *<i>P</i><0.05 versus Con A only control, ^#<i>P</i><0.05 versus Non-treatment control.</p

BE preferentially induces apoptosis in mitogen-activated splennocytes.

<p>Murine splenocytes were treated with indicated concentrations of BE for 24 h in the absence or presence of 5 µg/ml of Con A, and the percentages of apoptosis were detected using Annexin V/PI staining. (A) is a representative of three independent assays. (B) represents mean ± SEM of three independent experiments. *<i>P</i><0.05 versus untreated controls.</p

BE selectively induces apoptosis of LPS-activated CD19⁺ B cells through mitochondrial pathway.

<p>CD19⁺ B cells were isolated from murine splenocytes using Miltenyi MACS Purification and incubated with indicated concentrations of BE for 24 h in the absence or presence of 10 µg/ml of LPS. (A, B) The percentages of cells with low ΔΨm were analyzed by flow cytometry using JC-1 staining. (C, D) The percentages of active caspase-3+ cells were analyzed by flow cytometry using FITC-anti-active caspase-3 mAb staining. A and C demonstrate representative experiment of three assays. B and D show mean ± SEM of three independent experiments. (E) The release of cytochrome c (Cyto-c) from mitochondria in B cells after BE treatment in the presence of LPS was examined by Western blotting. (F) Protein levels of Bcl-2 and Bax in B cells after BE treatment in the presence of LPS were examined by Western blotting. The results shown in E and F are representative of three experiments. (G) The activities of caspase-3, 8, 9 in B cells after BE treatment in the presence of LPS was measured using colorimetric assay. Each column represents the mean ± SEM of 3 experimental values. *<i>P</i><0.05 versus untreated controls.</p

BE protected mice against Con A-induced hepatitis and increased the incidences of apoptosis in liver-infiltrating MNCs and splenocytes, as well as in CD3⁺ and CD19⁺ splenocytes.

<p>Murine hepatitis was induced by an intravenous injection of Con A at a dose of 15 mg/kg. BE (100 mg/kg) was administrated intraperitoneally immediately after Con A injection. Blood samples were collected at 0, 8, and 24 h after Con A injection. Livers and spleens were collected 24 h after Con A injection. (A) Serum ALT levels at indicated time points after Con A injection. (B) Serum levels of IFN-γ and TNF-α at indicated time points after Con A injection. (C) Photomicrographs of representative H&E stained liver sections (×200). (D) Cell numbers of MNCs, CD3⁺ T cell and CD19⁺ B cells in livers at 24 h after Con A injection. (E) Percentages of apoptosis in liver MNCs detected by flow cytometry using FITC-annexin V/PI staining. (F and G) Flow cytometry analysis of apoptosis in splenic T and B cells using PE-anti-CD3/staining and PE-anti-CD19/FITC-annexin V staining, respectively. F shows representative results of 10 mice in each experimental group. Data in A, B D, E, and G are mean ± SEM of 10 mice/group. *<i>P</i><0.05 versus Con A only control, ^#<i>P</i><0.05 versus Non-treatment control.</p

BE selectively induces apoptosis in activated lymphocytes.

<p>(A) Different types of immune cells as indicated were incubated with 10 µM of BE for 24 h in the absence or presence of activator, and the percentages of apoptosis were detected using Annexin V/PI staining. The increase in the percentages of apoptosis in each type of cells upon BE exposure was calculated. Following activators were used: 5 µg/ml of Con A for splenocytes and CD3⁺ splenocytes; 500 ng/ml of LPS for CD19⁺ splenocytes, BM-derived DCs, peritoneal macrophages, and RAW264.7 cells; 25 ng/mL of PMA and 1 µM of ionomycin for Jurkat T cells. Data are mean ± SEM of three independent experiments. *<i>P</i><0.05 versus BE only control. (B) CD3⁺ splenocytes, CD19⁺ splenocytes, and Jurkat T cells were treated with indicated concentrations of BE for 24 h in the absence or presence of respective activator, and the percentages of apoptosis were detected using Annexin V/PI staining. Data are mean ± SEM of three independent experiments. *<i>P</i><0.05 versus untreated controls.</p

An androgen response element driven reporter assay for the detection of androgen receptor activity in prostate cells

<div><p>The androgen receptor (AR) transcription factor plays a key role in the development and progression of prostate cancer, as is evident from the efficacy of androgen-deprivation therapy, AR is also the most frequently mutated gene, in castration resistant prostate cancer (CRPC). AR has therefore become an even more attractive therapeutic target in aggressive and disseminated prostate cancer. To investigate mechanisms of AR and AR target gene activation in different subpopulations of prostate cancer cells, a toolkit of AR expressor and androgen response element (ARE) reporter vectors were developed. Three ARE reporter vectors were constructed with different ARE consensus sequences in promoters linked to either fluorescence or luciferase reporter genes in lentiviral vector backbones. Cell lines transduced with the different vectors expressed the reporters in an androgen-dependent way according to fluorescence microscopy, flow cytometry and multi-well fluorescent and luminescence assays. Interestingly, the background reporter activity in androgen-depleted medium was significantly higher in LNCaP cells compared to the prostate transit amplifying epithelial cell lines, EP156T-AR and 957E/hTERT-AR with exogenous AR. The androgen-induced signal to background was much higher in the latter benign prostate cells than in LNCaP cells. Androgen-independent nuclear localization of AR was seen in LNCaP cells and reduced ARE-signaling was seen following treatment with abiraterone, an androgen synthesis inhibitor. The ARE reporter activity was significantly stronger when stimulated by androgens than by β-estradiol, progesterone and dexamethasone in all tested cell types. Finally, no androgen-induced ARE reporter activity was observed in tumorigenic mesenchymal progeny cells of EP156T cells following epithelial to mesenchymal transition. This underscores the observation that expression of the classical luminal differentiation transcriptome is restricted in mesenchymal type cells with or without AR expression, and presence of androgen.</p></div

Comparison of different AR expression vectors in 293FT cells.

<p>293FT cells were co-transfected with ARE reporter CCS-1019L and three separate AR expression vectors. The cells were grown with or without synthetic androgen 1 nM R1881 for 24 hours. The AR expression was detected by measuring Firefly luminescent signals. These values were normalized by Renilla luminescence values. The error bars show the standard error of the mean (SEM) from three independent experiments. Significance was confirmed by using unpaired two-tailed Student’s t-test. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.</p

ARE reporter response to different steroids.

<p>FACS analysis of mCherry signals in (A) EP156T-AR-241B, (B) 957E/hTERT-AR-241B and (C) LNCaP-241B cells. Cells were grown in androgen free medium and treated with different AR agonists (1 nM R1881 and 1 μM DHT), steroids (10 nM dexamethasone, 10 nM progesterone and 1 μM β-estradiol), AR antagonist (10 μM enzalutamide) and anti-androgen (10 μM abiraterone). The error bars show the standard deviation (SD) from three independent experiments.</p

ARE promoter response and sensitivity to AR activity in 293FT cells.

<p>(A) Three different ARE promoter sequences, 241B, 248B and 249B inserted in a Gaussia luciferase reporter vector, were co-transfected with AR expression vector pLenti6.3/AR-GC-E2325 into 293FT cells. Cells were treated with 1 nM R1881 for 24 hours and Gaussia luciferase values were detected. These values were normalized by the MTS assay. (B) The 241B ARE promoter mCherry reporter vector was co-transfected with AR expression vector pLenti6.3/AR-GC-E2325 in 293FT cells. Fluorescence microscopy was performed after stimulating the cells with 1 nM R1881 for 24 hours. The error bars show the standard error of the mean (SEM) from three independent experiments. Significance was confirmed by using unpaired two-tailed Student’s t-test. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.</p

Internal normalization of ARE reporter signals in prostate cells.

<p>(A) FACS analysis of mCherry fluorescent signals in LNCaP-207-01 and LNCaP-207-02 cells grown in androgen free medium. Cells were treated with different concentrations of androgen R1881, <i>i</i>.<i>e</i>. 0.1 nM, 1 nM and 10 nM, and 10 μM enzalutamide (enza) with 1 nM R1881 for 24 hours. The values were normalized by constitutively expressed GFP fluorescent signals. (B) Multi-well mCherry RFU measurements of LNCaP-207-01 and LNCaP-207-02 cells grown in androgen free medium. The cells were treated with different concentrations of androgen R1881, <i>i</i>.<i>e</i>. 0.1 nM, 1 nM and 10 nM, and 10 μM enzalutamide with 1 nM R1881 for 24 hours. The mCherry RFU values were normalized by GFP RFU. (C) Fluorescence microscopy of LNCaP-207-01 cells treated with ± 1 nM R1881 and different concentrations of enzalutamide, <i>i</i>.<i>e</i>. 5 μM, 10 μM and 20 μM for 24 hours. The error bars show the standard error of the mean (SEM) from three independent experiments. Significance was confirmed by using unpaired two-tailed Student’s t-test. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.</p