Additional file 1: of Peri-foci adipose-derived stem cells promote chemoresistance in breast cancer

Supplementary material. (XLS 1016 kb

Decrease of tumor volume by tamoxifen is not influenced by combination treatment of risperidone.

<p>In T47D-inoculated female nude mice, tumor masses had been recorded since day 21, and the individual or combination regimens of each drug [25 µg tamoxifen (Tam) per mouse or/and 2.5 µg risperidone (Ris) per mouse] were started at Day42 with intraperitoneal injection every 2 days. Tumor volume (tumor volume  =  length×width²×0.5) was recorded once a week until Day 91. Graph shows mean ± SEM of four mice in each group.</p

Regulation of anti-apoptotic and pro-apoptotic protein expression caused by tamoxifen are not affected by risperidone.

<p>Cells were treated with 1 µM tamoxifen with or without 3 µM risperidone or 0.3 µM fluoxetine for 72 hours. Treatment of tamoxifen with or without risperidone resulted in decreased protein expression of Bcl-2 (A) and Bcl-x_L (B). Protein expression of Bax (C) and Bak (D) were increased by tamoxifen with or without risperidone. Graphs show mean ± SEM of three or more independent experiments. *, <i>p</i><0.05 to control group; #, <i>p</i><0.05 to tamoxifen-treated group; t-test. Tam, tamoxifen; Ris, risperidone; Flx, fluoxetine.</p

Risperidone shows no interference in tamoxifen-induced cytotoxic effect in T47D cells.

<p>Cells were treated with risperidone (<b>A</b>) or its main metabolite paliperidone (0.01–10 µM) (<b>B</b>) for 7 days. Cells were treated with control vehicle (closed bar) or 1 µM tamoxifen (open bar) with or without risperidone (<b>C</b>) or fluoxetine (<b>D</b>) for 7 days. Cell viability of T47D cells was examined by crystal violet (CV) staining. Graphs show mean ± S.E.M. of at least three independent experiments. <b>*</b>, <i>p</i><0.05 to control vehicle group; #, <i>p</i><0.05 to control tamoxifen alone group; t-test.</p

Risperidone has no influence on tamoxifen-induced cleavage of caspases and PARP-1 in T47D cells.

<p>Cells were treated with 1 µM tamoxifen with or without 3 µM risperidone or 0.3 µM fluoxetine for 72 hours. Representative protein blotting images are shown in (<b>A</b>). Treatment of tamoxifen with or without risperidone resulted in increased protein expression of cleaved caspase 9 (<b>B</b>), caspase 7 (<b>C</b>), caspase 3 (<b>D</b>), and PARP-1 (<b>E</b>). Graphs show mean ± SEM of three or more independent experiments. <b>*</b>, <i>p</i><0.05 to control group; #, <i>p</i><0.05 to tamoxifen-treated group; t-test. Tam, tamoxifen; Ris, risperidone; Flx, fluoxetine.</p

Tamoxifen down-regulates cell cycle regulators pRb, cyclin D1 and oncoprotein c-Myc without disturbing by risperidone.

<p>Cells were treated with 1 µM tamoxifen with or without 3 µM risperidone or 0.3 µM fluoxetine for 48 hours. Protein expression of cell cycle regulators pRb (A) and cyclin D1 (B) and oncoprotein c-Myc (C) were measured by Western blotting. Graphs show mean ± SEM of three or more independent experiments. *, <i>p</i><0.05 to control group; #, <i>p</i><0.05 to tamoxifen-treated group; t-test. Tam, tamoxifen; 4-OHTam, 4-hydroxy-tamoxifen; Endx, endoxifen; Ris, risperidone; Pali, paliperidone; Flx, fluoxetine.</p

T47D human breast cancer cells exhibit tamoxifen-induced cytotoxic effect dose- and time-dependently.

<p>(<b>A</b>) T47D human breast cancer cells but not MCF-7 cells expressed significant amount of CYP2D6 protein. A549 human lung cancer was loaded as positive control which shows prominent protein expression. (<b>B</b>) Cells were treated with tamoxifen, 4-OH-tamoxifen, and endoxifen (0.1–3 µM) for 7 days. Cell viability of T47D cells was examined by crystal violet (CV) staining (<b>C, D</b>) Tamoxifen (1 µM), 4-OH-tamoxifen (1 µM), and endoxifen (1 µM) markedly inhibited cell viability from Day3 to Day7 in T47D cells, measured by both crystal violet (CV) staining and MTT assay. Graphs show mean ± S.E.M. of at least three independent experiments. <b>*</b>, <i>p</i><0.05 to control group; t-test. Tam, tamoxifen; 4-OHTam, 4-hydroxy-tamoxifen; Endx, endoxifen.</p

Tamoxifen-induced cell cycle arrest in G0/G1 phase is not interfered by risperidone in T47D cells.

<p>Cells were treated with tamoxifen (1 µM), 4-OH-tamoxifen (1 µM), endoxifen (1 µM), risperidone (3 µM), paliperidone (3 µM), tamoxifen with 3 µM risperidone, or tamoxifen with 0.3 µM fluoxetine for 7 days. Cell viability was examined by crystal violet staining (<b>B</b>), MTT assay (<b>C</b>), and SRB assay (<b>D</b>). Representative crystal violet staining was shown as (<b>A</b>) and quantified by spectrophotometry (<b>B</b>). (<b>E</b>, <b>F</b>) Tamoxifen-induced cytostasis analyzed by flow cytometry showed that cell cycle was arrested at G0/G1 phase, since the percentage of cells at G0/G1 phase was markedly increased, and percentage of cells at S and G2/M phase were decreased respectively by tamoxifen treatment for 2 days. Graphs show mean ± S.E.M. of at least three independent experiments. <b>*</b>, <i>p</i><0.05 to control group; #, <i>p</i><0.05 to tamoxifen-treated group; t-test. Tam, tamoxifen; 4-OHTam, 4-hydroxy-tamoxifen; Endx, endoxifen; Ris, risperidone; Pali, paliperidone; Flx, fluoxetine.</p

RNAi knockdown of CSK does not affect MCF-7 cell sensitivity to tamoxifen or paclitaxel.

<p>Cells were infected with empty lentivirus vector (pLKO.1) or two independent clones of lentiviruses expressing different shRNA species targeting CSK shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060889#pone-0060889-g001" target="_blank">Figure 1</a> (CSK KD#1 and #2) and then exposed to 1 µM 4-hydroxytamoxifen (4-OHT) for 10 days (A) or 1–1000 nM paclitaxel for 2 days (B). Cell viability was determined by crystal violet staining (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060889#pone.0060889.s003" target="_blank">Fig. S3</a>) and quantified by spectrophotometry (mean±SEM of three or more independent experiments).</p

Both fulvestrant and 17β-estradiol (E2) enhance proteasomal degradation of ERα protein in MCF-7 cells.

<p>(A–C) Fulvestrant (A) and E2 (B) caused time-dependent reduction in ERα protein expression: Western blotting. Intensities of ERα protein bands were determined by densitometry (C, mean±SEM of three independent experiments. Asterisks indicate statistical significance, p<0.05 to vehicle control). (D, E) E2 dose-dependent reduction in ERα protein expression. Cells were exposed to varying concentrations of E2 for 6 hours and subjected to Western blotting analysis of ERα protein (D). Intensities of ERα protein bands were determined by densitometry (E, mean±SEM of three independent experiments. Asterisk indicates t-test significance p<0.05 to vehicle control). (F–H), Pre-exposure to MG132 dose-dependently prevented reduction in ERα protein expression caused by fulvestrant (F) and E2 (G). Con, vehicle control (0.1% ethanol). Cells were exposed to varying concentrations of MG132 for 30 minutes and then exposed additionally to fulvestrant or E2 for 6 hours. Intensities of ERα protein bands were determined by densitometry (H, mean±SEM of three independent experiments. Asterisks indicate statistical significance, p<0.05).</p