Cell-Cycle analysis of RES, CUR and CC treated MCF-7 cells (Time-dependent and sensitized).

<p>Briefly, 0.2×10⁶ cells were pre-cultured for 48 h in phenol red-free DMEM then exposed for 3, 6 and 24 h with 10 µM/100 µM RES {A (MCF-7), B (MDA MB-231)}, 10 µM CC (C), 10 µM/30 µM CUR {D (MCF-7), E (MDA MB-231)} or preconditioned cells with polyphenols (RES: 10/100 µM and CUR: 10/30 µM) for 6 h and continued with CC (10 µM) for next 18 h (total of 24 h). Following incubations the MCF-7 (F) and MDA MB-231 (G) cells were harvested, permeabilized, stained with Propidium Iodide (40 µg/mL) and analyzed by Flow Cytometer (employing Modfit Software); % CV <10. Data shown are the mean ± S.E. of one of the three similar experiments each performed in triplicate. * P<0.05; ** P<0.01; *** P<0.001, calculated compared to control. $P<0.05;$$P<0.01;$$$ P<0.001 with respect to individual CC dose and # P<0.05; ## P<0.01; ### P<0.001 with respect to individual RES dose.</p

Ser-167 phosphorylation of AF-1 region of ER α.

<p>3×10⁶ MCF-7/MDA MB-231 cells were exposed to CC (10 µM)/polyphenols <i>per se</i> (RES: 10/100 µM and CUR: 10/30 µM) or sensitized with low/high dose polyphenols for 6 h and continued with CC (10 µM) for next 18 h. 50 µg of the whole cell lysate was separated on a 10% SDS-PAGE, probed with respective antisera following transfer to a nitrocellulose membrane with immunoblotting using β-actin as control. Data shown is one of three similar experiments each performed in triplicate.</p

Time-dependent ROS-generation and Mitochondrial Membrane Potential (ΔΨm).

<p>0.2×10⁶ MCF-7/MDA MB-231 cells were plated in 6-well plates, treated time-dependently (3, 6 and 24 h) with CC (10 µM) and polyphenols <i>per se</i> {(RES: 10/100 µM and CUR: 10/30 µM; A (ROS-generation) and B (ΔΨm)}; preconditioned cells with low and high dose polyphenols for 6 h and continued with CC (10 µM) for next 18 h {C (ROS-generation) and D (ΔΨm)}. Cells were then rinsed with PBS and then placed in a preheated 37°C fluorimeter for time-dependent measurement of generation of H₂O₂ (DCFDA). ΔΨm was determined on a Beckton–Dickinson Fluorescence Activated Cell Sorter. Flow Cytometry analysis of MCF-7 and MDA MB-231 cells stained with DCFDA was performed to confirm ROS results using N-Acetyl-l-Cysteine (L-NAC, 5 mM) 1 hr prior to treatment (E). Data shown are the Mean ± S.E. of one of the three similar experiments each performed in triplicate. * P<0.05; ** P<0.01; *** P<0.001, calculated compared to control. $P<0.05;$$P<0.01;$$$ P<0.001 with respect to individual CC dose and # P<0.05; ## P<0.01; ### P<0.001 with respect to individual RES dose.</p

Time-dependent JNK, p38 and p53 pathway inhibition studies induced by CC and polyphenols alone and JNK/p38-dependent apoptosis in sensitized cells.

<p>10⁴ MCF-7 (A)/MDA MB-231 (B) cells were pre-cultured for 48 h in phenol red-free DMEM then exposed to CC (10 µM)/polyphenols <i>per se</i> (RES: 10/100 µM and CUR: 10/30 µM) for MTT analysis. Percentage inhibition was determined per the formula {(Absorbance of Control – Absorbance of drug treated cells without and with inhibitor)/Absorbance of Control cells} ×100. For inhibitor experiments through Flow Cytometry, 0.2×10⁶ MCF-7 (C)/MDA MB-231 (D) cells were pre-cultured for 48 h in phenol red-free DMEM, exposed to CC (10 µM)/polyphenols <i>per se</i> (RES: 10/100 µM and CUR: 10/30 µM) or preconditioned with low/high dose polyphenols for 6 h and continued with CC (10 µM) for next 18 h. JNK (10 µM SP600125), p38 pathway (1 µM SB 203580), Pifithrin (15 µM Pif) and zVAD-fmk (30 µM) inhibitors were added during the last 2 hours prior to CC exposure, followed by the analysis of the Sub-G0/G1 fraction by FACS. Each data point is the Mean ± S.E. of one of three similar experiments each performed in triplicate. * P<0.05, ** P<0.01, *** P<0.001 drugs compared to drug treatment without inhibitor.</p

JNK/p38-dependent modulation of expression of Antioxidant enzymes, p53 and apoptogenic factors in MCF-7 cells`1.

<p>For expression analysis, 3×10⁶ MCF-7 cells were pre-cultured for 48 h in phenol red-free DMEM and exposed to CC (10 µM) and polyphenols <i>per se</i> {RES: 10/100 µM (A & B) and CUR: 10/30 µM (C & D)} or sensitized with low/high dose polyphenols for 6 h and continued with CC (10 µM) for next 18 h. Using 10 µM of SP600125 (JNK inhibitor)/1 µM of SB 203580 (p38 inhibitor) 2 hours prior to CC exposure, 50 µg of the whole cell lysate was separated on a 10% SDS-PAGE gel, probed with respective antisera following transfer to a nitrocellulose membrane and subsequent immunoblotting. β-actin was used as a protein loading control. (A) RES/CC+RES without and with SP600125; (B) RES/CC+RES without and with SB203580; (C) CUR/CC+CUR without and with SP600125; (D) CUR/CC+CUR without and with SB203580. However, for c-Jun and phospho ATF-2 detection, immunoprecipitation and detection was carried out as detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037736#s2" target="_blank">Materials and Methods</a>. Data shown is representative of one of the three similar experiments each performed in triplicate.</p