aaaConsumables, Chemicals and Kits for Study on “In Vitro and In Vivo

Anticancer Activity of Several Plants Methanolic Extract Against Human Breast

Adenocarcinoma”

In vitro study

 Human breast adenocarcinoma (MFC-7) cells (ATCC: Manassas, VA)

 RPMI-1640 medium supplemented with
 10% FBS, glutamine (2 raM),
 penicillin (100 units/mL)
 streptomycin (100 µg/mL)
 96-well plates
 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay
 Methanol
 DMSO
 malondialdehyde (MDA) kit (Cayman Chemical Company, USA) - DAH BELI
 Cell cycle Distribution Analysis - A Cycle TEST PLUS DNA Reagent Kit (BD Biosciences
Pharmingen (Franklin Lakes, NJ, USA)) - DAH BELI
 Apoptotic Cell Death - Annexin V-FITC Apoptosis Detection Kit I (BD Biosciences
Pharmingen (Franklin Lakes, NJ, USA)) 550911
 Caspase Glo 3/7 kit were purchased from Promega.

In Vivo study

Azoxymethane (AOM), 10 % (v/v) neutral buffered formalin, TRI Reagent®, and specific
primers were bought from Sigma (St. Louis, MO, USA). DAH BELI
Dulbecco’s Modified Eagle Medium (DMEM), RPMI-1640 medium, Mycoplex™ fetal
bovine serum (FBS), penicillin and streptomycin (100×), and trypsin EDTA (1×) were
obtained from PAA Laboratories GmBH (Pasching, Austria).
Colorimetric assay kit for caspase-3 and caspase-8 was bought from Genscript Corporation
Inc (Piscataway, NJ, USA).
Western blotting reagents were purchased from Bio-Rad (Hercules, CA, USA).
Primary antibodies included:
o β-catenin (E-5) mouse monoclonal antibody (1:50 dilution, catalogue number sc-7963;
Santa Cruz Biotechnology, Inc.),
o COX-2 (N-20) antibody (1:50 dilution, catalogue number sc-1746; Santa Cruz
Biotechnology, Inc.), and
o rabbit polyclonal to Ki-67 (1:200 dilution, catalogue number AB 66155, Abcam).
a rabbit polyclonal antibody against GST-α and µ (Santa Cruz Biotech, Santa Cruz, CA)
Primary antibodies against Proliferating Cell Nuclear Antigen (PCNA)
primary antibodies against cyclin D1 (diluted 1:100) and c-myc (diluted 1:50) (Abcam, UK)
Proteome Profiler Human Apoptosis Array Kit (ARY009) (R&D Systems)
Apoptosis using TUNEL assay - The DeadEnd™ Fluorometric TUNEL System (Promega (Madison,
WI, USA))

 Apoptosis
o Acridine orange (AO) and ethidium bromide (EB) double staining assay
 Analysis of cell morphology
o Annexin V/propidium iodide (PI) double staining assay
 Detection of early and late apoptotic cell death
o Western blot analysis
 Expression of cell cycle controlling proteins/cell signalling pathways
– P21, p27, p53, CDK2, SDK4, cyclin D1, Rb, pRb, caspase-7, PARP and E2F1
– Bax, Bcl-2, procaspase-9, procaspase-3, PARP, NAG-1, and -actin
o DNA fragmentation assay
o Effect on mitochondrial membrane potential and nuclear morphology